SYNTHESIS AND CHARACTERIZATION OF ALCOHOL-FREE TYROSINASE
ENCAPSULATED SILICA AEROGEL
NOR SURIANI BINTI HJ. SANI
UNIVERSITI TEKNOLOGI MALAYSIA

SYNTHESIS AND CHARACTERIZATION OF ALCOHOL-FREE TYROSINASE
ENCAPSULATED SILICA AEROGEL
NOR SURIANI BINTI HJ. SANI
A thesis submitted in fulfilment of the requirements for the award of the degree of
Master of Science (Chemistry)
Faculty of Science
Universiti Teknologi Malaysia
AUGUST 2009

iii

iv
ACKNOWLEDGEMENT
In the name of Allah the Almighty Lord of the world. Thanks to Him for giving me the opportunity and will to finish this thesis. Heartfelt thanks to my project supervisor, Prof. Dr. Halimaton Hamdan, who introduced me to the field of catalysis. Her patience, criticism and thoughtful guidance throughout this study are greatly appreciated. Without her continued support and interest, this thesis would not be the same as presented here. I would like to express my sincere appreciation and gratitude to Dr. Lee Siew Ling for her knowledge, guidance, evaluation and supporting me throughout the undertaking of this research. I am also very thankful to my research team, especially to Mrs. Fitri Hayati, Mrs. Rozana Abu Bakar and Ms.
Nurul Hidayah Yunus for giving me the motivation, knowledge, assistance and valuable advice in completing this project.
My appreciation also goes to all the staff at Faculty of Science, UTM and
Department of Chemistry, who in many ways contributes to the success of my study.
Not forgetting my beloved husband, Nik Ahmad Nizam Nik Malek for his endless support, encouragement, friendship, advice and understanding. I greatly appreciate it.
I want to extend my utmost gratitude and appreciation to my parents, family, fellow friends and those who provide assistance in this research either intentionally or unintentionally throughout the progress of this research. Finally, I would like to acknowledge MOSTI, IRPA and NSF for financial support and scholarship.

v
ABSTRACT
Encapsulation of tyrosinase enzyme into nanoporous silica aerogel via an alcohol-free colloidal sol-gel route using rice husk ash (RHA) as silica source was studied. Tyrosinase encapsulated silica aerogel (TESA) was synthesized with and without solvent extraction process at room temperature and neutral pH in order to study their effect on the enzyme activity and to minimize enzyme denaturation. The physicochemical properties of TESA was characterized by X-ray diffraction (XRD) technique, fourier transformed-infrared (FTIR) spectroscopy, field emissionscanning electron microscopy (FESEM), energy dispersive X-ray (EDX) analysis, transmission electron microscopy (TEM) and thermogravimetric analysis (TGA).
These characterizations confirmed tyrosinase in TESA was located inside the network of silica aerogel. Enzymatic activity of tyrosinase was assayed through the reduction of ascorbic acid using UV Visible spectrophotometer. Almost 98% of tyrosinase was successfully loaded into silica aerogel as determined by the leaching test of TESA. TESA without solvent extraction showed higher tyrosinase activity than TESA extracted by amyl acetate/acetone (v/v:1/1). The highest activities for both TESA were obtained with 10.00 mg/mL of enzyme loading that was aged for 2 days. The stability of tyrosinase in TESA was enhanced towards extreme temperature as well as acidic and basic conditions. Free tyrosinase was totally inactivated at pH < 4 and pH > 9 and at temperature exceeding 55 °C, while TESA showed a significant activity at these conditions. In the application of TESA, about
80% of phenol was removed after 3 hours contact with TESA. The reusability of tyrosinase in TESA was observed to be very high since TESA can be reused to remove phenol up to 10 times without significant loss. As a conclusion, nanoporous silica aerogel from RHA prepared with and without solvent extraction techniques can be used as suitable support for the improvement of the tyrosinase stability and it can be applied to remove phenol.

vi
ABSTRAK
Kajian terhadap enzim tirosinase yang dikapsulkan ke dalam aerogel berasaskan silika melalui kaedah koloid sol-gel tanpa melibatkan alkohol telah dijalankan. Pengkapsulan tirosinase ke dalam aerogel berasaskan silika (TESA) yang mempunyai liang bersaiz nano telah disintesis menggunakan abu sekam padi (RHA) sebagai sumber silika. Di dalam kaedah ini, TESA disintesis pada suhu bilik dan pH neutral melalui proses pengekstrakan dan tanpa pengekstrakan bagi mengkaji kesannya terhadap aktiviti enzim di samping mengurangkan penyahaslian enzim.
Sifat-sifat fiziko-kimia TESA telah dicirikan dengan kaedah XRD, FTIR, FESEM,
EDX, TEM dan TGA. Pencirian mengesahkan bahawa tirosinase telah berjaya dikapsulkan ke dalam silika aerogel dan ianya terletak di dalam rangkaian jaringan silika aerogel. Aktiviti tirosinase telah ditentukan dengan kaedah spektofotometer
UV Tampak melalui proses penurunan asid askorbik. Hasil Ujian Larut Lesap terhadap TESA menunjukkan hampir 98% tirosinase berjaya dimuatkan di dalam silika aerogel. TESA yang disintesis tanpa melibatkan pengekstrakan mempunyai aktiviti yang lebih tinggi berbanding TESA yang disintesis melalui pengekstrakan menggunakan pelarut amil asetat/aseton (v/v:1/1). Aktiviti yang tinggi bagi keduadua TESA diperolehi apabila sebanyak 10.00 mg/mL enzim dikapsulkan ke dalam silika aerogel pada suhu bilik selama 2 hari. Kestabilan tirosinase di dalam TESA terhadap asid dan alkali meningkat. Tirosinase tidak menunjukkan sebarang aktiviti pada pH < 4 dan pH > 9 juga pada suhu melebihi 55 °C tetapi tirosinase di dalam
TESA menunjukkan aktiviti pada keadaan tersebut. Kajian penggunaan TESA terhadap penyingkiran fenol menunjukkan 80% fenol berjaya disingkirkan selepas bertindak balas dengan TESA selama 3 jam. Kebolehan tirosinase di dalam TESA untuk menyingkirkan fenol berulang-kali adalah tinggi kerana TESA boleh digunakan sehingga 10 kali tanpa menunjukkan sebarang penyusutan yang ketara.
Kesimpulannya, silika aerogel daripada RHA, dengan liang bersaiz nano, yang disintesis sama ada melalui proses pengekstrakan atau tanpa proses pengekstrakan, mampu menjadi tapak kepada enzim bagi meningkatkan kestabilan tirosinase dan menyingkirkan fenol.

vii
1.4
1.5
1.6
Objectives of Research
Scope of Research
Outline of Research
CHAPTER
TABLE OF CONTENTS
TITLE PAGE
TITLE
DECLARATION
DEDICATION
ACKNOWLEDGEMENT
ABSTRACT
ABSTRAK
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF SYMBOLS
LIST OF ABBREVIATIONS
LIST OF APPENDICES xii xv xvi xvii v vi vii xi i ii iii iv
I INTRODUCTION
1.1 Background of Research
1
1
4
7
9
5
7

viii
2.1
2.3
2.4
Rice Husk Ash as a Silica Source
The Sol-Gel Process
2.3.1 Hydrolysis and Condensation
Immobilization of Enzyme
2.4.3 Immobilization of Enzymes by
Encapsulation via Sol-Gel Method
2.5.1 Immobilized Tyrosinase in the
Removal of Phenol
27
30
3.1 Raw Materials and Chemical Reagents
3.2 Synthesis of Tyrosinase Encapsulated Silica
Aerogel (TESA)
3.2.1
3.2.2
3.2.3
Preparation of Sodium Silicate
Synthesis of Wet Gel
Drying of Wet Gel
3.3 Characterization of Tyrosinase Encapsulated
Silica Aerogel (TESA)
3.3.1 X-Ray Diffraction (XRD) Technique
3.3.2 Fourier Transformed-Infrared (FTIR)
3.3.3
3.3.4
Spectroscopy
Field Emission-Scanning Electron
Microscopy (FESEM)
Energy Dispersive X-ray Analysis
(EDX)
33
33
34
34
35
35
36
37
40
43
44
10
10
12
14
15
19
19
22
23
24
25

IV ix
45
(TEM)
3.4 Optimization of Synthesis Condition
3.4.1 Effect of Solvent Extraction
3.4.2
3.4.3
Effect of Aging Period
Effect of Enzyme Loading
3.5 Assays of Enzymatic Activity
3.5.1 Assay of Free Tyrosinase Activity
3.5.2 Assay of Encapsulated Tyrosinase
Activity
46
47
47
48
48
49
50
51
3.5.4
3.5.5
Influence of Temperatures
Influence of pH
53
53
54
54
Aerogel (TESA)
3.6.1 The Removal of Phenol 54
55
RESULTS AND DISCUSSION
4.1 Synthesis of Tyrosinase Encapsulated Silica
Aerogel (TESA)
4.2 Characterization of Tyrosinase Encapsulated
Silica Aerogel (TESA)
4.2.2
4.2.4
4.2.5
56
56
58
X-ray Diffraction (XRD) Analysis
Field Emission Scanning Electron
Microscopy (FESEM)
Energy Dispersive X-ray Analysis
(EDX)
64
68
Microscopy 69
(TEM)
59
59
60

REFERENCES
APPENDICES
73
75
75
78
79
81
81 x
4.3 Optimization of Synthesis Conditions
4.3.1 Effect of Solvent Extraction
4.3.2
4.3.3
Effect of Aging Period
Effect of Enzyme Loading
4.4.1 Assay of Free Tyrosinase and
Tyrosinase Encapsulated Silica
Aerogel (TESA) Activity
4.4.3
4.4.4
Influence of Temperatures
Influence of pH
Aerogel (TESA)
4.5.1 The Removal of Phenol
V CONCLUSIONS RECOMMENDATIONS
83
83
85
87
87
88
91
91
94
96
111

TABLE NO.
3.1
4.1
4.2
4.3
LIST OF TABLES
TITLE
The assignments of the main FTIR bands for silica
FTIR wavenumbers and assignment for the functional group present in the tyrosinase
FTIR assignments of silica aerogel
Major elemental analysis (mass%
±
SD) of free tyrosinase, silica aerogel (with and without solvent extraction) and TESA (with and without solvent extraction)
PAGE
42
62
62
68 xi

LIST OF FIGURES
FIGURE NO. TITLE
1.1
1.2
2.1
2.2
The encapsulation of tyrosinase into silica aerogel network
Flow diagram of research activities
(a) Hydrolysis, (b) condensation and (c) polycondensation reactions, during the sol-gel process in the synthesis of silica aerogel
Polymeric structure of silica framework in acidic and basic conditions
2.3
2.4 Oxidation o -quinone
2.5
Monooxygenation reaction catalyzed by tyrosinase
3.1
Oxidation of phenol catalyzed by tyrosinase
Derivation of Bragg’s law for X-ray diffraction
3.2
3.3
4.1
4.2
The illustration of the X-ray powder diffraction method
Types of silanols exist in silica surface
Optical absorption spectra of (a) free tyrosinase,
λ max
= 289.14 nm, (b) TESA (with solvent extraction),
λ max
= 291.28 nm and (c) TESA (without solvent extraction),
λ max
= 292.91 nm
XRD diffractogram of (a) silica aerogel and (b)
TESA
PAGE
6
9
17
18
28
29
31
38
39
41
57
60 xii

4.3
4.4
The FTIR spectra of (a) silica aerogel and (b) free tyrosinase
FTIR spectra of (a) free tyrosinase, (b) silica aerogel,
(c) TESA with SE and (d) TESA without SE
4.5 FESEM showing the surface morphology of (a) silica aerogel with SE and (b)
TESA with SE
4.6
4.7
4.8
FESEM micrographs showing the surface morphology of (a) silica aerogel without SE and (b)
TESA without SE
FESEM micrograph showing the surface morphology of free tyrosinase
TEM micrographs showing the surface morphology of (a) silica aerogel with SE and (b) TESA with SE
4.9
4.10
TEM micrographs showing the surface morphology of (a) silica aerogel without SE and (b) TESA without SE
Thermogravimetry analysis (TGA) and Derivative thermogravimetry (DTG) curves of (a) silica aerogel without SE, weight loss = 21.102%, (b) silica aerogel with SE, weight loss = 16.284%, (c) TESA without
SE, weight loss = 10.696%, (d) TESA with SE, weight loss = 6.632% and (e) free tyrosinase, weight loss = 100%
4.11
4.12
4.13
4.14
4.15
Effect of solvent extraction to the enzymatic activity of TESA with SE
Enzymatic activities of (a) TESA with SE and (b)
TESA without SE; at different aging periods
Enzymatic activities of (a) TESA with SE and (b)
TESA without SE; of different enzyme loadings
Enzymatic activity of (a) free tyrosinase, (b) TESA without SE, (c) TESA with SE; extracted by amyl acetate/acetone (v/v:1/1) and (d) without tyrosinase; at pH 7
Enzymatic activities of (a) free tyrosinase, (b) TESA with SE; extracted by amyl acetate/acetone (v/v:1/1) and (c) TESA without SE; at different temperatures
61
64
65
66
67
70
71
74
76
78
80
82
84 xiii

4.16
4.17
4.18
Enzymatic activities of (a) free tyrosinase, (b) TESA with SE; extracted by amyl acetate/acetone (v/v:1/1) and (c) TESA without SE; at different pH
The effect of contact time on the percent removal of phenol by (a) free tyrosinase, (b) TESA without SE and (c) TESA with SE; extracted by amyl acetate/acetone (v/v:1/1)
Reusability studies of (a) TESA without SE and (b)
TESA with SE; extracted by amyl acetate/acetone
(v/v:1/1), in the removal of phenol
86
88
90 xiv

LIST OF SYMBOLS xv ppm - Part per million
μ g - gram
μ L - Liter
λ - Lambda
θ - Theta

APD
DDW
DTG
EDTA
EDX
FESEM
FTIR
HPLC
NADH
PFC
RHA
-
-
-
-
-
-
-
-
LIST OF ABBREVIATIONS
Ambient Pressure Drying
Double Distilled Deionized Water
Derivative Thermogravimetric Analysis
Ethylene Diamine Tetracetic Acid
Energy Dispersive X-ray Technique
Field Emission Scanning Electron Microscopy
Fourier Transform Infrared
High Performance Liquid Chromatography xvi
-
-
-
Nicotinamide Adenine Dinucleotide
Plug Flow Combustor
Rice Husk Ash
TESA - Tyrosinase Encapsulated Silica Aerogel

xvii
LIST OF APPENDICES
APPENDIX
A
B
C
D
E
Determination of Ascorbic Acid using UV-Vis
Spectrophotometer
EDX Elemental Analysis of Tyrosinase, Silica
Aerogel and TESA
Determination of Phenol using UV-Vis
Spectrophotometer
Paper for R&D Nanotechnology Symposium
2007, Malaysia
Paper for 26th International Symposium on
Space Technology and Science (ISTS), Japan
111
114
119
121
128

CHAPTER 1
INTRODUCTION
1.1
Research Background
Nanotechnology, the process to generate, manipulate and employ nanomaterials, represents an area holding significant promise for health care and biotechnology in many years to come [1-4]. Nanotechnology is now poised to revolutionize the biomedical field ranging from basic studies to disease diagnosis and treatment. Understanding the principle of nanotechnology may provide insight into critical biological system related to disease control, correction of genetic disorder and longevity [3].
Biosensing is one of the most emerging sectors of nanotechnology. Such growth is mainly derived from the expansion of R&D where nano dimension research is introduced to analyze living cell constituents and for efficient drug screening [5]. Biosensors are devices that incorporate biologically active element in intimate contact with a physico-chemical signal; utilize the high sensitivity and selectivity of biological sensing for analytical purposes in various fields of research and technology [6]. Many proteins are expected to play the role as biocomponents used in biosensors. Enzymes are large protein molecules that significantly increase

2 rates of reaction by lowering the activation energy. Enzymes specifically accelerate a huge number of chemical reactions at room temperature and normal pressure [7].
Enzymes offer three major advantages [8]:
•
Higher reaction rates: Up to a factor of 10
12
times greater than the uncatalyzed reactions
•
Reaction conditions: Enzymes operate at room temperatures less than
100 o
C, atmospheric pressures and near neutral pH
•
Reaction specificity: Enzyme specificity for both substrate and product produces fewer side reactions
Immobilizations of biological compounds into inorganic support are usually applied in various fields such as biosensing [9-11], affinity chromatography [12-14] and enzyme reactors [15-17]. One of the most challenging aspects in the development of these matrices is the integration of biological molecules in the host matrix and retaining the functionality of the biomolecules [10]. In biosensing, it is advantageous to use immobilized enzymes rather than free enzymes [18-19].
Following are the reasons for immobilization of enzyme:
•
To improve the stability of enzyme in adverse reaction conditions
•
To improve the stability of enzyme in the presence of organic solvents
•
To separate the enzyme from product stream
•
To allow a continuous flow operations and repetitive usage
The application of immobilized enzymes in analytical chemistry is not a new concept. The importance of immobilized enzymes as analytical reagents in clinical chemistry [20-21], food analysis [22-23] and the pharmaceutical industry [24-25] has

3 been steadily increasing. To simplify the enzymatic measurement of glucose, the principle of the litmus paper used for pH measurement has been implemented [6].
The first ‘enzyme test strip’ has been obtained by the impregnation of filter paper with the glucose-converting enzymes. It can be regarded as the predecessor of optoelectronic biosensors which initiated the development and application of ‘dry chemistry’ [26]. Apart from the application of complex biocatalytic system, intense effort are being made to broaden the spectrum of measurable substances and to improve the analytical parameters of biosensors by the immobilization of several enzymes in a silica host matrixes.
Silica host matrixes, made by the sol-gel process have emerged as a promising platform for immobilization of enzymes [19, 26]. The advantages of their usage in enzyme immobilization include:
•
The high surface area of silica matrix provide the possibility of high enzyme loadings in the matrix
•
The silica matrix consists of surface hydroxyl groups that can be readily attached by enzyme
•
The open pore morphology of silica matrix allows substrates to quickly move into the interior regions of the particle
•
Solvents used in the processing of the silica materials are environmentally benign thus avoid the denaturation of enzyme
Numerous techniques such as physical adsorption, covalent attachment, entrapment and encapsulation in polymer and inorganic matrixes have been explored over the years to achieve a high-yield, reproducible and robust immobilization technique that preserves the activity of the biological molecules [10, 27-29].
Enzymes find a more stable environment upon encapsulation in a silica host, because the polymeric framework grows around the biomolecules, creating a cage, thus

4 protecting the enzyme either from aggregation and unfolding or from microbial attack. Encapsulations also offer a protection for the enzyme against deterioration by the hydrophilic solvent, if the proper gel is selected [28]. Therefore, the encapsulated biomolecules often retain a sufficient level of activity and functionality presumably because of sufficient retention of their native state conformations. Moreover, the matrix pores allow the diffusion of reactant molecules and their reaction with the encapsulated biomolecules. Eventually, encapsulated enzyme can even improve the activity and storage stability of the enzymes and will be easier to be used because they can easily be recovered and washed [30].
In this research, tyrosinase was used as model enzyme because of its wide application in medicine, environmental and industrial systems [8, 24]. Tyrosinase is also suitable for the treatment of phenolic wastes [9, 11, 17]. Tyrosinase like most other enzymes, is expensive and thus the use of the soluble enzyme is not practical
[2]. Therefore, the encapsulation of tyrosinase is very attractive in order to exploit its catalytic properties and improve the cost effectiveness [5]. The improved stability of the encapsulated enzyme allows it to be highly reusable. Moreover, since enzyme does not dissolve in the solution, further purified process is not required and hence the encapsulated tyrosinase is economical to be used repetitively [8].
1.2
Problem Statement
In most of the reported applications, an orthosilicate such as tetramethyl orthosilicate (TMOS) or tetraethyl orthosilicate (TEOS) has been used as silica source in the synthesis of proteins encapsulated silica monoliths [30-33]. These silica sources present the advantages of relatively high purity sources of silica but lowering the enzymatic activity. 70% reduction of enzymatic activity was reported when lipase was encapsulated into TMOS-based silica matrix due to the presence of 5% volume of methanol in the reaction [27]. The usage of TMOS or TEOS as starting

5 materials lead to the generation of alcohol as a by-product and the presence of alcohol has been known to be detrimental to the activity of proteins by causing chain unfolding, aggregation, destruction of secondary and tertiary protein structures to a significant extent [28]. Moreover, such organic silicon precursors are usually too expensive. So the production of silica aerogel in an industrial scale is not economically practical.
Owing to their mesoporous structure, high specific surface area and extremely low thermal conductivity, aerogels are considered as an efficient candidate as a support for protein immobilization. However, the high level of sophistication and the risks involved in the supercritical drying of the gels prohibit the commercial production of the aerogels and their wide exploitation in various potential applications [34-36]. Supercritical drying process is too energy intensive and dangerous that real practice and commercialization are difficult. Therefore, it is necessary to synthesize silica aerogels by an ambient pressure drying (APD) technique at a reasonable cost [37].
1.3 Hypothesis
An alcohol-free aqueous colloidal sol-gel for the synthesis of silica monoliths with encapsulated biological entities that uses rice husk ash as the cheap silicon source for production of pure silicate solution has been developed [28, 38]. This approach completely avoids the generation of alcohol and it allows encapsulation to be carried out at neutral pH and ambient pressure in order to preserve biological activity of proteins. Thus, denaturation of the biomolecule caused by the undesirable interaction with alcohol molecules can be avoided and the degree of conformational change can be reduced.

