PROTEASE PRODUCTION FROM BACTERIAL ISOLATES OF
TRADITIONAL LEATHER PROCESSING PONDS FOUND AROUND
GONDAR TOWN
MSc THESIS
MIESHO HADUSH
October 2015
Haramaya University, Haramaya
PROTEASE PRODUCTION FROM BACTERIAL ISOLATES OF
TRADITIONAL LEATHER PROCESSING PONDS FOUND AROUND
GONDAR TOWN
A Thesis Submitted to the Postgraduate Program Directorate
(College of Natural and Computational Sciences)
HARAMAYA UNIVERSITY
In Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE IN
BIOTECHNOLOGY
Miesho Hadush
October 2015
Haramaya
HARAMAYA UNIVERSITY
Postgraduate Program Directorate
We hereby certify that we have read and evaluated this thesis titled “Protease Production from
Bacterial Isolates of Traditional Leather Processing Ponds found Around Gondar Town”
prepared under our guidance by Miesho Hadush. We recommend that it be submitted as fulfilling
the thesis requirement.
Berhanu Andualem (PhD)___________________ ___________
Major Advisor Signature Date
Ameha Kebede (PhD)
____________________________
Co-Advisor Signature Date
As member of the Board of Examiners of the M.Sc. Thesis Open Defense Examination, we
certify that we have read and evaluated the Thesis prepared by Miesho Hadush and examined the
candidate. We recommended that the Thesis be accepted as fulfilling the Thesis requirement for
the Degree of Master of Science in Biotechnology.
__________________________________ _______________
Chair person Signature Date
____________________________________ _______________
Internal Examiner Signature Date
____________________________________________________
External Examiner Signature Date
ii
DEDICATION
This piece of work is dedicated to my beloved family for making me what I am today.
iii
STATEMENT OF THE AUTHOR
I declare that this thesis is a result of my genuine work and that all sources of materials used for
this thesis have been duly acknowledged. This thesis has been submitted in partial fulfillment of
the requirements for M.Sc. degree at Haramaya University and will be deposited at University
Library to be available to borrowers under the rules of the library. I declare that this thesis is not
submitted to any other institution anywhere for the award of any academic degree, diploma, or
certificate.
Brief quotations from this thesis are allowable without special permission provided that accurate
acknowledgment of source is made. Requests for permission for extended quotation from or
reproduction of this manuscript in whole or in part may be granted by the head of the major
department or the Dean of the school of Graduate Studies when in his or her judgment the
proposed use of the material is in the interests of scholarship. In all other instances, however,
permission must be obtained from the author.
Miesho Hadush Signature: __________________
Place: Haramaya University, Haramaya
Date of submission:________________
iv
BIOGRAPHICAL SKETCH
The author was born on August 22, 1988 at Endabaguna, Westren Tigray from his father Hadush
Berhe and his mother Abrehet Asgedom. He attended his Elementary School education at
Endabaguna Elementary School and his secondary and preparatory school education at Aksum
Comprehensive High School.
After completing Senior Secondary School in 2008, he joined the University of Gondar in 2009
to pursue a study leading to the Degree of Bachelor of Sciences in Biology, and graduated with
BSc in July 2011. After graduation, he immediately joined Haramaya University to pursue a
study leading to the Degree of Master of Science in Biotechnology in September, 2012.
v
ACKNOWLEDGMENTS
Right from the outset, I would like to thank the supreme Almighty God for providing me the
patience and endurance to complete the study. After that I would like to express my deepest
heartfelt thanks to my major advisor, Dr. Berhanu Andualem for his unreserved guidance,
supervision, valuable suggestions and intensive help, for the freedom of discussion and generous
personal encouragement starting from the initial stage of thesis proposal development to the final
write up.
I would like to express my heartfelt gratitude to my Co advisor Dr. Ameha Kebede for his
excellent advice, guidance, critical reading, and valuable suggestions during the course of my
study.
