vii
TABLE OF CONTENTS
CHAPTER
I
TITLE
PAGE
TITLE
i
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGEMENT
iv
ABSTRACT
v
ABSTRAK
vi
TABLE OF CONTENTS
vii
LIST OF TABLES
xi
LIST OF FIGURES
xii
LIST OF SYMBOLS
xv
LIST OF ABBREVIATIONS
xvi
LIST OF APPENDICES
xvii
INTRODUCTION
1
1.1
Background of Research
1
1.2
Problem Statement
4
1.3
Hypothesis
5
1.4
Objectives of Research
7
1.5
Scope of Research
7
1.6
Outline of Research
9
viii
II
LITERATURE REVIEW
10
2.1
Rice Husk Ash as a Silica Source
10
2.2
Silica Aerogel
12
2.3
The Sol-Gel Process
14
2.3.1
Hydrolysis and Condensation
15
2.3.2
Gelation and Aging
19
2.3.3
Drying
19
2.4
Immobilization of Enzyme
22
2.4.1
Enzyme as Catalyst
23
2.4.2
Immobilization Techniques
24
2.4.3
Immobilization of Enzymes by
25
Encapsulation via Sol-Gel Method
2.5
Tyrosinase
2.5.1
27
Immobilized Tyrosinase in the
30
Removal of Phenol
III
RESEARCH METHODOLOGY
33
3.1
Raw Materials and Chemical Reagents
33
3.2
Synthesis of Tyrosinase Encapsulated Silica
34
Aerogel (TESA)
3.3
3.2.1
Preparation of Sodium Silicate
34
3.2.2
Synthesis of Wet Gel
35
3.2.3
Drying of Wet Gel
35
Characterization of Tyrosinase Encapsulated
36
Silica Aerogel (TESA)
3.3.1
X-Ray Diffraction (XRD) Technique
37
3.3.2
Fourier Transformed-Infrared (FTIR)
40
Spectroscopy
3.3.3
Field Emission-Scanning Electron
43
Microscopy (FESEM)
3.3.4
Energy Dispersive X-ray Analysis
(EDX)
44
ix
3.3.5
Transmission Electron Microscopy
45
(TEM)
3.3.6
3.4
3.5
Thermogravimetry Analysis
46
Optimization of Synthesis Condition
47
3.4.1
Effect of Solvent Extraction
47
3.4.2
Effect of Aging Period
48
3.4.3
Effect of Enzyme Loading
48
Assays of Enzymatic Activity
49
3.5.1
Assay of Free Tyrosinase Activity
50
3.5.2
Assay of Encapsulated Tyrosinase
51
Activity
3.6
3.5.3
Leaching Study
53
3.5.4
Influence of Temperatures
53
3.5.5
Influence of pH
54
Application of Tyrosinase Encapsulated Silica
54
Aerogel (TESA)
IV
3.6.1
The Removal of Phenol
54
3.6.2
Reusability
55
RESULTS AND DISCUSSION
56
4.1
56
Synthesis of Tyrosinase Encapsulated Silica
Aerogel (TESA)
4.2
Characterization of Tyrosinase Encapsulated
58
Silica Aerogel (TESA)
4.2.1
Physical Appearance
59
4.2.2
X-ray Diffraction (XRD) Analysis
59
4.2.3
FTIR Spectroscopy
60
4.2.4
Field Emission Scanning Electron
64
Microscopy (FESEM)
4.2.5
Energy Dispersive X-ray Analysis
68
(EDX)
4.2.6
Transmission Electron Microscopy
(TEM)
69
x
4.2.7
4.3
4.4
Thermal Analysis
73
Optimization of Synthesis Conditions
75
4.3.1
Effect of Solvent Extraction
75
4.3.2
Effect of Aging Period
78
4.3.3
Effect of Enzyme Loading
79
Enzymatic Activity
81
4.4.1
81
Assay of Free Tyrosinase and
Tyrosinase Encapsulated Silica
Aerogel (TESA) Activity
4.5
4.4.2
Leaching Study
83
4.4.3
Influence of Temperatures
83
4.4.4
Influence of pH
85
Application of Tyrosinase Encapsulated Silica
87
Aerogel (TESA)
V
4.5.1
The Removal of Phenol
87
4.5.2
Reusability
88
CONCLUSIONS AND RECOMMENDATIONS
91
5.1
Conclusions
91
5.2
Recommendations
94
REFERENCES
96
APPENDICES
111
xi
LIST OF TABLES
TABLE NO.
TITLE
PAGE
3.1
The assignments of the main FTIR bands for silica
42
4.1
FTIR wavenumbers and assignment for the
functional group present in the tyrosinase
62
4.2
FTIR assignments of silica aerogel
62
4.3
Major elemental analysis (mass% ± SD) of free
tyrosinase, silica aerogel (with and without solvent
extraction) and TESA (with and without solvent
extraction)
68
xii
LIST OF FIGURES
FIGURE NO.