6
In this research, after the synthesis is complete, tyrosinase molecule is expected to be encapsulated in the silica aerogel network since the tyrosinase was added into the silica sol before the gelation stage. As shown in Figure 1.1, after the addition of tyrosinase, the silica sol begins to link together in three-dimensional (3D) network and subsequently, creates a cage around the tyrosinase molecule. It is also known that when enzyme molecules are mixed with colloidal particles, the interaction between them may result in enzyme adsorption on the particle surface.
Furthermore, the preparation of silica aerogel using water glass precursor followed by an ambient pressure drying is the cheapest and safest method.
Enzyme
Support network
6
Figure 1.1
The encapsulation of tyrosinase into silica aerogel network
This research is proposed to develop an effective method for the immobilization of biomolecule into silica aerogel matrix. Unrevealing the interactions which take place in encapsulation of enzyme in the silica aerogel matrix will contribute to better understanding of various chemical and biochemical processes that occur when different synthesis conditions are applied. The fundamental understanding of the enzyme-silica support interactions can help in improving the fabrication of enzyme encapsulated silica aerogel as a potential

7 biosensor with enhanced thermal stability and enzymatic performance. Hence, this research enables new materials for biosensors to be developed.
The objectives of this research are to: i) Synthesize and characterize tyrosinase encapsulated silica aerogel ii) Investigate the influence of synthesis conditions on biocatalytic activities of tyrosinase encapsulated silica aerogel iii) Investigate the enzymatic activity of tyrosinase encapsulated silica aerogel iv) Study the application of tyrosinase encapsulated silica aerogel in the removal of phenol
1.5 Scope of Research
The project is conducted following various phases as outlined in the flow diagram in Figure 1.2. The tyrosinase encapsulated silica aerogel (TESA) was synthesized via alcohol-free aqueous colloidal sol-gel process according to the established method [28] but with some modification. TESA, which is synthesized from rice husk ash as a silica source, was synthesized with and without solvent extraction (SE) process in order to study their relationship with the enzyme activity.
The products were characterized for their textural properties by using X-ray diffraction technique (XRD) and Fourier transformed-infrared spectroscopy (FTIR).
The interactions of tyrosinase-silica aerogel and its surface morphology were studied

8 by using Field emission-scanning electron microscopy (FESEM), Energy dispersive
X-ray spectroscopy (EDX), Transmission electron microscopy (TEM) and
Thermogravimetric analysis (TGA).
Some of the synthesis parameters in a sol-gel process were investigated for their influences on biocatalytic activities of encapsulated enzyme namely effect of solvent extraction and enzyme loading. In order to obtain information regarding the tyrosinase-silica aerogel interaction, wet gels containing encapsulated tyrosinase was submitted to different aging periods before drying phase.
Meanwhile, biocatalytic activities of the free tyrosinase and TESA were assayed by examining the catecholase activity using UV-Vis spectrophotometer. The properties of TESA were evaluated by studying the activity of tyrosinase at different temperatures and pH ranges, as well as leaching test. TESA was used to remove phenol in aqueous solution and their efficiency in removing phenol and the stability of tyrosinase in TESA was determined through reusability study.

9
Synthesis of tyrosinase encapsulated silica aerogel (TESA)
TESA without solvent extraction (SE) TESA with solvent extraction (SE)
Characterizations of TESA
X-ray diffraction technique (XRD)
Fourier transformed-infrared spectroscopy (FTIR)
Field emission-scanning electron microscopy (FESEM)
Energy dispersive X-ray spectroscopy (EDX)
Transmission electron microscopy (TEM)
Thermogravimetry analysis (TGA)
Optimization of synthesis conditions
Effect of solvent extraction
Aging period
Enzyme loading
Enzyme assay: UV-Vis Spectrophotometer
Measurement of tyrosinase activity
Leaching test
Influence of temperatures
Influence of pH
Application of TESA
Removal of phenol
Reusability
Figure 1.2 Flow diagram of research activities

CHAPTER 1
INTRODUCTION
1.1
Research Background
Nanotechnology, the process to generate, manipulate and employ nanomaterials, represents an area holding significant promise for health care and biotechnology in many years to come [1-4]. Nanotechnology is now poised to revolutionize the biomedical field ranging from basic studies to disease diagnosis and treatment. Understanding the principle of nanotechnology may provide insight into critical biological system related to disease control, correction of genetic disorder and longevity [3].
Biosensing is one of the most emerging sectors of nanotechnology. Such growth is mainly derived from the expansion of R&D where nano dimension research is introduced to analyze living cell constituents and for efficient drug screening [5]. Biosensors are devices that incorporate biologically active element in intimate contact with a physico-chemical signal; utilize the high sensitivity and selectivity of biological sensing for analytical purposes in various fields of research and technology [6]. Many proteins are expected to play the role as biocomponents used in biosensors. Enzymes are large protein molecules that significantly increase

2 rates of reaction by lowering the activation energy. Enzymes specifically accelerate a huge number of chemical reactions at room temperature and normal pressure [7].
Enzymes offer three major advantages [8]:
•
Higher reaction rates: Up to a factor of 10
12
times greater than the uncatalyzed reactions
•
Reaction conditions: Enzymes operate at room temperatures less than
100 o
C, atmospheric pressures and near neutral pH
•
Reaction specificity: Enzyme specificity for both substrate and product produces fewer side reactions
Immobilizations of biological compounds into inorganic support are usually applied in various fields such as biosensing [9-11], affinity chromatography [12-14] and enzyme reactors [15-17]. One of the most challenging aspects in the development of these matrices is the integration of biological molecules in the host matrix and retaining the functionality of the biomolecules [10]. In biosensing, it is advantageous to use immobilized enzymes rather than free enzymes [18-19].
Following are the reasons for immobilization of enzyme:
•
To improve the stability of enzyme in adverse reaction conditions
•
To improve the stability of enzyme in the presence of organic solvents
•
To separate the enzyme from product stream
•
To allow a continuous flow operations and repetitive usage
The application of immobilized enzymes in analytical chemistry is not a new concept. The importance of immobilized enzymes as analytical reagents in clinical chemistry [20-21], food analysis [22-23] and the pharmaceutical industry [24-25] has

3 been steadily increasing. To simplify the enzymatic measurement of glucose, the principle of the litmus paper used for pH measurement has been implemented [6].
The first ‘enzyme test strip’ has been obtained by the impregnation of filter paper with the glucose-converting enzymes. It can be regarded as the predecessor of optoelectronic biosensors which initiated the development and application of ‘dry chemistry’ [26]. Apart from the application of complex biocatalytic system, intense effort are being made to broaden the spectrum of measurable substances and to improve the analytical parameters of biosensors by the immobilization of several enzymes in a silica host matrixes.
Silica host matrixes, made by the sol-gel process have emerged as a promising platform for immobilization of enzymes [19, 26]. The advantages of their usage in enzyme immobilization include:
•
The high surface area of silica matrix provide the possibility of high enzyme loadings in the matrix
•
The silica matrix consists of surface hydroxyl groups that can be readily attached by enzyme
•
The open pore morphology of silica matrix allows substrates to quickly move into the interior regions of the particle
•
Solvents used in the processing of the silica materials are environmentally benign thus avoid the denaturation of enzyme
Numerous techniques such as physical adsorption, covalent attachment, entrapment and encapsulation in polymer and inorganic matrixes have been explored over the years to achieve a high-yield, reproducible and robust immobilization technique that preserves the activity of the biological molecules [10, 27-29].
Enzymes find a more stable environment upon encapsulation in a silica host, because the polymeric framework grows around the biomolecules, creating a cage, thus

4 protecting the enzyme either from aggregation and unfolding or from microbial attack. Encapsulations also offer a protection for the enzyme against deterioration by the hydrophilic solvent, if the proper gel is selected [28]. Therefore, the encapsulated biomolecules often retain a sufficient level of activity and functionality presumably because of sufficient retention of their native state conformations. Moreover, the matrix pores allow the diffusion of reactant molecules and their reaction with the encapsulated biomolecules. Eventually, encapsulated enzyme can even improve the activity and storage stability of the enzymes and will be easier to be used because they can easily be recovered and washed [30].
In this research, tyrosinase was used as model enzyme because of its wide application in medicine, environmental and industrial systems [8, 24]. Tyrosinase is also suitable for the treatment of phenolic wastes [9, 11, 17]. Tyrosinase like most other enzymes, is expensive and thus the use of the soluble enzyme is not practical
[2]. Therefore, the encapsulation of tyrosinase is very attractive in order to exploit its catalytic properties and improve the cost effectiveness [5]. The improved stability of the encapsulated enzyme allows it to be highly reusable. Moreover, since enzyme does not dissolve in the solution, further purified process is not required and hence the encapsulated tyrosinase is economical to be used repetitively [8].
1.2
Problem Statement
In most of the reported applications, an orthosilicate such as tetramethyl orthosilicate (TMOS) or tetraethyl orthosilicate (TEOS) has been used as silica source in the synthesis of proteins encapsulated silica monoliths [30-33]. These silica sources present the advantages of relatively high purity sources of silica but lowering the enzymatic activity. 70% reduction of enzymatic activity was reported when lipase was encapsulated into TMOS-based silica matrix due to the presence of 5% volume of methanol in the reaction [27]. The usage of TMOS or TEOS as starting

5 materials lead to the generation of alcohol as a by-product and the presence of alcohol has been known to be detrimental to the activity of proteins by causing chain unfolding, aggregation, destruction of secondary and tertiary protein structures to a significant extent [28]. Moreover, such organic silicon precursors are usually too expensive. So the production of silica aerogel in an industrial scale is not economically practical.
Owing to their mesoporous structure, high specific surface area and extremely low thermal conductivity, aerogels are considered as an efficient candidate as a support for protein immobilization. However, the high level of sophistication and the risks involved in the supercritical drying of the gels prohibit the commercial production of the aerogels and their wide exploitation in various potential applications [34-36]. Supercritical drying process is too energy intensive and dangerous that real practice and commercialization are difficult. Therefore, it is necessary to synthesize silica aerogels by an ambient pressure drying (APD) technique at a reasonable cost [37].
1.3 Hypothesis
An alcohol-free aqueous colloidal sol-gel for the synthesis of silica monoliths with encapsulated biological entities that uses rice husk ash as the cheap silicon source for production of pure silicate solution has been developed [28, 38]. This approach completely avoids the generation of alcohol and it allows encapsulation to be carried out at neutral pH and ambient pressure in order to preserve biological activity of proteins. Thus, denaturation of the biomolecule caused by the undesirable interaction with alcohol molecules can be avoided and the degree of conformational change can be reduced.

6
In this research, after the synthesis is complete, tyrosinase molecule is expected to be encapsulated in the silica aerogel network since the tyrosinase was added into the silica sol before the gelation stage. As shown in Figure 1.1, after the addition of tyrosinase, the silica sol begins to link together in three-dimensional (3D) network and subsequently, creates a cage around the tyrosinase molecule. It is also known that when enzyme molecules are mixed with colloidal particles, the interaction between them may result in enzyme adsorption on the particle surface.
Furthermore, the preparation of silica aerogel using water glass precursor followed by an ambient pressure drying is the cheapest and safest method.
Enzyme
Support network
6
Figure 1.1
The encapsulation of tyrosinase into silica aerogel network
This research is proposed to develop an effective method for the immobilization of biomolecule into silica aerogel matrix. Unrevealing the interactions which take place in encapsulation of enzyme in the silica aerogel matrix will contribute to better understanding of various chemical and biochemical processes that occur when different synthesis conditions are applied. The fundamental understanding of the enzyme-silica support interactions can help in improving the fabrication of enzyme encapsulated silica aerogel as a potential

7 biosensor with enhanced thermal stability and enzymatic performance. Hence, this research enables new materials for biosensors to be developed.
The objectives of this research are to: i) Synthesize and characterize tyrosinase encapsulated silica aerogel ii) Investigate the influence of synthesis conditions on biocatalytic activities of tyrosinase encapsulated silica aerogel iii) Investigate the enzymatic activity of tyrosinase encapsulated silica aerogel iv) Study the application of tyrosinase encapsulated silica aerogel in the removal of phenol
1.5 Scope of Research
The project is conducted following various phases as outlined in the flow diagram in Figure 1.2. The tyrosinase encapsulated silica aerogel (TESA) was synthesized via alcohol-free aqueous colloidal sol-gel process according to the established method [28] but with some modification. TESA, which is synthesized from rice husk ash as a silica source, was synthesized with and without solvent extraction (SE) process in order to study their relationship with the enzyme activity.
The products were characterized for their textural properties by using X-ray diffraction technique (XRD) and Fourier transformed-infrared spectroscopy (FTIR).
The interactions of tyrosinase-silica aerogel and its surface morphology were studied

8 by using Field emission-scanning electron microscopy (FESEM), Energy dispersive
X-ray spectroscopy (EDX), Transmission electron microscopy (TEM) and
Thermogravimetric analysis (TGA).
Some of the synthesis parameters in a sol-gel process were investigated for their influences on biocatalytic activities of encapsulated enzyme namely effect of solvent extraction and enzyme loading. In order to obtain information regarding the tyrosinase-silica aerogel interaction, wet gels containing encapsulated tyrosinase was submitted to different aging periods before drying phase.
Meanwhile, biocatalytic activities of the free tyrosinase and TESA were assayed by examining the catecholase activity using UV-Vis spectrophotometer. The properties of TESA were evaluated by studying the activity of tyrosinase at different temperatures and pH ranges, as well as leaching test. TESA was used to remove phenol in aqueous solution and their efficiency in removing phenol and the stability of tyrosinase in TESA was determined through reusability study.

9
Synthesis of tyrosinase encapsulated silica aerogel (TESA)
TESA without solvent extraction (SE) TESA with solvent extraction (SE)
Characterizations of TESA
X-ray diffraction technique (XRD)
Fourier transformed-infrared spectroscopy (FTIR)
Field emission-scanning electron microscopy (FESEM)
Energy dispersive X-ray spectroscopy (EDX)
Transmission electron microscopy (TEM)
Thermogravimetry analysis (TGA)
Optimization of synthesis conditions
Effect of solvent extraction
Aging period
Enzyme loading
Enzyme assay: UV-Vis Spectrophotometer
Measurement of tyrosinase activity
Leaching test
Influence of temperatures
Influence of pH
Application of TESA
Removal of phenol
Reusability
Figure 1.2 Flow diagram of research activities

CHAPTER 2
LITERATURE REVIEW
2.1 Rice Husk Ash as a Silica Source
Rice husk is a major waste product of the rice-processing industries. Like most of the other biomass materials such as sugar, cane leaf, corn leaf, rice husk is also recognized as a potential source for energy generation from gasification or incineration processes. Acid leaching [39] and gasification [40] methods have been investigated for recovering silica from rice husk. Of all the plant residues, the ash of rice husk contains the highest amount of silica (SiO
2
). The burning of rice husk in air results in the formation of rice husk ash (RHA) with SiO
2 content of 85% to 98% depending on the burning condition, furnace type, rice variety, moisture content, weather and geographic area [41].
RHA is available in abundance at no cost. The presence of silica in RHA is known since 1938 and extensive literatures have reported the uses of RHA as silica replacement for the production of silicon based materials in industrial and technological applications [42-47]. Some small amounts of inorganic impurities are usually present in the ash together with unburned carbon. The unburned carbon can be removed from the ash by further heating at high temperature; however high

11 temperature usually leads to crystallization of the amorphous silica to cristobalite and/or tridymite [48]. The crystallization of silica in RHA occurs when the burning conditions of husk are uncontrolled [42]. The crystallization form of silica has drawbacks towards the preparation of silicon based materials since silica ash is inactive in its crystalline form [42, 49]. Highly reactive silica in RHA can be produced by maintaining the combustion temperature below 500 °C under oxidising conditions for relatively long period or high temperature up to 680 °C for less than one minute [43]. In addition, the combustion environment affects the specific surface area of silica, hence temperature and environment must be considered in the pyroprocessing of rice husk to produce ash with a maximum reactivity of silica [48].
However, silica in RHA can remain in amorphous form if the combustion temperatures up to 900 °C for less than an hour, whereas crystalline silica can be produced at 1000 °C when heated for more than 5 minutes [44].
However, silica can be easily extracted at low temperature of 40 °C because of the amorphous nature of silica in RHA. Amorphous silica in RHA can be extracted using low temperature alkali extraction since the solubility of amorphous silica is very low at pH below than pH 10 and it increases sharply above pH 10. The unique solubility behaviour of amorphous silica enables it to be extracted in pure form from RHA by dissolution under alkaline conditions [50]. This low energy method based on alkaline solubilization of amorphous silica can be cost effective compared to the current smelting method. Hence, it provides a lower alternative energy compared to the current high energy method. The development of a simple low energy chemical method for producing pure silica from rice husk has lead to a variety of industrial applications for RHA.
Silica is an excellent insulator, having low thermal conductivity, high melting point, low bulk density and high porosity. The use of silica as a support for catalysts is well known. Silica is widely used in glass, ceramics and cement as a major component and in pharmaceuticals, cosmetics, and detergents industries as a bonding and adhesive agents [51]. It has also been used as a major precursor for a variety of inorganic and organometallic materials which have the applications in synthetic

12 chemistry as catalysts and in thin films or coatings for electronic and optical materials [52-54]. Other solid forms of silica include the crystalline quartz, tridymite and cristabolite. These forms of silica are generally inappropriate as catalyst supports because they are non-porous. Pelleted diatomaceous earth is a naturally occurring form of siliceous material which is sometimes used as a catalyst support because it has a porous structure and relatively crush-resistant. However, it also contains alumina and iron impurities which may be harmful to many catalytic reactions [55].
Silica offers a number of advantages; the thermal stability of porous silica is excellent; having a glass transition temperature of 900 °C and no weight loss at
450 °C and the pore size can be controlled and easily limited to 10 nm [31].
Silica matrix with a mesoporous structure and high surface area was first reported in the preparation of aerogel by Steven Kistler in 1931. With the use of supercritical drying, he produced aerogel and shows that the gel morphology could be tuned by variation of drying conditions [56]. However, this method is not commercially viable because it is time consuming and requires a great deal of capital investment. In addition, high temperature and pressure that are required to obtain the supercritical condition can create a potentially explosive situation. Therefore, as alternative, ambient temperature and pressure are vital in the synthesis of aerogel.
What is an aerogel? According to Husing [57], there are two useful definitions of aerogel. The first one is defined according to the preparation method in which “all materials prepared from wet gels by a special drying process, the supercritical drying technique, were called aerogels irrespective of their structural properties”. The second definition addresses whether a product obtains an intact microstructure in its gel: “materials in which the typical of the pores and the network is largely maintained while the pore liquid of a gel is replaced by air are called

13 aerogels”. As overall, an aerogel is a porous solid material in which a very high percentage (95%) of its volume is filled with air [58].
Silica aerogel was first discovered in 1931 by Kistler [59-60]. In order to form the aerogel network, polymerization of silicic acid (Si(OH)
4
) was generated by acidic neutralization of sodium silicate in water. This process can be represented in
Equation 1.
Na
2
SiO
3
+ H
2
SO
4
+ H
2
O Si(OH)
4
+ Na
2
SO
4
(1)
According to Kistler’s method, the aqueous silica hydrogels were repeatedly rinsed with anhydrous methanol to remove all amounts of water. However, this method has numerous disadvantages particularly the toxicity of methanol and the flammable solvent which is needed to be handled at very high temperature and pressure [34]. Subsequently, ethanol based processing using silicon alkoxides derivatives (e.g. tetraethylorthosilicate or TEOS) are the preferred soluble silica source for the formation of silica aerogels over last few decades since water/alcohol solvent exchange could be avoided [61-63]. Via this method, alcohol is used as a homogenizing agent since water and alkoxysilanes are immiscible. Nevertheless, gels can be prepared from silicon-alkoxide-water mixtures without the addition of solvent because alcohol that is produced as a by-product of the hydrolysis reaction is sufficient to homogenize the initially phase separated system. Silicic acid that was produced from the reaction of silicon-alkoxide-water mixtures is very sensitively condensed with itself and rapidly generates sol particles. As condensation continues, a three dimensional gel network is formed which subsequently fill the mould dimension and entrain all of the liquid solvent.