I would like to thank the Ministry of Education of the Federal Democratic Republic of Ethiopia,
for giving me the opportunity to pursue my postgraduate study and sponsoring my in-country
scholarship. I wish also to express my sincere words of thanks to Haramaya University School of
Graduate Studies and Department of Biology for providing me with all the required support.
My special and grateful thanks go to University of Gondar, Department of Biotechnology for
providing me all the resources and facilities used in the study. I wish to express my special
thanks to Miss Tigst Alene, Mr. Tadele Tamru, Mr. Tekeba, Mr. Gebrekidan Woldegerima and
Mr. Wagaw for their great help during the laboratory work of the study.
Finally, I am grateful to my parents, sisters and brothers for their endless support and care during
the thesis preparation as in all stages of my life.
vi
LIST OF ABBREVIATIONS AND ACRONYMS
AA
Amino acids
CYP
Casein-Yeast extract- Peptone
OD
Optical density
OVAT
One Variable at a Time
rpm
revolutions per minute
SmF
Submerged fermentation
spp.
Species
SSF
Solid state fermentation
TCA
Ttrichloroacetic acid
TSI
Triple sugar iron
U
Unit
UV
Ultra Violet
V/v
Volume by volume
W/v
Weight by volume
U/ml
Units per milliliter
vii
TABLE OF CONTENT
Page
STATEMENT OF THE AUTHOR
IV
BIOGRAPHICAL SKETCH
V
ACKNOWLEDGMENTS
VI
LIST OF ABBREVIATIONS AND ACRONYMS
VII
LIST OF TABLES
XII
LIST OF FIGURES
XIII
ABSTRACT
XIV
1. INTRODUCTION
1
2. LITERATURE REVIEW
5
2.1.
Enzyme
5
2.2.
Proteases
5
2.3.
Occurrence of Protease Enzyme
6
2.4.
Classifications of Proteases
6
2.5.
Sources of Protease Enzyme
8
2.5.1. Microbial proteases
2.5.1.1.
Proteases from neutralophiles
2.5.1.2.
Proteases from alkaliphiles
2.6.
2.6.1.
2.6.2.
8
9
9
Protease Production Methods
10
Submerged fermentation (SmF)
Solid state fermentation (SSF)
10
10
2.7.
Protease Assay
11
2.8.
Factors Affecting Activity and Stability of Proteases
11
2.8.1.
2.8.2.
2.8.3.
2.8.4.
2.8.5.
2.8.6.
Effect of pH
Effect of temperature and incubation period
Effect of substrate
Effect of moisture content
Time course
Inoculum size
viii
12
12
12
13
13
14
2.9.
Industrial Application of Proteases
2.9.1.
2.9.2.
2.9.3.
2.9.4.
2.9.5.
2.9.6.
2.9.7.
2.9.8.
2.9.9.
2.9.10.
2.9.11.
2.9.12.
2.9.13.
2.9.14.
Application of proteases in leather industry
Food and feed industry
Dairy industry
Baking Industry
Brewing industry
Manufacture of soy products
Removal of bitter components of protein hydrolysates
Feed processing
Detergent industry
Medical and pharmaceutical industry
Photographic industry
Chemical industry
Textile industry
Management of industrial and household wastes
14
14
17
17
17
18
18
19
19
20
20
21
21
21
22
2.10. Status of Research and Potential Applications of Proteases in Ethiopia
22
3. MATERIALS AND METHODS
24
3.1.
Description of the Study Area
24
3.2.
Research Design
24
3.3.
Sample Collection and Isolation of Protease Producing Isolates
24
3.4.
Screening for Protease Production
25
3.5.
Preparation of Substrates for Solid State Fermentation
26
3.6.
Solid State Fermentation (SSF)
26
3.7.
Determination of the Protease Activity of Selected Isolates
26
3.7.1.
3.7.2.
3.8.