TITLE
PAGE
1.1
The encapsulation of tyrosinase into silica aerogel
network
6
1.2
Flow diagram of research activities
9
2.1
(a) Hydrolysis, (b) condensation and (c)
polycondensation reactions, during the sol-gel
process in the synthesis of silica aerogel
17
2.2
Polymeric structure of silica framework in acidic and
basic conditions
18
2.3
Monooxygenation reaction catalyzed by tyrosinase
28
2.4
Oxidation of o-diphenol to o-quinone
29
2.5
Oxidation of phenol catalyzed by tyrosinase
31
3.1
Derivation of Bragg’s law for X-ray diffraction
38
3.2
The illustration of the X-ray powder diffraction
method
39
3.3
Types of silanols exist in silica surface
41
4.1
Optical absorption spectra of (a) free tyrosinase, λmax
= 289.14 nm, (b) TESA (with solvent extraction),
λmax = 291.28 nm and (c) TESA (without solvent
extraction), λmax = 292.91 nm
57
4.2
XRD diffractogram of (a) silica aerogel and (b)
TESA
60
xiii
4.3
The FTIR spectra of (a) silica aerogel and (b) free
tyrosinase
61
4.4
FTIR spectra of (a) free tyrosinase, (b) silica aerogel,
(c) TESA with SE and (d) TESA without SE
64
4.5
FESEM micrographs showing the surface
morphology of (a) silica aerogel with SE and (b)
TESA with SE
65
4.6
FESEM micrographs showing the surface
morphology of (a) silica aerogel without SE and (b)
TESA without SE
66
4.7
FESEM micrograph showing the surface morphology
of free tyrosinase
67
4.8
TEM micrographs showing the surface morphology
of (a) silica aerogel with SE and (b) TESA with SE
70
4.9
TEM micrographs showing the surface morphology
of (a) silica aerogel without SE and (b) TESA
without SE
71
4.10
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%
74
4.11
Effect of solvent extraction to the enzymatic activity
of TESA with SE
76
4.12
Enzymatic activities of (a) TESA with SE and (b)
TESA without SE; at different aging periods
78
4.13
Enzymatic activities of (a) TESA with SE and (b)
TESA without SE; of different enzyme loadings
80
4.14
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
82
4.15
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
84
xiv
4.16
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
86
4.17
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)
88
4.18
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
90
xv
LIST OF SYMBOLS
°C
-
Degree Celsius
cm
-
Centi meter
g
-
Gram
K
-
Kelvin
kV
-
Kilo Volt
L
-
Liter
M
-
Molar
m
-
Meter
mg
-
Mili gram
min
-
Minute
mL
-
Mili Liter
mM
-
Mili Molar
mm
-
Mili meter
nm
-
Nano meter
ppm
-
Part per million
rpm
-
Rotation per minute
v
-
Volume
W
-
Watt
Å
-
Angstrom
μg
-
Micro gram
μL
-
Micro Liter
λ
-
Lambda
θ
-
Theta
xvi
LIST OF ABBREVIATIONS
APD
-
Ambient Pressure Drying
CaA
-
Calsium Aluminosilicate
DDW
-
Double Distilled Deionized Water
DTG
-
Derivative Thermogravimetric Analysis
EDTA
-
Ethylene Diamine Tetracetic Acid
EDX
-
Energy Dispersive X-ray Technique
FESEM
-
Field Emission Scanning Electron Microscopy
FTIR
-
Fourier Transform Infrared
HMDSO
-
Hexamethyldisiloxane
HPLC
-
High Performance Liquid Chromatography
IR
-
Infrared
NaA
-
Sodium Aluminosilicate
NADH
-
Nicotinamide Adenine Dinucleotide
PFC
-
Plug Flow Combustor
RHA
-
Rice Husk Ash
SE
-
Solvent Extraction
SD
-
Standards Deviation
TEM
-
Transmission Electron Microscopy
TESA
-
Tyrosinase Encapsulated Silica Aerogel
TEOS
-
Tetraethyl Orthosilicate
TMOS
-
Tetramethyl Orthosilicate
TGA
-
Thermogravimetric Analysis
UV-Vis
-
Ultra Violet-Visible
XRD
-
X-Ray Diffraction
xvii
LIST OF APPENDICES
APPENDIX
TITLE
PAGE
A
Determination of Ascorbic Acid using UV-Vis
Spectrophotometer
111
B
EDX Elemental Analysis of Tyrosinase, Silica
Aerogel and TESA
114
C
Determination of Phenol using UV-Vis
Spectrophotometer
119
D
Paper for R&D Nanotechnology Symposium
2007, Malaysia
121
E
Paper for 26th International Symposium on
Space Technology and Science (ISTS), Japan
128