14
An aerogel is one of the most fascinating solid materials because it is very light and highly transparent polymer material which is often called “frozen smoke” because of its hazy blue appearance [58]. Silica aerogel is a highly porous, open cell and low-density foam. Since its microstructure consists of nano-sized pore which is created from linked particles, it exhibits many desirable properties [64]. The applications of the silica aerogel have expanded into many fields [54] such as fillers for paints and varnishes, thermal and acoustic insulation materials, adsorbents and catalyst supports, as electronic materials such as Cherenkov detectors and sensor materials [65-66]. In addition, the highly hydrophobic of aerogel can be used to remove oil from water stream [54].
2.3 Sol-Gel Process in Aerogel Synthesis
A sol is defined as a colloidal dispersion of particles in a liquid while a gel is a substance that contains a continuous solid skeleton enclosing a continuous liquid phase. Thus, the formal definition of sol-gel processing is the growth of colloidal particles and their linking together to form a gel [59]. The sol-gel process is a wetchemical technique for the fabrication of materials (typically a metal oxide) starting either from a chemical solution (sol short for solution) or colloidal particles (sol for nanoscale particle) to produce an integrated network (gel) of silicon precursor through a change of interaction between the colloidal particles which is changing the systems characteristics from a liquid to a gel [60].
The most documented method for the preparation of silica aerogel is the solgel route [53, 67]. In sol-gel chemistry, the addition of water allows hydrolysis of the precursor to occur which is then followed by a condensation reaction. The completion of hydrolysis reaction depends on the water/precursor ratio and the catalyst added. Condensation can be occurred by the reaction of two silanol group reacted with an alkoxy group. The properties and structure of gels are highly

15 dependent upon the chemical environment during the reaction [60]. Conventional silica sol-gel process consists of hydrolysis of an alkoxide precursor such as tetramethoxysilane (TMOS) or tetraethoxysilane (TEOS) with the aid of a base or acid catalyst to form a homogenous colloidal suspension of silica which is known as a sol. The sol is then aged extensively for further polycondensation reaction and subsequent dried to remove the solvents and by-products. Gelation period varied depend on the precursors and catalysts. A gel network can be formed by polymeric formation of covalent bonds that are presented by the formation of particulate sols network which are held together by Van der Waals interaction [53].
Sol-gel processing is not only used for the preparation of silica gel but also for the synthesis of ceramic products, thin film and coatings over porous membranes
[59]. Sol-gel based materials have diverse applications in optics, electronics, energy, space, biosensors, medicine and separation technology [27-28, 56]. The wide application of sol-gel is due to its ability to form pure and homogenous products at very low temperature [60]. Thus, sol-gel technology can replace the old ceramic fabrication processes in which powders are shaped into objects and subsequently densified at temperature close to their liquids. The most fascination of sol-gel technology derives from the ability to control its composition and microstructure at the molecular level combined with the ability to shape the materials at room temperature for example by casting bulk gels in precision moulds, spinning fibres or dip coating thin film [67].
The chemistry involves in the sol-gel process consists of an initial hydrolysis of the silane precursors followed by the condensation of the hydrolyzed silicate monomers. The hydrolysis reaction comprises a series of three steps primarily by nucleophilic substitutions. In the first stage, nucleophilic attacks the positive charged

16 metal atom brought about by its interaction with negative charge of oxygen atom that is associated with water molecules. This reaction generates the increase of the coordination number of the metal ion. The next step involves the movement of a positively charge proton to a negatively charge –OR group of the metallo-organic precursor [68]. Finally, R-OH molecule is released as a product. The formation of gel in first stage undergoes polycondensation in the next stage. The nucleophilic substitutions step that was involves in the hydrolysis reaction and the chemical reactions for the polycondensation reaction are expressed by the sol-gel chemistry equations in Figure 2.1.
OR
OH
RO Si OR + 4 H
2
O
H / OH
HO Si OH
+ 4 ROH
OR
OH
R'
OR
Si OR + 3 H
2
O
H / OH
OR
R'
OH
Si OH
OH
+ 3 ROH
HO
OH
Si
OH
OH + R'
(a) Hydrolysis of silane precursors
OR
OH
H / OH
Si OR
HO Si
OH
O Si
OR
OH
(b) Condensation of the pre-hydrolyzed silane precursors
OH
R' + H
2
O

17 n HO
OH
Si O
OH
OH
Si R'
H / OH
OH
Si
R'
O
O
O Si
O
O
Si
O
O
Si
O
O
Si
O
R'
Si
O
OH
Si
O
R'
(c) Polycondensation reaction
Figure 2.1 (a) Hydrolysis, (b) condensation and (c) polycondensation reactions, during the sol-gel process in the synthesis of silica aerogel
Hydrolysis and condensation reactions occur simultaneously in the sol-gel process. The overview of sol-gel processing by Brinker [59] and Coltrain and Kelts,
[69] explained that the processing factors influencing the hydrolysis and condensation of silicates are catalyst, steric and inductive effects provided by alkyl groups, the ratio of water to precursor and the solvent. During the hydrolysis and condensation stages of sol-gel processing, each of these elements are shown to play an important role in determining the trends of gel structure. This trend was observed from the evolution of silicates derived from alkoxide precursors. Since silicon alkoxide precursor is insensitive to hydrolyse, its reactivity is commonly enhanced by catalyst which improves the strength of the nucleophile. Catalysts derived from acidic or basic conditions affected the sol-gel structure by influencing the hydrolysis and condensation kinetics [56].

18
After the initial and condensation reactions, the silicate monomers condense into dimeric and oligomeric species that form polymeric silica chains or clusters.
Due to the dissimilarity of catalysis mechanisms which form different particles, the growth of silicate species is also different. Thus, the shape and texture of these silicate dimers or oligomers are determined by the type of catalyst as shown in
Figure 2.2.
SOL-GEL BASED ON ACID-CATALYZED
•
Produced weak branched polymeric networks
•
Yield a highly dense polymer and microporous gel
SOL-GEL BASED ON BASE-CATALYZED
•
Produced high branched polymeric networks
•
Yield a loosely packing polymer and mesoporous gel
Figure 2.2
Polymeric structure of silica framework in acidic and basic conditions
In acidic condition, the hydrolysis reaction occurs at a faster rate than condensation reaction. The rate of condensation reaction decreases with the increasing number of siloxane linkages around the centre of silicon atom leads to

19 weak branched polymeric networks. The weakly branched polymers yield a highly dense and microporous gels with uniform distribution of small slit shaped pores and a narrow pore size distribution [68]. In a basic condition, on contrary, condensation reaction is accelerated compared to hydrolysis. The rate of condensation reaction increases with the increasing number of siloxane linkages. Thus, highly branched networks with ring structures are formed [70]. Highly branched system produces the mesoporous gels due to loose packing and more linear polymer [68].
2.3.2 Gelation and Aging
As the polymeric network extends all over the total volume, the sol thickens to a gel. The gelation point is defined as the time when an infinite, spanning polymers or aggregate first appears [59]. It is influenced by the size of container, solution pH, nature of the salt concentration, anion and solvent, type of initial alkoxy group and the amount of water [71-72]. After gelation, the next phase in sol-gel fabrication is the aging process. When the gel is kept in contact with the pore-filling liquid, its structure and properties keep changing as a function of time. This process is called aging. During this phase, some shrinkage and stiffness of the gel network is occurred along with a corresponding expulsion of solvent. Gels of desired densities and pore structures as well as surface areas can be obtained by simply varying the aging time, temperature, pH and added electrolyte [52, 73].
2.3.3 Drying
The drying step is extremely important in sol-gel processing. When solvent is removed from the gel, the presence of liquid in microporous regions can cause

20 capillary tension on the solid gel structure. If the stress is too severe, extensive cracking in monolithic gels will occur. To prevent this to happen, the capillary stress must be minimized by a number of techniques; reducing the solvent evaporation rate, adding a solvent of surfactant of lower surface area energy, hypercritically drying which removes the solid-liquid interface or forming a monodispersion of pore size
[63, 71, 73].
The gel can be dried at very high pressure and temperature corresponding to the supercritical conditions of the dispersed liquid in the gel to yield aerogels. The surface area of the gel may be retained to a great extent even after drying through the supercritical drying process. Supercritical drying was firstly used by Kistler in 1931 to make aerogel [59, 60]. The idea in using the supercritical drying was to eliminate liquid-vapour interface inside pores during drying and thus achieving zero capillary forces. In practice, the liquid solvent of the wet gel was first transformed into its supercritical conditions by increasing the temperature and pressure above the critical point of the solvent. At this stage, the solvent could be considered as supercritical fluid since both liquid and gas are presents. Subsequently, the solvent was released at constant temperature and passes from supercritical condition to gas phase without crossing any liquid-vapour interphase. Historically in this process, alcohol played a role as an extraction solvent. The extraction with alcohol has been termed as the high temperature supercritical drying since many alcohols reach their critical condition at very high temperature and high pressure. However, the high level of sophistication instruments, high risks and high cost involves in the supercritical drying of the gel prohibited the commercial production of the aerogel [34, 36, 74-75].
Therefore, the synthesis of aerogel via an ambient pressure drying (APD) is one way to reduce the production cost. APD has been considered as a promising technique to be applied on a large scale for industrial purposes. It does not require high pressure and high temperature conditions unlike the supercritical drying technique. Recently, many researchers focus their work on this route in an attempt to develop cost-effective and simple process for the production of silica aerogels [35-37,
75-76]. However, significant collapse of pores may occurred due to the differential

21 stresses induced in the gel matrix as a result of the capillary forces happens in the pores of varying radii by the liquid-vapour interface during the APD process of the pore liquid [77].
Nevertheless, the differential stresses of gel which induces the collapse of pores can be minimized by either synthesizing gel with wider pores or by exchanging the pore liquid of gel with a low surface tension solvent before the drying step. Since water has high surface tension, the water in the pores of gel can be exchanged with the solvent from aging media and hence minimizing the pore water content [73, 77]. The success in preventing the loss of surface area in aerogel which has been produced via APD, by using novel sol-gel routes is reported. Brinker and his co-workers [52] reported the synthesis of aerogel via APD using two step routes from tetraethoxysilane (TEOS) at pH 8 produced aerogel with surface area up to
1000 m
2
/g. Meanwhile, Komarneni and his team [78] reported a two step synthesis of aerogel with surface area up to 1447 m
2
/g from TEOS by a series of subsequent post-synthesis treatments involving solvent aging, silane coating and vacuum drying of the wet gel.
However, the techniques available to date suffer from the disadvantages such as huge requirement of the expensive chemicals for the solvent exchange and surface modification of the hydrogels. Thus, Schwertfeger and his team [36] reported the synthesis of silica aerogels using low-cost water-glass precursor without solvent exchange and supercritical drying of the wet-gels. However, the process involves huge consumption of expensive silylating agents such as trimethylchlorosilane
(TMCS) and hexamethyldisiloxane (HMDSO) for the surface modification of the wet-gels. This again makes the production process expensive and the use of the lowcost precursor such as waterglass becomes meaningless.

22
2.4 Immobilization of Enzyme
In the process of enhancing the stability and reusabilityof enzyme, enzymes are often immobilized by physical or chemical means to the surface of insoluble supports. An immobilized enzyme can be defined as any enzyme which is not freely soluble and whose movement in space is completely or partially restricted to a small region [26]. It is also known as the process of imprisoning an enzyme molecule in a distinct phase that allows for exchange but is separated from the bulk phase in which substrate effectors or inhibitor molecules is dispersed [25]. The immobilized enzyme is usually insoluble in water and the support that is usually used to immobilize enzyme is composed of a high molecular weight, hydrophilic polymer. Although its movement is partially or completely restricted within the microenvironment, the term “immobilization” does not imply that the enzyme can never move within its distinct phase [79].
Immobilization of enzymes on solid surface places them in a more natural environment and in many cases; allow them to function more efficiently [25]. When soluble enzyme is used for catalyzing a reaction, the reaction can only be terminated by deactivating the enzyme or by changing the environment. In the case of immobilized enzyme, the extent of reaction can be adjusted either by changing the residence time of the reactants or by removing the immobilized enzyme support from the reaction solution [26]. Immobilized enzymes also retain their catalytic properties for a longer period of time, thus making their use even more economical.
Other benefit of immobilized enzymes is the inhibition of enzyme activity whereby the excess of product can be minimized [8]. Immobilized enzymes can also be used for multi enzyme systems where several enzymes are placed in the same support thus; enabling it to catalyze a sequence reaction [5].
The appropriate matrix or support for the immobilization of enzyme is chosen based on several different properties which affect the production process [80].
One of the properties is that the materials need to have high surface area particularly

23 up to 100 m
2
/g for high enzyme loadings and high porosity to provide access for the substrate. The immobilization matrix must also be resistant to chemical degradations and mechanical stability. Microbial resistance of matrix is also an important property that needs to be considered since a major concern to any immobilized enzyme process is the presence of microbes. Furthermore, the durability of the carrier is often determined by its resistance to microbial degradation [79].
Enzyme technology is truly an ancient art which has been used in our lives since antiquity [7]. Enzyme has been used for thousands of years without a clear understanding of their nature. The first recorded use of enzyme involves the production of cheese from the stomach lining of calves, which contain chymosin.
Primitive herdsmen discovered that storing milk in the stomach of animals resulted in a tasty solid food called cheese. The action of chymosin which is a coagulating enzymes present in the stomach, induces gelation of the protein casein and changes the milk to curd and whey. The casein gel is cut and drained to remove the whey and the remaining solid, curd is pressed to produce cheese. This is one of the earliest recorded applications of enzymes to the processing of foods [79].
The properties and reactions of enzyme catalysis were first recognized by
Kirchhoff where he discovered that a component of wheat was capable of producing sugar from starch. Then, in 1833, Payen and Persoz obtained a malt extracted, amylase, which hydrolyzed starch into sugar [5]. In 1846, Dubonfout discovered the activity of invertase and Berthelot observed the same activity in an alcohol precipitate from yeast [7]. However, the term “enzyme” was coined by Kühne in
1878 and in 1893. Ostwald first classified enzymes as a catalyst. The next leap in the understanding of enzymes came in 1894 when Emil Fisher first proposed the “lock and key” theory to explain the specificity of enzymes [8]. By the beginning of the

24
20 th
century, the protein nature of enzymes was recognized [7]. Knowledge of the chemistry of protein drew heavily on the improving techniques and concept of organic chemistry in the second half of the 19 th
century. Modern enzyme chemistry was heralded by the proposed hypothesis for enzyme reactions reported by Michaelis and Menten and the isolation of an enzyme, urease by Sumner [5].
The immobilization process is well known having the potential to increase the enzyme stability. Tyrosinase has been immobilized on a variety of supports using different procedures by various researches. A variety of techniques have been developed to immobilize enzymes on the solid supports. The common immobilization technique can be classified into two general methods; chemical and physical methods. The former is based on the covalent bond formation and the latter depends on non-covalent bond formation where weaker interactions or containment of the enzymes are involved [26, 79]. The chemical methods involve covalent attachment and cross linking using multi reagents while the physical methods include adsorption, entrapment and micro-encapsulation of enzyme on solid support.
Enzymes can be adsorbed physically on a surface-active adsorbent by contact of an aqueous solution with an adsorbent. The advantages of the adsorption technique are the procedure being simple, inexpensive and possible in separating and purifying enzyme while being immobilized. However, the bonding strength of adsorbed enzyme is weak thus the immobilization yield is very low. It was demonstrated by Pialis and his co-workers [9] that only 70% of enzyme uptake was observed when tyrosinase was immobilized on nylon 6,6 membranes for the phenol production. Besides, the state of immobilization is very sensitive to solution pH, ionic strength and temperature.

25
Proteins and other bio-materials can be immobilized within the pore of support prepared from organic polymers. The entrapped molecule cannot escape from the support but materials which have low molecular weight can enter and leave the support by diffusion. The immobilized enzyme that is produced by this technique provides an extremely large surface area. Enzyme can also be entrapped within cross-linked polymers by forming a highly cross-linked network of polymer. A wide variety of cross linking agents have been proposed for immobilizing enzymes but only glutaraldehyde is extensively used. This method has a major advantage in the fact that there is no chemical modification of the enzyme; therefore the intrinsic properties of an enzyme are not distorted [26]. However, this technique has unappealing features. For instance, during the polymerization process, enzyme activity can be affected by the free radicals. The enzyme leakage and limitation of diffusion for the higher molecular weight reactants may occur.
Covalent bonding technique of enzyme immobilized on solid matrix is the most widely studied in the development of enzyme immobilization. This method has the advantages of high activity retention, hydraulic properties and temperature stability. Enzyme immobilization that is produced using covalent bonding technique results in high stability, ease in handling and simple preparation. The carrier can also be re-used after enzyme deactivation. However, immobilization technique renders the enzyme inactive if the active site is blocked. Possible leakage of the chemical used to activate the surface can also occur during the immobilization which may be harmful to enzyme [79].
2.4.3 Immobilization of Enzymes by Encapsulation Technique via Sol-Gel
Method
In the last decade, a new approach has been developed to achieve the immobilization of enzyme by using silica-based inorganic polymers. This method

26 was pioneered by Avnir and his co-workers [62] which are based on so-called sol-gel process. Mild processing conditions, excellent thermostability and chemical inertness of the support are the significant characteristics of this method. The silica alkoxide precursor has been extensively used in the conventional sol-gel method [60].
However, the use of TMOS or TEOS as starting materials lead to the generation of alcohol as a by-product, the presence of which can be detrimental to the protein activity. Therefore, it is important to develop an alcohol free sol gel process by encapsulation of the biological materials in a transparent matrix.
Sol-gel process offers an opportunity to develop one type of immobilization technique which is encapsulation in inorganic porous network. It is a useful method for biocatalyst entrapment because inorganic polymeric networks can be prepared even under mild ambient condition. Progressive improvements in the enzyme encapsulation technique indicates that enzyme remain catalytically active. Sol-gel encapsulation offers a number of desirable advantages [81]. The hydrophilicity of the matrix can be readily controlled. The inorganic matrix has high mechanical and chemical stability which allows enzyme to encapsulate in a rigid glass framework physically. The rigid glass framework of inorganic matrix provides stabilizing interaction with enzyme and keeps away from leaching problem. In addition, the high degree of biomolecules rigidity reduces the denaturizing phenomena. In other aspect, surface area of support from the sol gel method is controllable and can withstand the high range of temperature. Lastly, encapsulated biomolecules typically show improved resistance to thermal and chemical denaturation which can increase the storage and operational stability of enzyme.
Silica has been extensively used as carriers for enzyme [82]. One of the reason is that it possesses high mechanical strength at a wide range of operating pressures as proven by its use in High Performance Liquid Chromatography (HPLC)
[83]. Additionally, silica has relatively higher thermal and chemical stabilities and is resistant to microbial degradation. Since then, various biologically active species of interest such as enzymes, proteins, antibodies, virus and bacteria have been immobilized via sol gel process in different silica supports [10, 74, 84-85].

27
However, the disadvantage in sol gel process is during the drying technique, the drastic change of a gel texture can be occurred due to the capillary stresses [53,
72]. In addition, an enzyme consists of a proteidic chain which is folded onto itself, is very sensitive to drying stresses. In order to developed more effectively biocatalyst having improved activity, high storage stability and protection against deterioration by the hydrophilic solvent, the extensive encapsulation technique of enzyme on silica matrix with varieties of drying technique must be highlighted.
2.5
Tyrosinase
One of the “versatile” enzymes in nature is tyrosinase (monophenols monooxygenase, odiphenol: O
2
oxidorecductase, E. C. 1.14.18.1). Tyrosinase was discovered by Bertrand and Bourquelot almost 100 years ago [86]. Tyrosinase is a tetramer having weighs about 130 000 Daltons. Mushroom tyrosinase or fungal tyrosinase, bacterial tyrosinase and vertebrate tyrosinase consist of a binuclear copper active site which can exist in three states [87];
•
Met: the resting form of tyrosinase with an oxidized, anti ferromagnetically coupled bicupric complex in the active site
•
Oxy: an oxidized form of tyrosinase wherein the coordinated oxygen exists as peroxide and contains Cu
2+
in the active site
•
Deoxy: a reduced form of tyrosinase consisting of a univalent copper complex in the active site

28
Tyrosinase is also recognized as a key enzyme for melanin biosynthesis in plants and animals. Therefore, tyrosinase inhibitors should be clinically useful for the treatment of some dermatological disorders associated with melanin hyper pigmentation and also important in cosmetics for whitening and depigmentation after sunburn [88]. In plants, sponges and many invertebrates, they are important components of wound healing and the primary immune response [89]. In arthropods, they are also involved in sclerotization of the cuticle after molting or injury. In mammals, tyrosinases are found in melanocytes of the retina and skin [90]. In food industry, tyrosinase is responsible for the enzymatic browning reactions in damaged fruits during post-harvest handling and processing. The controlling of the enzymatic browning during processing is important in fruit pulp manufacturing [91].
Tyrosinase is usually used to catalyze two main reactions via separate active sites [92]. The first reaction catalyzed by tyrosinase is a monooxygenation where one atom of molecular oxygen is incorporated into aromatic structure and the reducing agent, AH
2
is oxidized. Although alternative reducing agents were used to increase reactions rates, the odiphenol generated from the above reaction can act as the reductant. Thus, costly external cofactors are not required by this enzyme [93]. The second reaction catalyzed by tyrosinase is the oxidation of odiphenol to oquinone.
Non-enzymatic polymerization of the oquinone by oxidative coupling then occurs by a series of oligomerization and polymerization reactions with the subsequent formation of melanin or melanin-like compounds [86, 94]. Mechanism reaction of monooxygenation and oxidation reaction catalysed by tyrosinase is illustrated in
Figures 2.3 and 2.4, respectively.
OH
OH
+ O
2
+ AH
2
+ H
2
O + A
R
R
Figure 2.3
Monooxygenation reaction catalyzed by tyrosinase
OH

29
OH
+ 1/2 O
2
O
+ H
2
O
OH
O
R
R
Figure 2.4 Oxidation of o -diphenol to o -quinone
Tyrosinase is very delicate. Vigorous shaking and temperature higher than
60 °C can cause denaturation to the protein and lead to the inactivation of tyrosinase.
Tyrosinase should be used at a pH range between 5 and 7, since it can loses its activity at pH values lower than 5. The optimum pH region for the activity of tyrosinase is between 6 and 7 [92]. The tyrosinase concentrated solution (
<
1 mg/mL at pH 7) shows little loss of activity when stored for several months while in dilute solution, tyrosinase can lose their activity within 20 minutes even when stored at low temperature (
<
5 °C) [18]. Most tyrosinase prepared from various sources share the same substrate specificity, so that this tyrosinase is similar in their reactivity and stability based on reaction inactivation [92, 95]. However, they might exhibit differences in other factors such as thermostability and deactivation by mechanical forces where there exist different molecular properties; structure and genetic expression [5].