Protease assay
Tyrosine calibration curve
26
27
Phenotypic Characterization of the Bacterial Isolates
3.8.1. Macroscopic characterization of isolates
3.8.2. Microscopic characterization of isolates
3.8.2.1.
Gram staining
3.8.2.2.
Motility test
3.8.2.3.
Endospore staining
3.8.3. Biochemical characterization of isolates
3.8.3.1.
Catalase test
3.8.3.2.
Starch hydrolysis
3.8.3.3.
Urea hydrolysis
3.8.3.4.
Carbohydrate fermentation tests
3.8.3.5.
Gas production using triple sugar iron test (TSI)
ix
27
27
27
27
28
28
28
28
29
29
29
29
3.9.
Seed Culture Medium
30
3.10. Optimization of the Growth Conditions for Production of Protease
3.10.1.
3.10.2.
3.10.3.
3.10.4.
3.10.5.
3.10.6.
3.10.7.
3.10.8.
Effect of time on the production of protease
Effect of temperature on the production of protease
Effect of pH on the production of protease
Effect of carbon source on the production of protease
Effect of nitrogen source on the production of protease
Effect of NaCl concentration on the production of protease
Effect of moisture level on protease production
Effect of inoculum size on protease production
3.11. Characterization of Protease
3.11.1.
3.11.2.
30
30
30
30
31
31
31
31
32
32
Effect of pH on the activity and stability of protease
Effect of temperature on the activity and stability of protease
32
32
3.12. Partial Purification of Crude Enzyme
33
3.13. Data Analysis
33
4. RESULTS AND DISCUSSION
34
4.1.
34
4.1.1.
4.1.2.
Isolation, Screening and Selection of Protease Producing Bacteria
Isolation and screening of protease producing bacteria
Selection of the best protease producing bacteria
34
34
4.2.
Phenotypic Characterization of the Bacterial Isolates
36
4.3.
Effect of Culture Conditions on Protease Production under SSF
37
4.3.1.
4.3.2.
4.3.3.
4.3.4.
4.3.5.
4.3.6.
4.3.7.
4.3.8.
4.4.
4.4.1.
4.4.2.
4.4.3.
4.4.4.
4.5.
4.5.1.
Effect of time course on protease production
Effect of temperature on the production of protease
Effect of initial pH on the production of protease
Effect of different carbon sources on the production of protease
Effect of nitrogen source on the production of protease
Effect of inoculum size on protease production
Effect of moisture content on protease production
Effect of NaCl concentration on the production of protease
Characterization of Protease
38
39
40
41
42
43
44
45
46
Effect of pH on the activities of proteases of the selected isolates
Effect of pH on the stability of proteases of the selected isolates
Effect of temperature on the activities of proteases of the selected isolates
Effect of temperature on the stability of proteases of the selected isolates
46
47
48
48
Partial Purification of Crude Enzymes for Hair Removal
49
Enzymatic cow hide dehairing
50
5. SUMMARY, CONCLUSION AND RECOMMENDATION
x
52
5.1.
Summary and Conclusion
52
5.2.
Recommendations
54
6. REFERENCES
55
7. APPENDICES
65
xi
LIST OF TABLES
Table
Page
1. Screening of the 15 protease producing isolates
35
2. Morphological and biochemical characteristics of the selected bacterial isolates
37
3. Partial purification of Protease produced from selected bacterial isolates
50
xii
LIST OF FIGURES
Figure
Page
1 . Zone of hydrolysis of casein by the three selected isolates
36
2 . Effect of incubation time on protease production
38
3. The effect of incubation temperature on protease production.
39
4. The effect of initial pH of the media on protease production
40
5. The effect of different carbon sources on protease production
41
6. Effect of different nitrogen sources on protease production
42
7. The effect of inoculum size on protease production
43
8. Effect of moisture level on protease production
44
9. Effect of NaCl concentration on the production of protease
46
10. Effect of pH on the activity of protease
46
11. EffectofpHonstabilityofprotease
47
12. Effect of incubation temperature on the activity of proteases
48
13. Effect of temperature on the stability of proteases
49
14 .Results of cow hide dehairing experiment: (a) & (b) raw hide before dehairing, and (c) and
(d) are enzyme treated cow hide dehairing with fingers.