30
2.5.1 Immobilized Tyrosinase in the Removal of Phenol
Aromatic compounds including phenols and aromatic amines constitute one of the major classes of pollutants that are of concern in many countries [96]. Some of the industrial area that discharge phenol include oil refineries, coke and coal conversion plants, plastics and petrochemical companies, dyes, textiles and paper industries. Almost all phenols are toxic. Furthermore, phenol and many of its derivatives are considered to be hazardous pollutants. Due to its toxicity and hazard, the concentration of phenol which is greater than 50 ppb is harmful to some aquatic species and the ingestion of 1 g phenol may be fatal to humans [97]. As a consequence, this may affect the ecosystem of water sources where phenols are discharged. Thus, the removal of phenol in water is important.
Conventional methods in the removal of phenol and aromatic compounds from industrial waste are solvent extraction, microbial degradation, adsorption on activated carbon and chemical oxidation [98-101]. Although these methods are effective and useful to remove phenol in water, they suffer from serious drawbacks such as high cost, incompleteness of purification, formation of hazardous byproducts and applicability to only a limited phenol concentration range [97].
Enzymatic treatment has been proposed by many researchers as a convenient method for removing phenol [84-85, 102]. Enzymes are highly selective and can effectively treat phenol even in dilute wastes [96]. Moreover, enzymes operate over a broad aromatic concentration range and require low retention times with respect to other treatment methods [103].
Tyrosinase was demonstrated to remove phenols and aromatic amines from phenolic industrial effluents [96-97, 102-103]. Atlow and his group [103] reported a successful application of soluble tyrosinase in the “cleansing” of pollutants. The oxidation of phenol catalyzed by tyrosinase is shown in Figure 2.5.

31
OH OH
OH
+ O
2
+ 2 H
+ Tyrosinase
O
OH
O
OH
+ O
2
Tyrosinase
Figure 2.5
Oxidation of phenol catalyzed by tyrosinase
In the presence of proton, the oxidation process generates phenoxy radicals which diffuse from the active centre into solution to react with phenol and form substances that are much less water soluble which can be known as quinones. These quinones are reactive and slowly undergo non-enzymatic conversion, oligomerization reactions, which ultimately yield high molecular weight; insoluble polyphenolics [103-106] and consequently inactivated the tyrosinase for further reaction. Since inactivation of tyrosinase was considered to be associated with the quinone attachment on the amino acid residue in proximity to the active site of enzyme [11, 104], Sun and his teams [104] suggested the use of chitosan to adsorb quinones which have been generated during phenol degradation. Thus, these insoluble polymers can be precipitated out of the solution and separated by simple filtration or flocculation [103].
Several techniques based on immobilized tyrosinase have been developed for the treatment of phenolic industrial wastewaters. The best and suitable support has

32 the ability to stabilize the enzyme activity and increase its half-life. Moreover, the method of the attachment of enzyme should not disturb the enzyme active centre.
The cost of immobilization needs to be low and only small amounts of enzyme need to be used [106]. If these requirements are met, an effective immobilized tyrosinase for phenol degradation may be achieved [10, 28-29]. Previous efforts to immobilize tyrosinase for the removal of phenol include adsorption techniques using inorganic adsorbents which are alumina and cation resin. 7000 units of tyrosinase were immobilized on 500 mg of resin. However, only 0.02 g/L of phenol conversion was detected. A similar problem is encountered with alumina. In addition, the weakness of the binding forces involved in adsorption might permit the enzyme to desorb from the support [94].
Silica matrix, besides being inexpensive, has been considered as a potential support material in enzyme immobilization for removal of phenol because of its chemical inertness, high mechanical strength, optical transparency, hydrophilicity, biocompatibility, biodegradability and anti-bacterial properties [27-28, 34, 107]. It can also be prepared under ambient conditions and exhibit tuneable porosity, high thermal stability and negligible swelling in both aqueous and non-aqueous solutions
[19, 81]. The numerous literatures on such stabilization described mostly success rather than failure. For instance, Seetharam and his teams [11] suggested the use of immobilized tyrosinase for phenol degradation. Tyrosinase immobilized on calcium aluminosilicate (CaA) and sodium aluminosilicate (NaA) was able to remove between 15% and 60% of the phenol in solution, depending upon the initial phenol concentration, the enzyme loading, pH, and duration of reaction. It could be reused repeatedly without any decrease in performance. In addition, it is advantageous to use a porous support such as aerogel so that the enzyme can be spread on a large surface area [27, 81].

CHAPTER 3
RESEARCH METHODOLOGY
3.1
Raw Materials and Chemical Reagents
Rice husk ash (RHA) was used as a source of silica in the synthesis of silica aerogel. It was prepared according to Hamdan [108] via physical combustion using a custom-made Plug Flow Combustor (PFC) machine which is available at the Zeolite and Nano-Structured Materials Laboratory, Universiti Teknologi Malaysia (UTM).
Rice husk was obtained from a paddy field in Perak, Malaysia. It was initially washed by immersing them in distilled water to eliminate unwanted materials such as sand, dust, mites and other agricultural wastes. After the rice husk was completely dried under sunlight, it was then burnt using the PFC at a constant temperature of
550 °C and at ambient pressure to ensure that the product is in an amorphous silica phase. The product obtained was white powder and readily used as a silica source for the synthesis of silica aerogel.
Enzyme tyrosinase extracted from mushroom; Ludox HS-30% and ethylene diamine tetracetic acid (EDTA) were supplied by Sigma Chemical Company.
Potassium phosphate dibasic, sulphuric acid (97%), catechol and ascorbic acid were purchased from Merck. All chemicals used were of analytical grade. Double distilled

34 deionized water (DDW) was used throughout the experiment in order to minimize the presence of impurities.
3.2
Synthesis of Tyrosinase Encapsulated Silica Aerogel (TESA)
The synthesis of tyrosinase encapsulated into silica aerogel (TESA) was carried out via alcohol-free aqueous colloidal sol-gel process, according to the established method [28] but with some modification. The sol-gel technique which was reported by Bhatia and Brinker [28] involved the acidification process by acidic cation-exchange resin together with hydrochloric acid (HCl) and in the drying technique, the gel was aged at 4 ° C for 24 hours prior to use. In this study, the acidification process only by using HCl and the gel was aged at room temperature for 24 hours, wash in buffer and lastly dried using Ambient Pressure Drying (APD) technique. The process consists of three stages namely the preparation of sodium silicate, the synthesis of wet gel and lastly, gel drying.
3.2.1
Preparation of Sodium Silicate
Teflon bottles were used in order to prevent contamination to the sodium silicate. Teflon bottles were cleaned by immersing them in hydrofluoric acid (Merck;
5%) and were left overnight prior to the preparation of silicate solution. The sodium silicate solution was prepared by dissolving RHA in sodium hydroxide (Merck; 99%) and double distilled water with SiO
2
/Na
2
O ratio of 3.25. Sodium silicate was then stirred and heated in an oil bath at boiling water temperature of 100 °C for 24 hours.
After that, sodium silicate was filtered through Whatman filter paper (125 mm) to

35 remove undissolved residues. Finally, the filtrate was stored in capped Teflon bottle and was ready to be used in the next step of synthesis route.
3.2.2
Synthesis of Wet Gel
In order to synthesize wet gel, silica sol was prepared by acidifying colloidal silica sol (Ludox LS-30; Sigma Aldrich; 30 wt% suspensions in water; pH 8.2) with sufficient amount of concentrated sulphuric acid (Merck; 97%) in a Teflon beaker.
Glass vessels were avoided as glass participates in the reaction since silica would leach out from the glass. The sodium silicate solution prepared previously was added drop wise into the silica sol until pH 6.5 is achieved. Tyrosinase (Sigma; 5.00-30.00 mg/mL) in potassium phosphate buffer (GCE Laboratory Chemicals; 50 mM, pH 6.5) was then inserted into the silica sol under constant stirring. A transparent, solid-like, tyrosinase-containing gel (aqueous tyrosinase-silica gel) was ultimately formed. The aqueous tyrosinase-silica gel was then aged at room temperature at different aging periods to allow the formation of silica frameworks around the enzyme molecule.
3.2.3
Drying of Wet Gel
In the drying phase, the tyrosinase-silica gel was firstly washed with phosphate buffer in order to remove an excessive non-encapsulated tyrosinase and to restore the initial condition of the encapsulated tyrosinase. Thus, 100 mL of phosphate buffer (50 mM, pH 6.5) was added to the tyrosinase-silica gel followed by stirring to make slurry. The slurry was then centrifuged for 10 minutes at 3000 rpm.
The clear supernatant liquid was discarded and the tyrosinase-silica gel in the

36 centrifuge tube was transferred into a Teflon beaker. The beaker was then labelled as tyrosinase encapsulated silica aerogel without solvent exchange (TESA without SE).
Solvent extraction (SE) was performed on the gel in order to examine the effect of solvent extraction on the enzyme activity in TESA. In the solvent extraction process, water in the gel was extracted by solvent extraction technique. The extracted gel was identified as tyrosinase encapsulated silica aerogel with solvent exchange (TESA with SE). Finally, both TESA were dried by ambient pressure drying technique (APD) at 36 °C until the constant weights of dried products were obtained. The dried powder of TESA was then collected and stored in an airtight
Teflon bottle at 5 °C.
3.3 Characterization of Tyrosinase Encapsulated Silica Aerogel (TESA)
Activity of enzyme tyrosinase in aerogel may be influenced by the changes in the gel texture, i.e., pore-shape characteristics, or in the surface structure, such as the nature of functionalities present on the aerogel pore surface. Thus, a few characterization techniques related to the structure and activity of TESA were carried out. The synthesized TESA was characterized by various characterization techniques including X-ray diffraction (XRD) technique, fourier transformed-infrared (FTIR) spectroscopy, field emission-scanning electron microscopy (FESEM), energy dispersive X-ray technique (EDX), transmission electron microscopy (TEM) and thermogravimetry analysis (TGA).

37
3.3.1 X-ray Diffraction (XRD) Technique
X-ray powder diffraction technique is commonly used in the characterization of solids in order to determine and to identify structure of material as well as to monitor the effects of post-synthesis modification in powder forms. This technique is unique since it is the only analytical methods capable of providing qualitative and quantitative information about crystalline or amorphous compounds presents in a solid sample without destroying the samples. It is also used to determine whether the catalyst is amorphous, crystalline or quasi-crystalline [109-110] through the comparison of the positions of the diffraction lines and their intensities with a large data bank since each crystalline solid has its own characteristics of powder diffraction pattern [111]. The diffraction patterns may be obtained from the powder diffraction file (PDF), maintained by the International Centre for Diffraction Data
(ICDD) and contains information of about 50 000 crystalline phases. Thus, the chemical identity can be confirmed if an exact match is found between the pattern of an unknown and an authentic sample.
The X-ray diffraction technique is based on the Bragg’s Law which hypothesized that any crystal material is built up of layers or planes such that each acts as a semi-transparent mirror. The derivation of Bragg’s law is illustrated in
Figure 3.1. When the X-rays penetrate the crystal material, some are reflected of a plane with the angle of reflection equal to the angle of incidence and the rest are transmitted to be subsequently reflected by succeeding planes.

38
θ
θ d
: angle d : reflection
Figure 3.1
Derivation of Bragg’s law for X-ray diffraction
The relation between lattice planes with a distance d , the angle of reflection,
θ and measured at wavelength, λ can be described by Bragg’s law in Equation (1). n λ = 2 d sin θ (1)
The primary use of Bragg’s law is for the determination of the spacing between the layers in the lattice. Once the angle, θ corresponding to a reflection is determined, d may readily be calculated [112]. The principles of the powder diffraction technique are shown in Figure 3.2. When a monochromatic beam of Xray strikes a finely powdered sample, it is dispersed randomly in every possible orientation. The diffracted beams may be detected by surrounding the sample with a detector [113].

39
Source
Filter
Sample
Detector
Figure 3.2
The illustration of the X-ray powder diffraction method
The most important use of the powder diffraction method is in the qualitative identification of crystalline or amorphous phases. While most chemical methods of analysis give information about the elements present in a sample, powder diffraction is very different and perhaps unique that it determines which crystalline compound or phase is present with no direct information about their chemical composition
[109]. Besides, X-ray diffractogram provides information about the degree of crystallinity and the presence of other phases or impurities in the crystal structure. It also provides valuable informations on any changes happening to unit cell parameters.
In this research, the XRD technique was applied to determine the formation of silica aerogel and its crystallinity before and after encapsulation. The phase identification of silica in the silica aerogel and TESA was determined using X-ray
Diffraction (XRD) method using a Bruker AXS GmbH (German) instrument. X-Ray diffraction patterns were recorded with a CuK α radiation at λ = 1.5418 Å at 40 kV and 20 mA in the range of 2 θ = 5° to 45° with a scanning speed of 0.05° per second.
For each analysis, 1 g of ground sample was carefully pressed on a sample holder to get a thin layer. The analysis was performed at the 2 θ scale of 1.5 to 10 ° with a step interval of 0.025
° and counting time of 1 s per step.

40
3.3.2 Fourier Transformed Infrared (FTIR) Spectroscopy
The infrared (IR) spectroscopy technique is generally used for qualitative analysis, mainly to determine the presence of functional groups. The characteristic bond and group frequencies obtained from infrared spectroscopic technique can be used for the identification of gases, liquids and solids, molecular structural analysis and checking the purity of substances [111]. The principle theory of the IR can be explained by the infrared radiation that will promote transitions in a molecule between rotational and vibrational energy levels of the ground (lowest) electronic energy state. The vibrational modes involving pairs or groups of bonded atoms can be excited at higher energy states by the absorption of radiation at appropriate frequency.
The frequencies of the normal vibrations of molecules, i.e., the position of the spectrum bands obtained (expressed in wavelengths or in wavenumbers) are determined by the masses of the atoms of the molecule. The slightest difference in structure resulted in the variation of the IR spectrum [114]. In the infrared spectroscopy technique, the frequency of the incident radiation is varied and the quantity of radiation absorbed or transmitted by the sample is obtained. The resulting spectrum represents the molecular absorption and transmission, creating a molecular fingerprint of the sample. This makes infrared spectroscopy useful for several types of analysis.
Infrared spectroscopy has been widely applied to study the silica, especially the nature of hydroxyl groups on the surface. The surface structure of amorphous silica is highly disordered, so one cannot expect a regular arrangement of hydroxyl groups. The surface of silica can be formed as either a siloxane group ( ≡ Si-O-Si ≡ ) with the oxygen on the surface or silanols groups ( ≡ Si-OH) [115]. Figure 3.3 illustrates the types of silanols that exist on the silica surface.

41
O
Si
H
O
Si
H
O
Si
H
H
O
Si
O
H
Isolate or terminal silanol Geminal silanols Vicinal silanols
Figure 3.3
Types of silanols exist in silica surface
The silanols groups on the silica surface can be divided into three types. The first type of silanol is isolated terminal groups (free silanols), where the surface silicon atom has three bonds into the bulk structure and the fourth bond is attached to a single OH group. The second type of silanols are vicinal silanols (bridging silanols), where two single silanols groups attached to different silicon atoms, are lose enough to hydrogen bond. A third type of silanols, geminal silanols, consists of two hydroxyl groups, attached to one silicon atom. The geminal silanols are close enough to form hydrogen bond with each other, whereas the free hydroxyl groups are separated too far from one to another. Hence the surface of amorphous silica gel may be covered by isolated as well as vicinal hydroxyl groups [116]. The original assignments of the main IR bands are described in Table 3.1 [71].

Table 3.1 The assignments of the main FTIR bands for silica
STRETCHING O-H VIBRATIONS
Frequencies (cm -1 ) Assignments
42
3400 - 3500 Molecular adsorbed H
2
O
1625 Bending O-H (molecular water)
VIBRATIONS OF SILICA
Frequencies (cm -1 ) Assignments
2000 - 1870
1250 – 1020
970
800
Si-O-Si (overtone) vibrations
Asymmetrical Si-O-Si stretching
Asymmetrical Si-OH stretching
Symmetrical Si-OH stretching
500 Si-O bending
Fourier transformed infrared (FTIR) spectroscopy may provide various informations such as the ability to identify unknown materials and to determine the quality or consistency of a sample as well as the amount of components in a mixture.
FTIR spectroscopy was developed in order to overcome the limitations encountered with dispersive instruments. FTIR is preferred compared to the dispersive or filter methods of infrared spectral analysis because several reasons [115]:

43
• It is a non-destructive technique
• It provides a precise measurement method which requires no external calibration
• It can increase speed, collecting a scan every second
FTIR analysis was applied to determine the structure of silica aerogel network and tyrosinase encapsulated silica aerogel (TESA) as well as their functional group on the silica surface. The infrared spectra were recorded on FTIR
Spectrophotometer (model FTIR-8300, Shimadzu, Japan) using the KBr method.
Approximately 0.001 g of the solid sample was used as a representative amount of the overall sample. A sufficient amount of KBr in the sample:KBr ratio of 1:100 were mixed thoroughly and ground using a mortar. The mixture was then pressed at a pressure of 7 tonnes to form a KBr disk. The disk was put in the sample holder and the FTIR spectrum of the sample was recorded in the wavenumber range of 350 cm -1 to 4000 cm -1 .
3.3.3 Field Emission Scanning Electron Microscopy (FESEM)
Field emission scanning electron microscopy (FESEM) is a technique to study the surface morphology including shape, size of the particulate also surface defect during synthesis of samples [115]. Prior to analysis, the samples were coated with a thin layer of platinum in a customized coating machine. FESEM can produce very high resolution images at very high magnification, showing particle shapes and sizes. FESEM can view a greater range of distances while maintaining resolution.
Thus, it can be able to produce a 3D image. FESEM can show interface behavior between atoms which have different atomic number, and regional boundaries in materials [110]. FESEM does not work with light (photons) but with electrons.
Electrons are generated in a 'source' (emission) and accelerated under the influence

44 of a strong electrical voltage gradient (field). Electromagnetic coils in FESEM formed an electron beam and the electron beam then scans the surface of sample.
Interaction between electron beam and the atom at the surface of sample will produce the secondary electrons [111, 117]. These electrons contain valuable informations on topography of the surface of the sample.
The procedure for taking the images using FESEM technique consists of three main techniques. First, the solid powder sample was put in the sample holder
(12.5 mm) which had been previously covered by a carbon conductive tape (8 mm wide) purchased from Ted Pella. Second step involves coating the sample on the sample holder with platinum (SB 1361, Pt 99.99%). The coating was done using
Auto-fine Coater (model JFC-1600, JEOL). The coating time was one minute with
20 mA sputtering current using the magnetron-type sputtering method. The platinum coated sample was inserted in the FESEM instrument’s specimen holder. Finally, the micrograph was taken by FESEM (model JSM 6701F, JEOL) at an emission current of 2.00 kV with working distance of 3.0 mm and probe current of 8 kV.
3.3.4 Energy Dispersive X-Ray Spectroscopy (EDX)
Energy dispersive X-ray (EDX) spectroscopy is an analytical technique used for the elemental analysis or chemical characterization of a sample. As one type of spectroscopic techniques, it relies on the investigation of a sample through the interaction between electromagnetic radiation and matter, analyzing X-rays emitted by the matter in response to being hit with the electromagnetic radiation [112].
Energy dispersive X-ray (EDX) spectroscopy is a chemical microanalysis technique performed together with a field emission scanning electron microscope

45
(FESEM). FESEM is used to record the image of sample surface at a specific area, then, the elemental analysis of the recorded sample surface is analyzed by EDX analyzer. When the surface of sample is bombarded by the electron beam from
FESEM, electrons will be ejected from the atoms to produce a vacant area. A resulting electron vacancy will then be filled by an electron from a higher shell and an X-ray is emitted to balance the energy difference between the two electrons. The
X-ray detector in EDX instrument measures the number of emitted X-rays as a function of their energy [118]. Energy of the X-ray is characteristic of each element from which the X-ray emits. A spectrum of the energy as a function of relative counts of the detected X-rays is obtained and evaluated for qualitative and quantitative determinations of the elements present in the sample volume [117].
The elemental analysis of samples was determined using Energy Dispersive
X-ray (EDX) spectrometer (model EX-2300 BU, JEOL). The procedure prior to the analysis was similar to the procedure for the characterization of silica aerogel by
FESEM as discussed in Section 3.3.3. The procedure for acquiring the value of the element in the samples was by defining the area for analysis with FESEM operation, image acquisition, spectrum acquisition, spot analysis and lastly the qualitative and quantitative analysis. The EDX analysis was carried out with emission current of
15.0 kV with working distance of 8.0 mm and probe current of 14 kV.
3.3.5 Transmission Electron Microscopy (TEM)
The transmission electron microscopy (TEM) is heavily used in both material science or metallurgy and the biological sciences. TEM provides information about the size, shape and arrangement of the particles which make up the specimen as well as their relationship to each other on the scale of atomic diameters. As well as the arrangement of atoms in the specimen and their degree of order, detection of atomicscale defects in areas a few nanometers in diameter [118]. There are a number of

46 drawbacks to the TEM technique. Many materials require extensive sample preparation to produce a sample thin enough to be electron transparent, which makes
TEM analysis a relatively time consuming process with a low throughput of samples.
The structure of the sample may also be changed during the preparation process.
Also the field of view is relatively small, raising the possibility that the region analyzed may not be characteristic of the whole sample. There is potential that the sample may be damaged by the electron beam, particularly in the case of biological materials [117-118].
For the preparation of sample, powder sample was firstly dispersed in acetone. A drop of suspension was then deposited on a thin carbon film supported by a copper grid. After the acetone evaporated, a thin layer of sol-gel powder was left on the carbon film sample holder which was used for image processing. The sample was then analyzed and the image was recorded by using JEOL JEM 2100.
Thermogravimetry Analysis (TGA) is usually used to determine the changes in weight in relation to change in temperature. Such analysis relies on a high degree of precision in three measurements: weight, temperature, and temperature change. A derivative weight loss curve can be used to tell the point at which weight loss is most apparent [115]. TGA is commonly employed in research to determine characteristics of materials such as polymers, to determine degradation temperatures, absorbed moisture content of materials, the level of inorganic and organic components in materials, decomposition points of explosives, and solvent residues. It is also often used to estimate the corrosion kinetics in high temperature oxidation [110]. The
TGA analyzer usually consists of a high-precision balance with a pan (generally platinum) loaded with the sample. The pan is placed in a small electrically heated oven with a thermocouple, thus able to measure the temperature accurately. The atmosphere may be purged with an inert gas to prevent oxidation or other undesired reactions. A computer is used to control the instrument.

47
Thermal analysis was performed using TG Analyzer (model TA 4000,
Mettler Toledo) consisting of an electronic microbalance, furnace, temperature controller and the recorder. A few mg of silica aerogel and TESA were weighed in a standard alumina crucible and the crucible was then sealed. The sample pan and reference pan, which was empty, were put into an analyzer chamber and were heated through air at a temperature beginning at 30 °C and increasing at a rate of 10 °C/min to 1000 °C. The power (energy per unit time) differential between the sample and reference was measured during the programmed heating and cooling periods.
3.4 Optimization of Synthesis Conditions
The synthesis condition of TESA was optimized in order to obtain a higher activity of tyrosinase in silica aerogel. The effect of synthesis parameters such as aging period, type of solvent extraction and amount of enzyme loading on enzymatic activity was investigated.
3.4.1 Effect of Solvent Extraction
The effect of solvent extraction on the enzyme activity in TESA was studied by extracting TESA in different solvents at the final stage of the preparation of
TESA. Acetone, amyl acetate and combination between acetone and amyl acetate were selected as solvent to extract water inside TESA during the synthesis route. The organic solvents used in this study have lower surface tensions than that of water in order to prevent pore structure of TESA to collapse. After that, the tyrosinase-silica gels were directly dried at 37 °C until a constant weight was obtained. Finally, the activity of enzyme in TESA was examined as mentioned above.

48
3.4.2 Effect of Aging Period
The effect of aging period on the activity of TESA during the synthesis route was investigated from 1 to 12 days at 25 °C. The Teflon beaker containing the aqueous tyrosinase-silica gel was firstly sealed by parafilm during the aging period study in order to avoid contamination of sample. Aging period was from 1, 2, 4, 6, 8 and 12 days. After aging at ambient temperature and pressure, the wet gels were then extracted using organic solvent. Finally, the tyrosinase-silica gels were dried at
37 °C until constant weight of dried products was obtained. The dried product
(TESA) was then collected and stored in an airtight Teflon bottle. The enzyme assay of the product was done according to Section 3.5.2.
3.4.3 Effect of Enzyme Loading
The relation between enzyme loadings and TESA activities was examined by varying enzyme loading into silica aerogel. Quantity of tyrosinase used for the encapsulation ranged from 5.00 mg/mL to 30.00 mg/mL. The different quantities of tyrosinase (5.00, 10.00, 20.00 and 30.00 mg/mL) was added into silica sol suspension. Tyrosinase was added into silica sol was done before the gelation of gels.
Sample from the wet tyrosinase-silica gels was processed in the same manner as described in Section 3.2.3.

49
3.5 Assay of Enzymatic Activity
The determination of the catecholase activity of tyrosinase was carried out based on the standard method recommended by Sigma Chemical Company [2]. The catecholase activity was determined by monitoring the depletion of chemical reductor. In the assay, the reduction of ascorbic acid as chemical reductor was monitored. The catecholase activity of tyrosinase (T) and the role of chemical reductor (NADH) that plays in the reduction of obenzoquinone (Q) into catechol (C) are represented in the equation below:
T + C + ½ O
2
↔ TCO → TQ + H
2
O (2)
TQ → T + Q (3)
Q + NADH → C (4)
Tyrosinase (T) catalyzes the conversion of catechol (C) to o -benzoquinone
(Q). However, it is difficult to measure the amount of o -benzoquinone produced in the reaction accurately due to the instability of o -benzoquinone. It can be easily polymerized to form stable compounds. In order to overcome this problem, the production of o -benzoquinone was measured based on the reduction of chemical reductor [9]. Accordingly, o -benzoquinone is immediately reconverted into catechol when the chemical reductor such as ascorbic acid is consumed.

50
3.5.1 Assay of Free Tyrosinase Activity
Potassium phosphate buffer (50 mM) was prepared by dissolving potassium phosphate monobasic anhydrous (QRec) (5.5055 g) in 100 mL DDW. The buffer solution was adjusted to pH 7 by the addition of NaOH (Merck; 2 M). The pH of solution was measured using a CyberScan pH/Ion 510 pH meter (Eutech
Instruments). This buffer solution was used as a solvent in the preparation of catechol solution (Sigma-Aldrich; 5 mM; pH 7) which act as a substrate, ascorbic acid (Acros Organics; 2.1 mM; pH 7) and ethylene diamine tetraacetic acid solution
(EDTA) (Riedel-de-Haän®; 0.065 mM; pH 7) as well as mushroom tyrosinase solution (Sigma; 5.00-30.00 mg/mL; pH 7). All solution was freshly prepared during the analysis.
For the enzyme assay study, 0.10 mL of catechol (5.0 mM), 2.60 mL phosphate buffer (50.0 mM), 0.1 mL ascorbic acid (2.1 mM) and 0.1 mL EDTA
(0.065mM) were pipetted into cuvette and were mixed by inversion. The mixed reagents (will be called as buffered substrate solution) was then monitored in an
Ultra Violet-Visible (UV-Vis) Spectrophotometer (Perkin Elmer, model Lambda 25) at 265 nm until the absorbance readings constant. After the absorbance readings of buffered substrate solution was steady, about 0.10 mL of tyrosinase solution (5.00-
30.00 mg/mL) was added to the solution. The mixture in the cuvette was mixed by inversion and subsequently, the absorbance values were taken every 30 seconds for 5 minutes. Reference solution was prepared similar to the preparation of buffered substrate solution as stated previously but without the addition of tyrosinase. This was done in order to determine whether the substrate catechol is oxidized nonenzymatically at a perceptible rate. The cuvette was washed before and after each run with denatured alcohol (Rinting Sceintific) and was rinsed by DDW so that the cuvette would not be contaminated by enzyme.
The enzyme activity was calculated by the determination of the amount of units of activity per milligram of enzyme left in the solution. This value of enzyme

51 activity was obtained by dividing the slope of the absorbance measurements with the mass of enzyme in the reaction mixture (Equation 5). One unit of enzyme activity was defined as the changes of absorbance detected in 3.0 mL reaction mix at pH 7 at
25 o C.
Units/mg enzyme =
Δ A
265 nm
/ min
0 .
001 test
( ) ( mg
− Δ A enzyme
265
/ nm
RM
/
) min blank
(5)
0.001 = the change in A
265 nm
per unit of tyrosinase in a 3.00 mL
reaction mixture at pH 7 at 25 o C
RM = reaction mix
3.5.2 Assay of Encapsulated Tyrosinase Activity
The enzymatic activity of TESA was determined by monitoring the amount of depletion ascorbic acid similar to the previous method but with some additional procedure. It is based on the formation of dehydro-ascorbic acid and obenzoquinone from the reaction between catechol, oxygen and ascorbic acid. Buffered substrate solution containing the mixture of buffer solution (50 mM; pH 7), catechol solution
(5.0 mM; pH 7), ascorbic acid (2.1 mM; pH 7) and EDTA acid solution (0.065 mM; pH 7) was incubated at room temperature and was stirred gently. The buffered substrate solution was then monitored at 265 nm until the absorbance readings were constant. After that, TESA (5.00-30.00 mg/mL) was placed in a beaker containing buffered substrate solution and the oxidation of catechol (substrate) was initiated.
Subsequently, the suspension was filtered after 60 minutes and the filtrate was analyzed by UV-Vis spectrophotometer.

52
The procedure was repeated with silica aerogel without the existence of tyrosinase. It was designed to confirm that the oxidation of catechol was due to the encapsulated tyrosinase rather than other materials presents in the reaction beaker.
The enzyme activity of TESA was calculated accordingly to the dry weight of support. The unit of enzyme activity per gram support (A) is defined in Equation (6).
A (U/g) =
Uact
Wdry
(6)
U act
= the activity of immobilized enzyme
W dry
= the weight of dry support (g)
In order to validate the accuracy of the TESA assayed activity, the activity of encapsulated tyrosinase was also assessed. This was done by comparing the enzyme activity in a freshly-made tyrosinase solution with the enzyme activity left in the supernatant after the centrifugation which was carried out under the phase of gelation synthesis (refer to Section 3.2.3). Therefore, the fraction of residual of enzyme (R) can be defined in Equation (7):
R =
Re action
Re rate action of rate of tyro sin ase tyro sin ase assay in a assay stock after gelation solution of tyro sin ase
(7)
The activity of encapsulated enzyme in silica matrix (I) was then calculated by using
Equation (8):

53
I = 1 − R (8)
Control and leaching studies were designed to confirm that the recorded enzymatic activity in TESA was due to the encapsulated tyrosinase rather than other species presented in the reaction vessel. It was undertaken by assessing the enzyme activity upon the exposure of substrate to the support alone and to determine if tyrosinase leaches out from the support. Sample from an immobilized tyrosinase was assayed in the usual way using the continuous spectrophotometric method described in Section 3.5.2. The encapsulated tyrosinase was then filtered using a Whatman filter paper (125 mm) and the filtrate was analyzed for the depletion of ascorbic acid.
The filtrate was then re-monitored under an UV-Vis Spectrophotometer (Perkin
Elmer, model Lambda 25) at 265 nm after 48 hours. The decrease in absorbance of ascorbic acid was taken every 30 seconds for 5 minutes.
3.5.4 Influence of Temperatures
The enzymatic activity of encapsulated tyrosinase in silica aerogel and free enzyme at different temperature were studied. For this purpose, tyrosinase and encapsulated tyrosinase were incubated in buffered substrate solution at temperature ranging from 5 °C to 70 °C at pH 7. Encapsulated enzyme activity in the incubation

54 mixture was then measured as a function of time using the continuous spectrophotometric method.
3.5.5 Influence of pHs
Free tyrosinase and encapsulated tyrosinase were incubated in buffered substrate solution and pH ranges 4 to 9 at 25 o C for ability studies at different pH values. The adjustment of the pH was carried out by the addition of NaOH (Merck; 2
M) or HNO
3
(Merck; 2 M) solution to obtain the desired pH values. Encapsulated enzyme activity in the incubation mixture was then measured as a function of time using the continuous spectrophotometric method.
3.6 Application of Tyrosinase Encapsulated Silica Aerogel (TESA)
In this study, tyrosinase encapsulated silica aerogel (TESA) was used to remove phenol in aqueous solution. The efficiency of TESA in removing phenol and the stability of tyrosinase in TESA was determined through reusability study.
3.6.1 The Removal of Phenol
Free tyrosinase (10.00 mg/mL in 50 mM phosphate buffer, pH 7) and TESA
(10.00 mg/mL of enzyme loading) was put in phenolic solution (10 mg/L in 50 mM

55 phosphate buffer, pH 7) for 3 hours. The reaction mixture was then incubated under aerobic conditions using a stirrer. After the prescribed time, the sample was withdrawn and assayed for phenols by UV-Vis Spectroscopy and the difference absorption spectra of reaction solutions were measured.
3.6.2 Reusability
For the reusability study, TESA was recycled for a different number of batches. For each batch, TESA was immersed in artificial phenolic waste water and the phenol degradation for each batch was monitored using UV-Vis
Spectrophotometer. Samples were collected at regular intervals for each batch.

CHAPTER 5
CONCLUSIONS AND RECOMMENDATIONS
5.1 Conclusions
Tyrosinase encapsulated silica aerogel (TESA) was successfully prepared via alcohol-free aqueous colloidal sol-gel route. The encapsulation process was carried out at room temperature and at neutral pH to further minimize the denaturation of enzymes. Sodium silicate from rice husk ash (RHA), an agricultural waste, was used as silica source since RHA contained high quantity of the amorphous silica (more than 95%). TESA was synthesized with and without solvent extraction (SE) process in order to study the relationship to the enzyme activity. TESA and silica aerogel were prepared by drying the samples via ambient conditions. In this study, tyrosinase was chosen as a model enzyme for encapsulation due to their potential application in the treatment of phenolic wastes. Moreover, tyrosinase is only active at narrow pH and temperature range. Therefore, the enhancement in its stability after the encapsulation process can be used as an indicator to the achievement of the encapsulation process.
The physicochemical properties of TESA were determined using X-ray diffraction (XRD) technique, fourier transformed-infrared (FTIR) spectroscopy, field

92 emission-scanning electron microscopy (FESEM), energy dispersive X-ray (EDX) analysis, transmission electron microscopy (TEM) and thermogravimetry (TGA) analysis. XRD analysis reveals tyrosinase molecules were well-dispersed into silica aerogel as the amorphous structure remained even after the encapsulation process.
The structure of the TESA was not converted to other phases and there were no impurities in the product after the encapsulation process. FESEM and TEM revealed that silica aerogel and TESA were successfully synthesized in nano-sized particles in the range of 20 to 25 nm, via an alcohol-free aqueous colloidal sol-gel route.
Moreover, these particles became larger by the utilization of solvent extraction process. The morphological studies also indicate the achievement of the encapsulation process as the presence of tyrosinase molecules in the spherical particles networks of silica aerogel and the absence of tyrosinase molecules on the spherical particles networks of silica aerogel surface. Therefore, it validates the encapsulation theory of this research which is, silica aerogel network is built around the tyrosinase molecules as the molecule size of tyrosinase is larger than silica aerogel, around 10
μ m.
TGA thermograms revealed that silica aerogel is thermally stable as only loss a small percentage of its weight after 900 °C of heating treatment meanwhile, free tyrosinase is easily denatured at higher temperature without left any residue.
However, thermograms of TESA indicated that the stability of tyrosinase towards direct heating treatment was enhanced after the encapsulation process since the silica aerogel networks is built a cage around the tyrosinase molecules thus acts as an insulator to the tyrosinase molecules. Hence, these thermograms validated that the use of alcohol-free aqueous colloidal sol-gel route does provide a promising approach for enzyme stabilization. The absence of tyrosinase molecules at the silica aerogel surface can also be observed via elemental analysis using EDX and the presence of tyrosinase in TESA can be evident via FTIR spectra. FTIR spectra of
TESA showed a presence of characteristic absorption peak which assigned to amine groups of tyrosinase.

93
Optimization of the encapsulation procedure was carried out in order to obtain a higher activity of encapsulated tyrosinase in TESA. The highest activity for
TESA was obtained with 10.00 mg/mL of enzyme that was aged for 2 days. The increment of enzyme loading did not necessarily improve the performance of enzyme which implies that only sufficient amounts of enzyme are needed for the enzymatic reaction.
In order to confirm that the enzymatic activity of tyrosinase still remained after the encapsulation, the enzymatic activity of free tyrosinase and tyrosinase in
TESA were assayed by examining the catecholase activity using UV-Vis
Spectrophotometer. Free tyrosinase with the concentration of 10 mg/mL and TESA
(with and without SE) was from the tyrosinase concentration of 10 mg/mL in 30 g of silica aerogel that was aged for 2 days were used in the evaluation of tyrosinase activity. TESA without SE showed higher tyrosinase activity of 5% compared to
TESA with SE that was extracted by amyl acetate/acetone (v/v:1/1). However, free tyrosinase has a higher enzymatic activity value compared to that of for TESA.
Silica aerogel networks limits the accesses of substrate to the encapsulated tyrosinase thus, lower the rate of the enzymatic reaction catalyzed by the TESA. The conformational stability of the tyrosinase seems to be retained upon encapsulation as the identical spectra between free tyrosinase and TESA which was demonstrated by
UV spectra. The leaching test showed no significant enzymatic activity observed in the filtrate of TESA which suggested that most of the tyrosinase was effectively loaded in silica aerogel. Meanwhile, UV-Vis spectra of TESA revealed the remarkable enhancement of encapsulated tyrosinase which is active at wider temperature and pH range. The maximum catalytic activity was observed at pH 7 and significant activity was also observed at pH ranging from 4 to 9 and at temperature ranging from 5 to 80 °C. In contrast, free tyrosinase was totally inactive at these pH values and at temperature exceed than 55 o
C. This indicates that the stability of the tyrosinase towards extreme temperature as well as acidic and basic conditions is significantly improved after the encapsulation process.

94
The efficiency of TESA in the removal of phenol is remarkable. TESA was able to remove phenol up to 80%, which is 10% lower than that of free tyrosinase after 3 hours of contact time. The successful degradation of phenol was accomplished at pH 7 using TESA with 10.00 mg/mL of enzyme loading which was aged for 2 days. This study also showed that the stability and reusability of tyrosinase in TESA was very high since TESA can be reused to remove phenol up to
10 times without significant loss. The removal of phenol profile consistently levelled off after 5 times of reuse. It is due to the product inhibition. This research has demonstrated that a potential biosensor based on tyrosinase encapsulated silica aerogel may be developed. With better understanding of the fundamental reaction, the degree of response can be increased, which eventually leads to a potential biosensor with enhanced thermal stability and improved enzymatic performance.
5.2 Recommendations
The experimental results in this thesis demonstrate that the alcohol-free aqueous colloidal sol-gel method led to the encapsulated tyrosinase with high activity retention, enhanced thermostability and operational stability with the reduction of production cost. The information gathered from the reactions catalyzed by the encapsulated tyrosinase provided with a promising picture about heterobiocatalysts in the phenol biosensor industries. However, continues studies are essential to offer more details interpretation which could lead to the large scale application of the encapsulated tyrosinase. It is suggested that future work can be done towards two directions:
•
The improvement of the sol-gel encapsulation procedure and the expansion of encapsulation method to other enzymes for medical application and protein purification

95
•
The development of enzyme encapsulated aerogel as an electro-analytical biosensor device for the determination of phenol either in aqueous or organic media
Current study mainly focuses on the activity and stability of the enzyme.
However, from the practical point of view, the physical properties of the enzyme play an important role in the process design and control. The encapsulated enzyme produced in this study was in the form of a powder, which is suitable for small batch reactor operation. However, the powder is not suitable when utilize in fixed bed continuous operation due to the creation of large pressure drops. Indeed, sol-gel based procedures offer the possibility of making product in different physical forms, such as monolith, film, fibre and powder.
Besides that, research can also be done in the development of enzymatic reactions. The bench level batch studies of the enzymatic reaction have been optimized with more than 95% conversion in 1 hour. However, the half-life of the biocatalyst and other kinetic parameters need to be determined for the scale-up process. In the study of the removal of phenol, it is difficult to get 100% removal in
1 hour, a process need to be developed to enhance the removal up to 100% and a separation protocol needs to be designed to purify and recycle the biocatalyst. Thus, more works in analytical and optimization need to be done in the study of the removal of phenol.

4.
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111
Determination of Ascorbic Acid using UV-Vis Spectrophotometer
1.90
1.8
1.7
1.0
A
0.9
0.8
1.6
1.5
1.4
1.3
1.2
1.1
0.3
0.2
0.1
0.0
0.7
0.6
0.5
0.4
280.0
(c)
(e)
(g)
(h)
(i)
(j)
(f) (k)
(l)
(d) (m)
(a)
(b)
(a) 0.2 mM ascorbic acid
(b) 0.4 mM ascorbic acid
(c) 0.6 mM ascorbic acid
(d) 0.8 mM ascorbic acid
(e) 1.0 mM ascorbic acid
(f) 1.2 mM ascorbic acid
(g) 1.4 mM ascorbic acid
(h) 1.6 mM ascorbic acid
(i) 1.8 mM ascorbic acid
(j) 2.0 mM ascorbic acid
(k) 2.2 mM ascorbic acid
(l) 2.4 mM ascorbic acid
(m) 2.6 mM ascorbic acid
285 290 295 300 305
Wavelength, nm
310 315
Figure A-1 UV spectra of ascorbic acid with different concentrations
320 325.0

112
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0 0.5
1 1.5
2
C once ntrations of Ascorbic Acid (mM)
2.5
3
Figure A-2 Relation of concentration of ascorbic acid with its absorbance at 290 nm
1.6
1.4
1.2
1
0.8
0.6
0.4
y = 1.2578x
R
2
= 0.9982
0.2
0
0 0.2
0.4
0.6
0.8
1 1.2
1.4
C once ntrations of Ascorbic Acid (mM)
Figure A-3 Standard calibration graph for the determination of ascorbic acid

113
30
20
10
0
0
90
80
70
60
50
40
20 40 60
Time (min)
80 100
Figure A-4 Activity of free tyrosinase as a function of time
120 140

EDX Elemental Analysis of Tyrosinase, Silica Aerogel and TESA
114
(a)
9600
8800
8000
7200
6400
5600
4800
4000
O
Si (b)
3200
2400
Pt
1600
800
C Na
Pt
Pt
0
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
keV 46
Figure B-1 EDX of silica aerogel (with and without solvent extraction)

(a)
115
5500
5000
C
(b)
4500
4000
3500
3000
2500
Pt
2000
1500
1000
500
N
O
Cu
Mg
Pt
Pt
Ca
Pt Cu
Pt
0
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
keV
47
Figure B-2 EDX of free tyrosinase

(a)
116
8800
8000
7200
6400
5600
4800
Si (b)
4000
3200
O
2400
1600
Pt
C
Na
Pt
Pt Pt
800
0
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
keV
49
Figure B-3 EDX of TESA (with and without solvent extraction)

Table B-1 Calculation of EDX elemental analysis for silica aerogel, free tyrosinase and TESA
TYROSINASE
TYROSINASE
TYROSINASE
TYROSINASE
Average
S Deviation
Element (weight%)
AEROGEL WITH SE
AEROGEL WITH SE
AEROGEL WITH SE
AEROGEL WITH SE
Average
S Deviation
Element (weight%)
AEROGEL WITHOUT SE
AEROGEL WITHOUT SE
AEROGEL WITHOUT SE
AEROGEL WITHOUT SE
Average
S Deviation
Element (weight%)
C
45.04
44.13
9.48
43.78
42.75
N
9.17
9.20
9.64
O
19.20
19.37
19.62
19.59
Na
0.00
0.00
Mg
0.41
0.42
0.00
0.44
0.00
0.42
Si
0.00
0.00
0.00
0.00
Pt
26.24
27.74
Ca
0.69
0.69
Cu
3.25
2.17
Fe
0.00
0.00
27.40
1.00
2.56
0.00
26.98
1.01
2.61
0.00
Total
77.76
76.26
76.60
76.02
43.9250 9.3725 19.4450 0.0000 0.4225
0.0000 27.0900 0.8475 2.6475 0.0000
76.6600
0.95
0.23
0.20
0.00
0.01
0.00
0.65
0.18
0.45
0.00
2.66
Total
57.30
12.23
25.37
0.00
0.55
0.00
0.00
1.11
3.45
0.00
76.66
100
5.37
0.00
4.77
0.00
5.16
0.00
4.79
0.00
35.00
34.82
35.70
34.06
1.33
0.00
1.27
0.00
1.02
0.00
1.08
0.00
30.33
28.47
0.00
0.00
0.00
30.70
29.77
0.00
0.00
0.00
29.60
29.96
0.00
0.00
0.00
29.75
30.21
0.00
0.00
0.00
5.0225 0.0000 34.8950 1.1750 0.0000 30.0950 29.6025 0.0000 0.0000
0.00
0.29
0.00
0.67
0.15
0.00
0.51
0.78
0.00
0.00
0.00
7.06
0.00
49.02
1.65
0.00
42.27
0.00
0.00
0.00
0.00
5.91
5.32
5.73
4.75
0
0
0
0
31.96
31.44
32.05
31.37
1.37
1.36
1.31
1.01
0
0
0
0
31.28
39.34
30.27
38.21
30.38
37.98
31.19
33.63
0
0
0
0
0
0
0
0
0
0
0
0
72.03
71.56
71.48
69.68
71.19
1.63
Total
71.19
100
70.52
68.39
69.47
68.32
5.4275 0.0000 31.7050 1.2625 0.0000 30.7800 37.2900 0.0000 0.0000 0.0000
69.1750
0.51
0.00
0.35
0.17
0.00
0.53
2.51
0.00
0.00
0.00
1.56
Total
7.85
0.00
45.83
1.83
0.00
44.50
0.00
0.00
0.00
0.00
69.18
100
117

TESA WITH SE
TESA WITH SE
TESA WITH SE
TESA WITH SE
Average
S Deviation
Element (weight%)
TESA WITHOUT SE
TESA WITHOUT SE
TESA WITHOUT SE
TESA WITHOUT SE
Average
S Deviation
Element (weight%)
C
3.00
2.49
3.00
2.29
2.6950
N
0
0
0
O
39.98
41.26
Na
2.41
2
0 40.84
1.92
0 39.82
1.84
40.475 2.0425
Mg
0.36
0.00
0.69
0.25
0.00
3.95
0.00
59.28
2.99
0.00
0
0
Si
22.42
23.67
0.46
33.78
Pt
27.27
27.07
0
0
22.93
23.24
26.96
26.33
0 23.065 26.9075
0.41
0.00
Ca
0
0
0
0
0
0.00
0.00
Cu
2.87
3.46
2.52
3.07
0 36.12
2.97
0 36.2
2.98
0 35.69
3.7
0 35.56
3.23
0
0
0
0
31.98
30.28
31.91
38.54
30.12
41.42
31.12
37.02
2.9800
0 35.8925
3.22
0.39
0.00
0.32
0.34
0 31.2825
0.00
0.87
36.815
4.72
0
0.00
4.06
0.00
48.92
4.39
0.00
42.63
0.00
0.00
0
0
0
0
0
0
0
0
0
0
0
Fe
0
0
0
0
Total
67.81
69.42
68.69
67.19
0
0
0 68.2775
0.00
1.76
Total
0 0.00
68.28
100
0
0
0
73.94
74.55
72.03
0 0 72.98
0
0
0 73.3750
0.00
1.92
Total
0 0.00
73.38
100
118

119
Determination of Phenol using UV-Vis Spectrophotometer
0.800
0.75
0.70
0.65
0.60
0.55
0.50
0.45
A
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.001
225.0
230
(a) 0.2 mM phenol
(b) 0.4 mM phenol
(c) 0.6 mM phenol
(d) 0.8 mM phenol
(e) 1.0 mM phenol
235 240
(e)
(d)
(c)
(b)
(a)
245 250 255 260 265 270
Wavelength, nm
275 280 285 290 295 300.0
Figure C-1 UV spectra of phenol with different concentrations

120
0.3
0.2
0.1
0
0
0.8
0.7
0.6
0.5
0.4
y = 7.1587x
R
2
= 0.9974
0.02
0.04
0.06
0.08
Concentration of Phenol (mM)
0.1
Figure C-2 Standard calibration graph for the determination of phenol
0.12

121
Paper for R&D Nanotechnology Symposium 2007, Malaysia
Nor Suriani Sani, Lee Siew Ling, Halimaton Hamdan
*
Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia
(UTM), 81300 Skudai, Malaysia
* hali@kimia.fs.utm.my
Abstract: Enzyme (tyrosinase) is successfully encapsulated into nano-porous silica xerogel via an alcohol-free colloidal sol-gel route. The encapsulation process was carried out at room temperature and at neutral pH to further minimize denaturation of enzymes. The leaching test showed that no significant enzymatic activity was observed in the filtrate of tyrosinase encapsulated nano-porous silica xerogel, which indicated that most of the enzyme was effectively loaded. The enzymatic activity of tyrosinase encapsulated nano-porous silica xerogel which was assayed through reduction of ascorbic acid using UV-Vis spectrophotometer reveals remarkable enhancement which is active at wider temperature and pH range. At pH 4 and pH 9, tyrosinase encapsulated nano-porous silica xerogel show the activity up to 30% after
60 minutes of reaction and it retains its enzymatic activity at 80 o
C. In contrast, free o tyrosinase was totally inactive at these pH values and at temperature exceeding 55
C. This indicates that the stability of the tyrosinase towards extreme temperature as well as acidic and basic conditions is significantly improved with good reusability after the encapsulation process.

122
1. Introduction
Biosensing is one of the most tetramethyl orthosilicate (TMOS) or tetraethyl orthosilicate (TEOS) has been nanobiotechnologies. Many proteins are chosen as biocomponents in biosensors. monoliths containing encapsulated proteins [5]. Despite the fact that these
Enzymes are large protein molecules that chemicals contain relatively pure silica, are extensively used in industrial unfortunately, 70% reduction of enzymatic relatively expensive reaction components encapsulated into TMOS-based silica and therefore increase production cost.
They are also extremely sensitive to matrix, in a presence of 5% volume of methanol [6]. The presence of alcohol is environment conditions and can be easily denatured. Free enzymes can be used only once in an enzymatic process as they are generally soluble in aqueous solutions and known to be detrimental to the activity of proteins by causing chain unfolding, aggregation, destruction of secondary and tertiary protein structures to a significant are very difficult to recover in useable form.
In order to increase reusability and enhance stability in process applications, extent [3]. Moreover, such organic silicon precursors are usually too expensive; hence the production in an industrial scale is not economically viable. enzymes are often immobilized by physical or chemical means to the surface
In order to overcome these problems, a newly modified aqueous route of insoluble supports [1-2]. Silica xerogel, for the synthesis of silica monoliths with made by the sol gel process has emerged encapsulated biological entities using rice as a promising platform for the husk ash as the cheap silicon source for encapsulation in a silica xerogel, the polymeric framework grows around the precursor has been developed [3,7]. This approach completely avoids the generation biomolecules, creating a cage, thus of alcohol and it allows the encapsulation protecting the enzyme either from to be carried out at neutral pH which aggregation and unfolding or from preserves biological activity of proteins. microbial attack [3]. Therefore, the The enzyme selected is tyrosinase from biomolecules encapsulated often retain a mushroom. It is a multifunctional copperfunctionality presumably because of distributed in plants and animals. sufficient retention of their native state Tyrosinase is well known as a key enzyme conformations. Meanwhile, the matrix for treatment of phenolic wastes [8]. pores allow the diffusion of reactant molecules and their reaction with the encapsulated biomolecules. Eventually 2. Experimental encapsulated enzyme can even improve the activity and storage stability of the 2.1 Chemicals and Equipment enzymes and will be easier to be used because they can easily be recovered and washed [4].
Mushroom tyrosinase, Ludox
HS-30% and ethylene diamine tetracetic acid (EDTA) were obtained

123 acid (97%) and ascorbic acid were purchased form Merck. All chemicals used were of analytical grade. The enzymatic activity for identifying the encapsulated tyrosinase was measured with continuous spectro-photometric method by using DR-UV-Vis
Spectrophotometer, Perkin Elmer
Lambda 25. monitored at 265 nm until the readings were constant. The encapsulated tyrosinase (10.00 mg/mL) was placed in the beaker containing buffered substrate solution and this initiated the reduction of ascorbic acid. The encapsulated tyrosinase was then filtered and the filtrate was finally analyzed for every 10 minutes for
60 minutes. The procedure was repeated
2.2 Enzyme Encapsulated Silica with silica xerogel without the existence of tyrosinase. It was designed to confirm that
Xerogel
Encapsulation of tyrosinase in the encapsulated tyrosinase rather than other materials presents in the reaction silica xerogel was carried out via the aqueous sol gel process. In this research, rice husk ash was used as a silica source in producing sodium silicate solution. The silica sol, sulfuric acid and the silicate the reduction of ascorbic acid was due to beaker.
2.3 Enzymatic Ability Test
The enzymatic activity of encapsulated tyrosinase and free enzyme solution were mixed and the resultant solution has a pH value of ~7. Then, a mixture of tyrosinase and phosphate buffer at different temperature and pH range were studied. For ability studies at different temperature ranges, tyrosinase solution was added to the solution to form gel. The gel was aged at room temperature before it is washed with pH 7 phosphate and encapsulated tyrosinase were incubated in buffered substrate solution of temperature ranging from 5 o
C to 70 o
C at buffer to desorb any enzyme molecules that do not encapsulate in the pores of the pH 7. Tyrosinase and encapsulated tyrosinase were incubated in buffered gel. Finally, the enzyme encapsulated silica xerogel was produced by drying the enzyme-silica gel at 36 o
C for 3 days. substrate solution in pH ranging from 4 to
9 at 25 o
C for ability studies at different pH values.
2.3 Assays of Enzymatic Activity 2.4 Leaching Study
The catecholase activity of encapsulated tyrosinase was determined
Control studies were designed to confirm that the reduction of ascorbic acid by monitoring the depletion of ascorbic was due to the encapsulated tyrosinase acid. It is based on the formation of dehydro-ascorbic acid and o -benzoquinone rather than other species present in the reaction vessel. It was undertaken to assess from the reaction between catechol, the depletion of ascorbic acid upon oxygen and ascorbic acid. Buffered exposure to the support alone and to substrate solution containing the mixture determine if tyrosinase desorbed from the of buffer solution (50 mM; pH 7), catechol solution (5.0 mM; pH 7), ascorbic acid
(2.1 mM; pH 7) and EDTA acid solution
(0.065 mM; pH 7) was incubated at room support and contribute to reduction of ascorbic acid. Sample from an encapsulated tyrosinase was processed in the usual way using DR-UV-Vis temperature and stirred gently. The Spectrophotometer. The filtrate was rebuffered substrate solution was then monitored 48 hours later.

124
3. Results and Discussion
3.1 Enzymatic Activity
3 .2 Measurement of Tyrosinase Activity
Figure 3.2 shows the enzymatic activity of free tyrosinase and encapsulated tyrosinase at pH 7.
Figure 3.1 shows the absorption spectra of tyrosinase encapsulated silica xerogel and free tyrosinase. The UV-Vis spectrum of tyrosinase encapsulated silica
70%
60%
50% xerogel shows a band centered at 290 nm while the band of commercial tyrosinase
40%
30% and isolated tyrosinase are centered at 289
20% nm and 292 nm, respectively. The absorption bands are preserved upon
10% encapsulation suggesting that the enzyme
0%
0 20 40
Time (min)
60 80 conformation is well-maintained to a considerable extent after the encapsulation. Therefore, it suggests that the use of an aqueous sol-gel encapsulation route does provide a Figure 3.2: Enzymatic activity of at pH 7 promising approach for enzymes stabilization [3]. The identical absorbance It encapsulated tyrosinase free tyrosinase
is clearly seen that free tyrosinase has spectra of tyrosinase in solution and in silica xerogel indicated the success of higher reduction value compared to the encapsulated tyrosinase. This phenomenon encapsulation protein (95%) into porous can be explained by the diffusion silica materials via sol-gel process. resistance of substrate in the silica matrix network [3]. Since the diffusion rate of the
1.000
0.95
0.90
0.85
(a) Enzyme encapsulated silica xerogel
(b) Commercial tyrosinase
(c) Isolated tyrosinase substrate was sufficiently slow compared to enzymatic catalysis, thus only the enzyme molecules which were
0.80
0.75
0.70
0.65
(b)
289
(a)
290 encapsulated close to the surface of silica matrix could reduce the ascorbic acid.
0.60
0.55
A A
0.50
0.45
0.40
(c)
Besides, small amount of the enzymes could have been denatured during the encapsulation process, hence led to lower
0.35
0.30
0.25
0.20
reduction of ascorbic acid in encapsulated tyrosinase.
0.15
0.10
0.05
0.000
280.0
285 290 295 300 nm
Wavelength, nm
305 310 315 320 325.0
Figure 3.1: Optical absorption spectra tyrosinase encapsulated silica xerogel
3 .3 Enzymatic Activity of Tyrosinase at
Different pH
The p H profiles for the enzymatic activity of free tyrosinase and encapsulated tyrosinase within silica xerogel in the pH range of 4 to 9 is depicted in Figure 3.3.

125
90%
80%
70%
60%
50%
40% these polymerization products may be the key to improve free tyrosinase performance at these pH values. Similarly, the enzymatic performance of the free tyrosinase was further decreased at pH 5.
Only 5% of ascorbic acid was detected by free tyrosinase at pH 5. However, up to
30% 30% of activity was achieved when the protection was given upon encapsulation
20%
10% into xerogel. At pH 4 and pH 9, tyrosinase encapsulated silica xerogel successfully
0%
3 5 7 9 11 showed its activity from 15% to 40%, pH w hile free tyrosinase was completely encapsulated tyrosinase free tyrosinase inactive at these pH values. It demonstrates that tyrosinase encapsulated
Figure 3.3: Enzymatic activity of free silica xerogel is active at a wider pH range tyrosinase and tyrosinase encapsulated than free tyrosinase. This further silica xerogel at different pH. demonstrates that the stability of
F igure 3.3 reveals that the tyrosinase conditions is significantly enhanced after encapsulated silica xerogel and free encapsulation. tyrosinase achieved their maximum tyrosinase towards acidic and basic activity level at pH 7. This finding is in 3 agreement with previous study which Different Temperature reported that the optimum activity of Experiments w ere performed to tyrosinase occurred near pH 7 [9]. determ ine the optimum temperature and to
However, the enzymatic activity of examine the thermal stability of tyrosinase was slightly decreased upon encapsulated tyrosinase in the reduction of encapsulation into xerogel due to the ascorbic acid. The enzymatic activity as a diffusion limitation. Only 50% of function of temperature subjected to 60 enzymatic activity was detected at pH 8 by minutes of reaction time is presented in using free tyrosinase compared to Figure 3.4. It shows that the long-term encapsulated tyrosinase; while 80% of thermostability of tyrosinase was enzymatic activity was showed by enhanced after the encapsulation process. encapsulated tyrosinase. This is explained by the fact that some of the free enzymes
The optimum temperature for both free and encapsulated tyrosinase was 25 o
C. were denatured at pH 8. Besides, the decrease in reduction of ascorbic acid at
The encapsulated tyrosinase was stable up to 70 o
C at all incubation periods however, pH 8 may be attributed to the some loss of activity was observed at 80 polymerization of quinones [9]. It was o
C. For the free tyrosinase, incubation at reported that polymerization of quinones temperature above 40 o
C was detrimental may easily occur at pH 8. During to enzyme activity and the activity began polymerization process, more stable and insoluble intermediates were produced. to decrease sharply when temperature went beyond 35 o
C. The observed
Accumulation of the intermediates could enhancement in thermostability of the deactivate tyrosinase, thus, limited further encapsulated tyrosinase is attributed to the reduction of ascorbic acid. Removal of

126 tight confinement of tyrosinase in the silica xerogel matrix which acts as an insulator for tyrosinase.
80%
70%
60%
90%
50%
80%
40%
70%
30%
60%
20%
50%
10%
40%
0%
30%
1 6 11
20%
Re usability
10%
0%
Figure 3.5: Assessment of the stability of tyrosinase encapsulated silica xerogel.
5 55
Te mpe rature ( o C )
105 free tyrosinase encapsulated tyrosinase
Figure 3.4: Enzymatic activity of free tyrosinase and tyrosinase encapsulated observed when the support alone was silica xerogel at different temperature.
3.6 Leaching Study
No enzymatic activity was immersed in the substrate, confirming that reduction was solely due to encapsulated
3.5 Reusability tyrosinase encapsulated silica xerogel is tyrosinase. Encapsulated tyrosinase showed a slight change in the activity.
The enc apsulated tyrosinase was recycle d 10 times, in each case; the enzymatic activity was studied after 60 minutes of reaction. The enzymatic reaction. Therefore it suggests that most of the enzyme molecules are sterically activity over 10 times of recycles is shown in Figure 3.5. The average activity over 10
However it was negligible since the activity was only 0.2% after 60 minutes of confined in the silica gel network. Since the enzyme was added prior to gelation, it batches was 60%. It is concluded that the is possible that a silica gel network was formed around the enzyme. stable since each cycle gave essentially the same conversion. It is clear that the encapsulated tyrosinase is initially active which then seems to level off due to were observed on the recovered enzyme
4. Conclusions product inhibition [9]. The accumulation
The enzymatic activity of of the product on the support prevents the encapsulated tyrosinase has been assayed encapsulated enzyme to undergo further through the reduction of ascorbic acid. It reaction. Upon filtration of the immobilized enzyme from the substrate shows that at optimum conditions (pH 7 and 35 o
C), 10% loss in enzymatic activity solution of each cycle, brown particles was detected upon encapsulation of enzyme into xerogel. It is also thus, suggesting production of quinones or demonstrated that tyrosinase encapsulated polyphenolics species which are known to silica xerogel is active at a wider pH and be colored. temperature range compared to the free tyrosinase. This also demonstrates that the

127 stability of tyrosinase encapsulated silica xerogel towards acidic and basic
[4] S. Maury, P. Buisson, A.Perrard, and
A. C. Pierre, J. Mol. Catal. B: conditions is significantly enhanced with good reusability.
Enzymatic
296, 2000.
, 32: 193, 2005.
[5] I. Gill, and A. Ballesteros, Tibtech, 18:
5. Acknowledgements
We would like to thank Ministry of
Science, Technology and Innovation for the financial support of this work and
[6] B. Dunn, J. M. Miller, B. C. Dave, J. S.
Valentine and J. I. Zink, Acta Mater,
46(3): 737, 1998.
[7] D.M. Liu and I.W. Chen, Acta Mater,
Faculty of Science, UTM for assisting the research.
References
[1] P Monsan, and D. Combes, Methods in
Enzymology , 137: 584, 1998.
[2] L. Bergogne, and S. Fennouh, Mol.
Cryst. Liq. Cryst, 354:667, 2000.
[3] R. B. Bhatia, C. J. Brinker, A.K.
Gupta, and A. K. Singh, Chem. Mater,
12: 2434, 2000.
[8] G. B. Seetharam and B. A. Saville,
47(18): 4535, 1999.
Water Res, 37 436, 2003.
[9] S. C. Atlow, L. Bonadonna-Aparo and
A. M. Klibanov, Biotechnol. Bioeng ,
26: 599, 1984.

128
Paper for 26th International Symposium on Space Technology and Science
(ISTS), Japan
Degradation of Waste in Space Using Alcohol-Free Enzyme Encapsulated
Silica Maerogel
By Nor Suriani Sani, Lee Siew Ling and Halimaton Hamdan
Department of Chemistry, Faculty of Science, University Technology of Malaysia (UTM),
81300 Skudai, Malaysia
Enzyme (tyrosinase) was successfully encapsulated into nano-porous silica maerogel via an alcohol-free colloidal sol-gel route using organic-silica source. The encapsulation process was carried out at room temperature and at neutral pH to further minimize the denaturation of enzymes. The leaching test of tyrosinase encapsulated nano-porous silica maerogel (TESA) showed no significant enzymatic activity in the filtrate indicating that most of the enzyme was effectively loaded. The enzymatic activity of TESA was assayed through the reduction of ascorbic acid. The activity of TESA shows remarkable enhancement which is active at a wider temperature and pH range. At pH 4 and pH 9, the oxidation of catechol (phenol substitute) by
TESA was up to 30% after 60 minutes of reaction and enzymatic activity was retained at
80 °C. In contrast, free tyrosinase was totally inactive at these pH values and at temperature exceeding 55 °C. This indicates that the stability of tyrosinase towards extreme temperature as well as acidic and basic conditions is significantly improved with good reusability after the encapsulation process. TESA was further used to remove phenol in aqueous solution and about
90% of phenol was removed in aqueous solution after three hours of contact time with TESA.
Thus, combination of enzyme and maerogel may be used to decompose waste in space efficiently.
Key Words: Silica aerogel, Encapsulation, Enzyme, Tyrosinase, Phenol
1. Introduction
Aromatic compounds including phenols and aromatic amines, constitute one of the major classes of pollutants that was stated as one of the main pollutants that is of concern in many countries.
1
Due to its toxicity, the concentration of phenol which is greater than 50 ppb is harmful to some aquatic species and the ingestion of 1 g phenol can be fatal in humans
2
. Hence, the removal of phenol in water is important.
Conventional methods for removing phenol and aromatic compounds from industrial waste are solvent extraction, microbial degradation, adsorption on activated carbon and chemical oxidation.
3-6
These methods, although effective and useful to remove phenol, suffer from serious drawbacks such as high cost, incompleteness of purification, formation of hazardous by-products and applicability to only a limited phenol

129 concentration range.
2
Enzymatic functionality presumably because of treatment has been proposed by many researchers as a convenient method for removing phenol.
7
Enzymes are highly sufficient retention of their native state conformations. Meanwhile, the matrix pores allow the diffusion of reactant molecules and their reaction with the selective and can effectively treat phenol even in dilute wastes.
1
Moreover, enzymes operate over a broad aromatic concentration range and require low encapsulated biomolecules. Eventually encapsulation may improve the activity retention times with respect to other treatment methods.
8
Tyrosinase was demonstrated to and storage stability of the enzymes and facilitate its application, recovery and washing.
14
In most of the reported applications of silica aerogel, orthosilicate such as remove phenols and aromatic amines from phenolic industrial effluents.
1-3,7
Tyrosinase is also known as polyphenol oxidase, phenolase or catecholase. This tetramethyl orthosilicate (TMOS) or tetraethyl orthosilicate (TEOS) has been enzyme oxidizes numerous phenols, generating corresponding phenoxy radicals which diffuse from the active centre into solution to react with phenol used as the silica source in preparing the silica monoliths containing encapsulated proteins.
15
Despite the fact that these chemicals contain relatively pure silica,
70% reduction of enzymatic activity was and form substances that are much less water soluble. These insoluble polymers then precipitate out of the solution and can be separated by simple filtration or flocculation.
9 reported when lipase was encapsulated into TMOS-based silica matrix, in the presence of 5% volume of methanol.
16
The presence of alcohol is known to be detrimental to the activity of proteins by
However, there has been almost no discussion about the contamination due to the remaining soluble enzyme and non-precipitated products in the aquatic solution after enzymatic treatment. It is very important to ensure that, especially in drinking water, the water after the treatment is free of enzyme and such products. Furthermore, enzymes are causing chain unfolding, aggregation, destruction of secondary and tertiary protein structures to a significant extent.
13
Moreover, such organic silicon precursors are usually too expensive; hence the production in an industrial scale is not economically viable.
In order to overcome these problems, a newly modified aqueous route for the components which contribute to the increase in the production cost. They are also extremely sensitive to the environmental conditions and can be easily denatured. Free enzymes can be used only once in an enzymatic process as they are generally soluble in aqueous solutions and are very difficult to recover in usable form.
10
In order to increase reusability and enhance stability, enzymes are often immobilized to the surface of insoluble supports by physical or chemical means.
11-12
Silica maerogel, made by the sol gel process is a promising platform for the encapsulation of enzymes. Upon encapsulation in silica maerogel, the polymeric framework grows around the biomolecules, creates a cage and protects the enzyme either from aggregation and unfolding or from microbial attack.
13
Therefore, the biomolecules encapsulated often retain a sufficient level of activity and encapsulated biological entities using rice husk ash (maerogel) as cheap silicon source for the production of pure silicate solution as a precursor has been developed.
13,17-18
This approach completely avoids the generation of alcohol and it allows the encapsulation to be carried out at neutral pH to preserve biological activity of proteins.
The aim of this study is to develop the bio-materials which have the tendency to degrade waste, suitable to be used in space.
The bio-material consists of enzyme tyrosinase encapsulated in silica aerogel and was applied to degrade phenol as a model waste.
2. Experimental
2.1. Chemicals and equipments
Mushroom tyrosinase, Ludox
HS-30% and ethylene diamine tetracetic acid (EDTA) were obtained

130 from Sigma Chemical Company.
Potassium phosphate dibasic, sulphuric acid (97%) and ascorbic acid were purchased form Merck. All chemicals used were of analytical grade. The enzymatic activity to identify the encapsulated tyrosinase was measured with continuous spectrophotometric method by using
UV-Vis Spectrophotometer, Lambda
25, Perkin Elmer.
2.2. Preparation of Enzyme encapsulated silica maerogel
(TESA)
Encapsulation of tyrosinase in silica maerogel was carried out via the aqueous sol gel process. In this research, rice husk ash was used as a silica source in producing sodium silicate solution.
The silica sol, sulphuric acid and the silicate solution were mixed and the resultant solution has a pH value of ~7.
Then, a mixture of tyrosinase and phosphate buffer solution was added to the solution to form gel. The gel was aged at room temperature before it was washed with phosphate buffers to desorb any enzyme molecules that did not encapsulate in the pores of the gel.
Finally, the enzyme encapsulated silica maerogel (TESA) was produced by drying of the enzyme-silica gel at 36 °C.
2.3. Assays of Enzymatic Activity
The catecholase activity of TESA was determined by monitoring the amount of depletion ascorbic acid. It is based on the formation of dehydro-ascorbic acid and o -benzoquinone from the reaction between catechol, oxygen and ascorbic acid. Buffered substrate solution containing the mixture of buffer solution
(50 mM; pH 7), catechol solution (5.0 mM; pH 7), ascorbic acid (2.1 mM; pH
7) and EDTA acid solution (0.065 mM; pH 7) was incubated at room temperature and stirred gently. The buffered substrate solution was then monitored at 265 nm until the readings were constant. TESA (10.00 mg/mL) was placed in a beaker containing buffered substrate solution and the oxidation of catechol (substrate) was initiated. The suspension was then filtered after 60 minutes and the filtrate was finally analyzed. The procedure was repeated with silica maerogel without the existence of tyrosinase. It was designed to confirm that the oxidation of catechol was due to the encapsulated tyrosinase rather than other materials presents in the reaction beaker.
As to the activity of TESA, it was calculated accordingly to the dry weight of support. The active unit per gram of support (A) is defined as stated in Eq.
(1).
A ( U / g )
= U act ...……(1)
W dry where U act
is the activity of immobilized enzyme, W dry
is the weight of dry support
(g).
2.3. Enzymatic Ability Test
The enzymatic activity of TESA and free enzyme at different temperature and pH range were studied. For the ability studies at different temperature ranges, free tyrosinase and TESA were incubated in buffered substrate solution of temperature ranging from 5 °C to
70 °C at pH 7. In order to study the stability in acidic and basic conditions, tyrosinase and encapsulated tyrosinase were incubated in buffered substrate solution in pH ranging from 4 to 9 at
25 °C.
2.4. The removal of phenol
TESA and phosphate buffer (0.1 M, pH7) were put in an artificial phenolic waste water (2.0 mM). The reaction mixture was incubated under aerobic conditions using a stirrer. After the prescribed time, the sample was withdrawn and assayed for phenols by
UV-Vis Spectroscopy and the disappearance absorption spectra of reaction solutions were measured.
2.5. Reusability
TESA was recycled for a different number of batches. For each batch,
TESA was immersed in artificial phenolic waste water and the phenol degradation for each batch was monitored using UV-Vis
Spectrophotometer. Samples were collected at regular intervals for each batch.
2.6. Leaching study
Control studies were designed to

131 confirm that the activity was due to
TESA rather than other species present in the reaction vessel. It was undertaken to assess the oxidation of catechol upon exposure to the support alone and to determine if tyrosinase was desorbed from the support and contributed to the oxidation of catechol. Sample from
TESA was analysed in the same way using UV-Vis Spectroscopy. The filtrate was re-monitored 48 hours later at 265 nm.
3. Results and discussion
3.1. Absorption spectra of TESA
Fig. 1 shows the optical absorption spectra of TESA and free tyrosinase.
3.2 Measurement of tyrosinase activity
Fig. 2 shows the enzymatic activity upon oxidation of catechol using free tyrosinase and TESA at pH 7.
70
60
50
40
30
20
10
0
0 20 40 time (min)
60 80
T ESA without catalyst free tyrosinase
1.000
0.95
(a) TESA
(b) Commercial tyrosinase
0.90
0.85
0.80
0.75
0.70
0.65
0.60
0.55
A 0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
(b)
289
(a)
290
(c) Isolated tyrosinase
(c)
Fig. 2. Enzymatic activity of free tyrosinase and TESA at pH 7.
It is evident that the free tyrosinase has a higher enzymatic activity compared to the TESA and without the existence of catalyst. This phenomenon can be explained by the diffusion resistance of phenol in the silica maerogel network.
13
Since the diffusion
0.05
0.000
280.0
285 290 295 300 nm
Wavelength, nm
305 310 315 320 325.0
Fig. 1. Optical absorption spectra of
TESA and free tyrosinase
The UV-Vis spectra of TESA show an absorption band at 290 nm while the band of commercial tyrosinase and isolated tyrosinase are at 289 nm and
292 nm, respectively. The absorption bands are not changed upon encapsulation suggesting that the enzyme conformation is well-maintained to a considerable extent after the encapsulation. Therefore, it suggests that the use of an aqueous sol-gel encapsulation route does provide a promising approach for enzymes stabilization.
13
The identical absorbance rate of the substrate was sufficiently slow compared to enzymatic catalysis, thus only the enzyme molecules which were encapsulated close to the surface spectra of free tyrosinase and in TESA indicate the success of encapsulation of tyrosinase (95%) into porous silica maerogel via sol-gel process. of silica maerogel matrix could oxidise the catechol. Besides, a small amount of the enzymes could have been denatured during the encapsulation process, which led to the lower activity in the encapsulated tyrosinase.
3.3 Enzymatic activity of tyrosinase at different pH
The pH profiles for the enzymatic activity of free tyrosinase and TESA in the pH range of 4 to 9 is depicted in Fig.
3. It reveals that the TESA and free tyrosinase achieved their maximum activity level at pH 7. This finding is in agreement with previous study which reported that the optimum activity of tyrosinase occurred near pH 7.
9
However, the enzymatic activity of tyrosinase was slightly decreased upon encapsulation into maerogel due to the diffusion limitation. Only 50% of

132 activity was detected at pH 8 by using free tyrosinase compared to TESA; while 80% of activity was showed using
TESA. This is explained by the fact that some of the free enzymes were denatured at pH 8. Besides, the decrease in activity at pH 8 may be attributed to the polymerization of quinones.
It was reported that polymerization of quinones may easily occur at pH 8. During polymerization process, more stable and insoluble intermediates were produced.
Accumulation of the intermediates could deactivate tyrosinase, thus, limit further activity of tyrosinase. Removal of these polymerization products may be the key to improve free tyrosinase performance at these pH values.
9
3.4 Enzymatic activity of tyrosinase at different temperature
Experiments were performed to determine the optimum temperature and to examine the thermal stability of
TESA. The relative activity as a function of temperature subjected to 60 minutes of reaction time is presented in
Fig. 4. It shows that the long-term thermostability of tyrosinase was enhanced after the encapsulation process.
The optimum temperature for both free tyrosinase and TESA was 25 °C. TESA was stable up to 70 °C at all contact periods; however loss of some activity was observed at 80 °C. For free tyrosinase, incubation at temperature above 40 °C was detrimental to enzyme activity and the activity began to decrease sharply when temperature was beyond 35 °C. The enhancement in the thermostability of the TESA is attributed to the tight confinement of tyrosinase in the silica maerogel matrix which acts as an insulator for tyrosinase. 50
40
30
20
10
0
90
80
70
60
3 5
T ESA
7 pH
9 free tyrosinase
1 1
9 0
8 0
7 0
6 0
5 0
4 0
3 0
Fig. 3. Enzymatic activity of TESA and free tyrosinase at different pH.
Similarly, the enzymatic performance of the free tyrosinase was further decreased at pH 5. Only 5% of activity was detected by using free tyrosinase at pH 5. However, up to 30% activity of tyrosinase was achieved when it was protected by encapsulation into maerogel. At pH 4 and pH 9, TESA successfully showed activity from 15% to 40%, while free tyrosinase was completely inactive at these pH values.
It demonstrates that TESA is active at a wider pH range than free tyrosinase.
This further indicates that the stability of tyrosinase in acidic and basic conditions is significantly enhanced after encapsulation.
2 0
1 0
0
5 2 5 4 5 te m pe ra tu re ( o C ) free t y ro sin ase
6 5
T E SA
8 5 1 0 5
Fig. 4. Enzymatic activity at different temperature.
3.5 Removal of phenol by TESA
Studies were conducted at pH 7, using
TESA with 10.00 mg/mL of enzyme loading. Fig. 5 shows the percentage removal of phenol by the tyrosinase treatment as a function of contact time with TESA.

133
100
90
80
70
60
50
40
TESA to undergo further reaction. Upon filtration of TESA from the substrate solution of each cycle, brown particles were observed on the recovered TESA suggesting the production of quinones or polyphenolics species which are known to be coloured.
18
30
80
20
70
10
60
0
50
0 20 40 60 80 100 120 140 160 180 200 time (min)
40
30
Fig. 5. The effect of contact time on removal percentage of phenol
Almost 70% of phenol was removed after an hour of contact with TESA. As discussed earlier, encapsulation of tyrosinase could lead to a mass transfer limitations or changes in substrate affinity.
13
Thus; the intra-particle mass transfer resistance affects the percentage of phenol removal. The slight percentage removal of phenol after 80 minutes of contact with TESA might has resulted from either the chemical coupling of free radicals or generation of quinones catalyzed by tyrosinase.
19
The occurrence of chemical coupling of free radicals during the reaction could prevent the intermediate complex of the enzyme-substrate for degrading phenol.
The excess generation of quinones, which is the product of phenol degradation would interfere the activity of free tyrosinase to degrade phenol in the aqueous solution.
3.5 Reusability
TESA was reused 10 times, in each case; the removal of tyrosinase was studied after 60 minutes of reaction. The reduction over 10 times of recycles is shown in Fig. 6. The average activity over 10 batches was 60%. It is concluded that TESA is stable since each cycle gave essentially the same activity. It is clear that TESA is initially active which then levelled off due to product inhibition.
9
The accumulation of the product on the support prevents
20
10
0
1 3 5 reusability
7 9 11
Fig. 6. Assessment of the stability of
TESA
3.6 Leaching study
No activity was observed when the silica maerogel alone was immersed in the substrate, confirming that the identified activity was solely due to
TESA. In contrast, TESA showed a slight change in the activity of the filtrate due to oxidation of catechol.
However it was negligible since the activity was only 0.2% after 60 minutes of reaction. Therefore it suggests that most of the enzyme molecules are sterically confined in the silica maerogel network. Since the enzyme was added prior to gelation, it is possible that a silica maerogel network was formed around the enzyme, indicating that most of the enzyme was effectively loaded.
4. Conclusions
The optimum condition of enzyme tyrosinase encapsulated in silica aerogel
(TESA) is at pH 7 and 35 °C. From the ability test, tyrosinase in silica maerogel is active at a wider pH and temperature range compared to the free tyrosinase.
Enhancement of the stability towards acidic and basic conditions was observed when tyrosinase was

encapsulated in silica maerogel. TESA was remove phenol up to 70% in an hour and shows high reusability.
5. Acknowledgements
Thanks to Ministry of Science,
Technology and Innovation for the financial support of this work.
References
1) Girelli, A. M., Mattei, E. and
Messina, A.: Phenols Removal by
Immobilized Tyrosinase Reactor in On-line High Performance
Liquid Chromatography, Anal.
Chim. Acta, 580 (2006), pp.
271-277.
2) Aitken, M. D.: Waste Treatment
Applications of Enzymes:
Opportunities and Obstacles,
Chem Eng. J., 52 (1993), pp.
B49-B58.
3) Britto, J. M., Rabelo, D. and
Rangel, M.: Catalytic Wet
Peroxide Oxidation of Phenol
From Industrial Wastewater on
Activated Carbon, Catal. Today ,
133-135 (2008), pp. 582-587.
M.,
Vijayaraghavan, K., Binupriya,
A.R., Stephan, A.M., Choi, J.G. and Yun, S.E.: Porogen Effect on
Characteristics of Banana Pith
Carbon and the Sorption of
Dichlorophenols, J. Colloid
Interface Sci .
, 320 (2008), pp.
22-29.
5) Adak, A., Pal, A. and
Bandyopadhyay, M.: Removal of
Phenol From Water Environment by Surfactant-Modified Alumina through Adsolubilization,
Colloids Surf., A , 277 (2006), pp.
63-68.
6) González, P. S., Capozucca, C.
E., Tigier, H. A., Milrad, S. R. and Agostini, E.:
Phytoremediation of Phenol from
Wastewater by Peroxidases of
Tomato Hairy Root Cultures,
Enzyme Microb. Technol., 39
(2006), pp. 647-653.
7) Karam, J. and Nicell, J. A.:
Potential Application of Enzymes in Waste Treatment, J. Chem.
Tech. Biotechnol., 69 (1997), pp.
141-153.
8) Siddique, M. H., St. Pierre, C. C.,
Biswas, N., Bewtra, J. K. and
Taylor, K. E.: Immobilized
Enzyme Catalyzed Removal of
4-Chlorophenol from Aqueous
Solution, Water Res., 27 (1993), pp. 883-890.
9) Atlow, S. C., Bonadonna-Aparo,
134
L. and Klibanov, A. M.:
Dephenolization of Industrial
Waste Waters Catalyzed by
Polyphenol Oxidase, Biotechnol.
Bioeng., 26 (1984), pp. 599-603.
10) Messing, R. A.: Immobilized
Enzymes for Industrial Reactors,
Academic Press, New York,
1975, pp. 2-4.
11) Monsan, P. and Combes, D.:
Enzyme Stabilization by
Immobilization, Methods
Enzymol ., 137 (1998), pp.
584-590.
12) Bergogne, L. and Fennouh S.:
Bio-Encapsulation within Sol-Gel
Glasses, Mol. Cryst. Liq. Cryst.,
354 (2000), pp. 667-670.
13) Bhatia, R.B., Brinker, C. J.,
Gupta, A.K. and Singh, A.K.:
Aqueous Sol-Gel for Protein
Encapsulation. Chem. Mater, 12
(2000), pp. 2434-2440.
14) Maury, S., Buisson, P., Perrard,
A. and Pierre, A. C.: Compared
Esterification Kinetics of the
Lipase From Burkholderia
Cepacia Either Free or
Encapsulated in a Silica Aerogel,
J. Mol. Catal. B: Enzymatic , 32
(2005), pp. 193-198.
15) Gill, I. and Ballesteros, A.:
Bio-Encapsulation within
Synthetic Polymers (Part 1):
Sol-Gel Encapsulation
Biologicals. Tibtech., 18 (2000), pp. 296-305.
16) Dunn, B., Miller, J. M., Dave, B.
C., Valentine, J. S. and Zink, J. I.:
Strategies for Encapsulation
Biomolecules in Sol-Gel
Matrices, Acta Mater., 46 (1998), pp. 737-742.
17) Liu, D.M. and Chen, I.W.:
Encapsulation of Protein
Molecules in Transparent Porous
Silica Matrixes via an Aqueous
Colloidal Sol-Gel Process, Acta
Mater., 47 (1999), pp. 4535-4541.
18) Seetharam, G. B. and Saville, B.
A.: Degradation of Phenol Using
Tyrosinase Immobilized on
Siliceous Supports, Water Res.,
37 (2003), pp. 436-442.
19) Dec, J. and Bollay, J. M.:
Detoxification of Substituted
Phenols by Oxide-reductive
Enzymes through Polymerization
Reactions, Arch. Environ.
Contam. Toxicol., 19 (1990), pp.
543-550.

CORRECTION THESIS AFTER VIVA (EXTERNAL EXAMINER)
ABSTRACT
Page/Section/Chapter Comments/original Correction
Page v enzyme tyrosinase effect to the enzyme activity
UV-Vis spectrophotometer tyrosinase enzyme effect on the enzyme activity
UV Visible spectrophotometer
ABSTRAK
Page/Section/paragraph Before
Page vi (Abstrak)
Correction dicirikan dengan XRD, FTIR, FESEM, EDX, dicirikan dengan kaedah XRD, FTIR, FESEM,
TEM dan TGA EDX, TEM dan TGA
Aktiviti tyrosinase ditentukan ditentukan oleh spektofotometer UV-Vis
Aktiviti tyrosinase telah ditentukan ditentukan dengan kaedah spektofotometer UV
Tampak
Hasil ujian larut lesap terhadap TESA Hasil Ujian Larut Lesap terhadap TESA
Kebolehan tyrosinase di dalam TESA untuk Kebolehan tyrosinase di dalam TESA untuk digunakan berulang-kali adalah tinggi kerana menyingkirkan fenol berulang-kali adalah tinggi
TESA boleh menyingkirkan fenol sehingga 10 kerana TESA boleh digunakan sehingga 10 kali kali
1

CHAPTER 1
Page/Section/paragraph Comments/original
Page 2/section1.1/para 2 Enzymes offer four major advantages
Correction
Enzymes offer three major advantages
Page 2/section1.1/para 3
Page 4/section 1.1/para 5
Page 4/section 1.1/para 6 development of these matrixes reason for immobilization enzyme development of these matrices reason for immobilization of enzyme promising platform for immobilization enzymes promising platform for immobilization of enzymes
Page 7/section 1.5/para 1
Eventually encapsulated enzyme can even Eventually, encapsulated enzyme can even improve the activity improve the activity
The tyrosinase encapsulated into silica aerogel The tyrosinase encapsulated silica aerogel (TESA)
(TESA)
Page 7/section 1.5/para 3 which is from rice husk ash
Energy dispersive X-ray spectroscopy (EDX)
Transmission electron microscopy (TEM) which is synthesized from rice husk ash
Energy dispersive X-ray spectroscopy (EDX),
Transmission electron microscopy (TEM) pH ranges as well as leaching test pH ranges, as well as leaching test
2

CHAPTER 2
Page/Section/paragraph Comments/original Correction
Page 17/section 2.3.1/para 1 Figure 2.1 (a) Hydrolysis, (b) condensation Figure 2.1 (a) Hydrolysis, (b) condensation and and (c) polycondensation reactions, during the (c) polycondensation reactions, during the sol-gel sol-gel process process in the synthesis of silica aerogel
CHAPTER 3
Page/Section/paragraph Comments/original
Page 35/section 3.2.1/para 1 heated in the oil bath heated in an oil bath
Correction
Page 38/section 3.3.1/fig 3.1
θ
(Not complete) d
θ
θ
: angle d : reflection d
Page 40/section .3.2/para 2 wavelengths or in wave numbers wavelengths or in wavenumbers
3

CHAPTER 4
Page/Section/paragraph Comments/original
Page 61/section 4.2.3/fig 4.3
(b)
1401.24
1101.64
2965.71
(a)
1548.98
1642.46
3466.37
2093.33
968.98
(b)
3466
2966
(a)
2093
Correction
1549
1401
1102
1642
969
799 798.50
1639 1638.81
3424.04
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800
Wavenumber (cm -1 )
600
466.30
350.0
31
3424
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800
Wavenumber (cm -1 )
600
466
350.0
31
(Omit the decimal point
Page 61/section 4.2.3/para 2 peaks at 3424.04 cm
-1 and 1638.81 cm
-1 appear at 798.50 cm
-1 and 466.30 cm
-1 absorption at 968.98 cm
-1
correspond to peaks at 3424 cm
-1 appear at 799 cm
-1 and 1639 cm
-1 and 466 cm
-1 absorption at 969 cm
-1
corresponds to
Page 62/section 4.2.3/para 3 Meanwhile, IR spectrum of tyrosinase shows Meanwhile, FTIR spectrum of tyrosinase shows an the intense and broad peak beyond 3000 cm
-1 intense and broad peak beyond 3000 cm
-1
which is which is assigned to retain water and N-H
2 assigned to water and N-H
2
bonds [120]. bonds [120].
The shoulder peak at 1548.98 cm
-1
The shoulder peak at 1549 cm
-1
The IR bands present and their assignments in The FTIR bands present and their assignments in
4

IR spectrum of tyrosinase and silica aerogel
Page 62/section 4.2.3/ Table 4.1 FTIR wavenumbers and assignment caption table 4.1 assignment for the functional group present in for the functional group present in the tyrosinase the tyrosinase
Page 62/section 4.2.3/table
4.1 Secondary Alcohol
3500 – 3200 cm
-1
FTIR spectrum of tyrosinase and silica aerogel
Secondary Alcohol
3500 – 3200 cm
-1
O-H stretching
1101.64 cm
-1
1102 cm
-1
3350 cm
-1 and
3150 cm
-1
2965.71 cm
1642.46 cm
-1
-1
1548.98 cm
-1
Primary Amide
Asymmetrical and nonasymmetrical N-H stretching
N-H
2
bending
3350 cm
-1 and
3150 cm
-1
Primary Amide
Asymmetrical and nonasymmetrical N-H stretching
2966 cm
-1
1642 cm
-1
1549 cm
-1
N-H
2
bending
5

Page/Section/paragraph Comments/original
Page 62/section 4.2.3/table
4.2
Vibrations of O-H
3500 – 3300 cm
-1
O-H stretching of silanol
1638.81 cm
-1
(molecular adsorbed
H
2
O)
Correction
Vibrations of O-H
3500 – 3300 cm
-1
O-H stretching of silanol
1639 cm
-1
(molecular adsorbed
H
2
O)
Page 63/section 4.2.3/ table
4.2 (cont)
2093.33 cm
-1
Vibrations of Silica vibration
1250 - 1020 cm
-1 stretching
968.98 cm
-1 stretching
798.50 cm
-1 stretching
466.30 cm
-1
2093 cm
969 cm
799 cm
466 cm
-1
-1
-1
-1
Vibrations of Silica
1250 - 1020 cm
-1
Si-O-Si (overtone) vibration stretching stretching stretching
6

Page/Section/paragraph Comments/original Correction
Page 63/section 4.2.3/para 4 The IR spectra of free tyrosinase, silica aerogel The FTIR spectra of free tyrosinase, silica aerogel and TESA and TESA
Page 64/section 4.2.3/fig 4.4
(a) (a)
NH
2
NH
2
1102
1401
(b) (b)
1549
1642
(c) (c)
1553
1634
(d) (d)
1640
1550
1800 1700 1600 1500 1400 1300
Wavenumber (cm -1 )
1200 1100 1000.0
34
1800
1636
1700 1600 1500 1400 1300
Wavenumber (cm -1 )
1200
(Insert the wavenumbers in each significant peak)
Page 69/section 4.2.5/para 2 and eventually presented in silica aerogel and eventually existed in silica aerogel
1100 1000.0
34
7

Page/Section/paragraph Comments/original
Page 72/section 4.2.6/para 3 Figure 4.8 b and Figure 4.9 b prove that
Correction
TEM micrograph of silica aerogel was found to tyrosinase is located inside the silica aerogel differ from micrograph of TESA regarding their network rather than at the surface of silica appearance where micrograph of TESA is observed aerogel. It shows that the encapsulation process as more opaque than that of for silica aerogel. This of tyrosinase in silica aerogel via an alcoholis because the networks of silica aerogel surround free colloidal sol-gel route was successful. the tyrosinase molecules in TESA which is resulted
Enzyme-sol interaction which resulted from the in the increment of the thickness of silica aerogel. addition of tyrosinase into the sol before the Hence, it prove that tyrosinase is located inside the aging process promotes the encapsulation of silica aerogel network rather than at the surface of enzyme into the growing silica network. The silica aerogel. It shows that the encapsulation large number of hydrogen bonding groups on process of tyrosinase in silica aerogel via an the surface of tyrosinase promotes the extensive alcohol-free colloidal sol-gel route was successful. interaction with the silicate polymer containing Enzyme-sol interaction which resulted from
Si-O(H)-Si and Si-OH fragments during the the addition of tyrosinase into the sol before the initial stages of network formation. The existent aging process promotes the encapsulation of of hydrogen bonding which serves as a nucleus enzyme into the growing silica network. The large enables the condensation and polymerization of number of hydrogen bonding groups on the surface the sol to occur [27]. Thus, the gel network can of tyrosinase promotes the extensive interaction tyrosinase can acts as a template and eventually Si-OH fragments during the initial stages of form a porous inorganic polymer cage surround network formation. The existent of hydrogen
8

the tyrosinase. bonding which serves as a nucleus enables the condensation and polymerization of the sol to occur
TEM micrograph of silica aerogel was [27]. Thus, the gel network can develop around the found to differ from micrograph of TESA tyrosinase because tyrosinase can acts as a template regarding their appearance where micrograph of and eventually form a porous inorganic polymer
TESA is observed as more opaque than that of cage surround the tyrosinase. for silica aerogel. This is because the networks of silica aerogel surround the tyrosinase molecules in TESA which is resulted in the increment of the thickness of silica aerogel.
(Restructured)
Page 73/section 4.2.7/ para 2 a minor change of percent mass in the samples a minor change of percentage mass in the samples
Page 75/section 4.3/para 1 Several synthesis conditions TESA were Several synthesis conditions of TESA were optimized optimized
Page 75/section 4.3.1/ para 2 as they strongly distorte the essential water as they strongly distort the essential water layer layer
Solvents having polarity indexes of 5.8 and Solvents with a polarity index of 5.8 and above above are suitable solvents for activity of were documented to be suitable solvents for tyrosinase because it does not denature the tyrosinase because they do not denature the tyrosinase [128].
(Rewrite) tyrosinase and retain its enzymatic activity [128].
9

Page/Section/paragraph Comments/original
Page 76/section 4.3.1/ caption fig 4.11
Figure 4.11 Effect of solvent extraction to the enzymatic activity of TESA with SE
Figure 4.11
Correction
Effect of solvent extraction on the enzymatic activity of TESA with SE
Page 83/section 4.4.2/para 1
Page 87/section 4.5.1/para 1
Page 89/section 4.5.2/para 2 free tyrosinase presented in the assay system
Figure 4.17 shows the percent removal
The average percent removal of phenol free tyrosinase exist in the assay system
Figure 4.17 shows the percentage removal
The average percentage removal of phenol
10

CORRECTION THESIS AFTER VIVA (INTERNAL EXAMINER)
CHAPTER 1
Page/Section/paragraph Comments/original Correction
Page 4/section 1.1/para 7 Include statements that portray the originality and importance of using tyrosinase
In this research, tyrosinase was used as model enzyme because of its wide application in medicine, environmental and industrial systems [8,
24]. Tyrosinase is also suitable for the treatment of phenolic wastes [9, 11, 17]. Tyrosinase like most other enzymes, is expensive and thus the use of the soluble enzyme is not practical [2]. Therefore, the encapsulation of tyrosinase is very attractive in order to exploit its catalytic properties and improve the cost effectiveness [5]. The improved stability of the encapsulated enzyme allows it to be highly reusable. Moreover, since enzyme does not dissolve in the solution, further purified process is
Page 4/section 1.2/
Page 6/section 1.3/para 2
Paragraph 1
As shown in Figure 4.6, not required and hence the encapsulated tyrosinase is economical to be used repetitively [8].
Eliminate
As shown in Figure 1.1,
1

CHAPTER 3
Page/Section/paragraph Comments/original Correction
Page 35/section 3.2/para 1 established method [28] but with some established method [28] but with some modification.
(Elaborate) modification. The sol-gel technique which was reported by Bhatia and Brinker [28] involved the acidification process by acidic cation-exchange resin together with hydrochloric acid (HCl) and in the drying technique, the gel was aged at 4
°
C for
24 hours prior to use. In this study, the acidification process only by using HCl and the gel was aged at room temperature for 24 hours, wash in buffer and lastly dried using Ambient Pressure Drying (APD) technique.
Page 36/section 3.2.3/para 1 Solvent extraction was performed on the In the drying phase, the tyrosinase-silica gel gel in order to remove water from the aqueous was firstly washed with phosphate buffer in order tyrosinase-silica gel before drying. In the to remove an excessive non-encapsulated solvent extraction process, 100 mL of tyrosinase and to restore the initial condition of the phosphate buffer (50 mM, pH 6.5) was added to encapsulated tyrosinase. Thus, 100 mL of the tyrosinase-silica gel followed by stirring in phosphate buffer (50 mM, pH 6.5) was added to the order to make slurry. The slurry was then tyrosinase-silica gel followed by stirring to make centrifuged for 10 minutes at 3000 rpm. The slurry. The slurry was then centrifuged for 10 clear supernatant liquid was discarded and the minutes at 3000 rpm. The clear supernatant liquid
2

tyrosinase-silica gel in the centrifuge tube was was discarded and the tyrosinase-silica gel in the transferred into a Teflon beaker. Then, water in centrifuge tube was transferred into a Teflon the gel was extracted by solvent extraction beaker. The beaker was then labelled as tyrosinase technique to produce tyrosinase-silica gel. encapsulated silica aerogel without solvent
Finally, the tyrosinase-silica gel was dried by exchange (TESA without SE). ambient pressure drying technique (APD) at 36 Besides, solvent extraction (SE) was also
°C until the constant weight of dried product performed on the gel in order to examine the effect was obtained. The dried powder of TESA was of solvent extraction on the enzyme activity in then collected and stored in an airtight Teflon TESA. In the solvent extraction process, water in bottle at 5 °C. the gel was extracted by solvent extraction technique. The extracted gel was identified as
(Method for SE and non SE not clearly stated) tyrosinase encapsulated silica aerogel with solvent exchange (TESA with SE). Finally, both TESA were dried by ambient pressure drying technique
(APD) at 36 °C until the constant weights of dried products were obtained. The dried powder of TESA was then collected and stored in an airtight Teflon bottle at 5 °C.
3

Page 55/section 3.6.2/para 1 Repetition For the reusability study, TESA was recycled for a different number of batches. For each batch, TESA was immersed in artificial phenolic waste water and the phenol degradation for each batch was monitored using UV-Vis Spectrophotometer.
Samples were collected at regular intervals for each batch.
CHAPTER 4
Page/Section/paragraph Comments/original Correction
Page 56/section 4.1/para 1 was synthesized with and without solvent was synthesized with and without solvent extraction extraction (SE)
TESA without solvent extraction (TESA TESA without SE and TESA with SE without SE) and TESA with solvent extraction
(TESA with SE)
Page 88/section 4.5.2/para 1 the removal of tyrosinase was studied the removal of phenol was studied
4

CHAPTER 5
Page/Section/paragraph Comments/original
Page 92/section 5/ Include suggestion for further work
Correction
The experimental results in this thesis demonstrate that the alcohol-free aqueous colloidal sol-gel method led to the encapsulated tyrosinase with high activity retention, enhanced thermostability and operational stability with the reduction of production cost. The information gathered from the reactions catalyzed by the encapsulated tyrosinase provided with a promising picture about hetero-biocatalysts in the phenol biosensor industries. However, continues studies are essential to offer more details interpretation which could lead to the large scale application of the encapsulated tyrosinase. It is suggested that future work can be done towards two directions:
•
The improvement of the sol-gel encapsulation procedure and the expansion of encapsulation method to other enzymes for medical application and protein purification
5

•
The development of enzyme encapsulated aerogel as an electro-analytical biosensor device for the determination of phenol either in aqueous or organic media
Current study mainly focuses on the activity and stability of the enzyme. However, from the practical point of view, the physical properties of the enzyme play an important role in the process design and control. The encapsulated enzyme produced in this study was in the form of a powder, which is suitable for small batch reactor operation.
However, the powder is not suitable when utilize in fixed bed continuous operation due to the creation of large pressure drops. Indeed, sol-gel based procedures offer the possibility of making product in different physical forms, such as monolith, film, fibre and powder.
Besides that, research can also be done in the development of enzymatic reactions. The bench level batch studies of the enzymatic reaction have been optimized with more than 95% conversion in
6

1 hour. However, the half-life of the biocatalyst and other kinetic parameters need to be determined for the scale-up process. In the study of the removal of phenol, it is difficult to get 100% removal in 1 hour, a process need to be developed to enhance the removal up to 100% and a separation protocol needs to be designed to purify and recycle the biocatalyst. Thus, more works in analytical and optimization need to be done in the study of the removal of phenol.
7

Thesis Checking
Title: SYNTHESIS AND CHARACTERIZATION OF ALCOHOL-FREE
TYROSINASE ENCAPSULATED SILICA AEROGEL
No. Subject Checking
1 Blank
2 Declaration of the status of Thesis
6 Dedication
7 Acknowledgement
9 Abstrak (bahasa melayu)
10 Table of contents
11 List of tables
12 List of figures
13 List of symbols
14 List of abbreviations
15 List of appendices
17 Chapter 2: Literature Review
18 Chapter 3: Experimental
19 Chapter 4: Results and Discussion
20 Chapter 5: Conclusions and Suggestions
21 References