xiii
51
PROTEASE PRODUCTION FROM BACTERIAL ISOLATES OF
TRADITIONAL LEATHER PROCESSING PONDS FOUND AROUND
GONDAR TOWN
ABSTRACT
Microbial proteases are hydrolytic enzymes widely used in many industrial processes and management of
wastes. This study was conducted with the aim of screening for potent protease-producing bacteria from
soils and water samples, determining optimal production conditions and partially characterizing the
stability and activity of the protease with regards to some physicochemical parameters. The
experimental design used for the optimization of cultivation conditions was conducted following
the OVAT (One Variable At a Time) method. Thus, plating with heat-shocked macerated samples on
casein agar media resulted in 3 potent proteolytic bacterial isolates which were later identified as
Bacillus species on the basis of their morphological and biochemical characteristics. The optimum
protease production time for these 3 isolates was found to be 48 h corresponding to a protease activity of
3.5 U/ml for Ds-7, 2.7 U/ml for Ew-9, and 4.2 U/ml for Sw-11 isolated from traditional leather
processing ponds.. The optimum temperature of protease production for both Ew-9 and Sw-11 was 37°C,
corresponding to 10.1 and 9.0 U/ml, respectively. Whereas 40°C was the optimum for Ds-7 which
resulted in a protease activity of 9.3 U/ml. In all cases, pH 7 was the optimum for production of protease
with activities corresponding to 13.8 U/ml, 12.5 U/ml and 10.2 U/ml for Ds-7, Ew-9 and Sw-11,
respectively. Among the various carbon sources tested, wheat bran gave maximum activity for isolates
Dw-7 (20.0 U/ml) and Ew-9 (12.9 U/ml), while glucose gave the highest only for Sw-11 (12.8 U/ml).
Regarding nitrogen sources, casein gave maximum activity for Ds-7 (33.5 U/ml) and Ew-9 (37.6 U/ml).
Whereas yeast extract was the best for Sw-11 (43.0 U/ml). Furthermore, 0.2 M NaCl concentration was
found to give better protease for isolates Ds-7 and Sw-11 corresponding to 6.8 U/ml and4.9U/ml
respectively.whereas,0.4M was the optimum for Ew-9 which resulted in a protease activity of 4.9 U/ml.
The best percentage of inoculum level for maximum production of protease was 10% in all isolates.
Regarding the moisture content of the medium 1:3 w/v bran to moistening agent ratio was found to be the
best for maximum protease production in all isolates. Studies on the effect of pH on the activity and
stability of protease enzymes revealed that the crude enzyme had a maximum activity and stability at pH
9.0 for isolates Ds-7 and Ew-9 (6.5 and 5.1 U/ml; and 3.4 and 4.7 U/ml for maximum activity and
stability at pH 9.0, respectively), while for isolate Sw-11 maximum activity and stability was achieved at
pH 8 with values corresponding to 4.5 and 4.4 U/ml, respectively. These results generally indicate that
the proteases obtained in this study belong to the class of alkaline protease. These proteases are also
active and stable at 50оC for Ds-7 and Sw-11 (41.8 and 32.3 U/ml; 43.8 and 42.8U/ml, respectively).
Whereas, isolate Ew-9 showed maximum activity and stability at 40оC corresponding to 21.9 and 21.5
U/ml, respectively. Pre-incubation at temperatures above 70°C for all isolates resulted in reduction of
enzyme activity, indicating that the proteases are thermally unstable. Application of the enzyme at the
inner side of cow skin at a dosage of 12.8U/ml, pH 7.5 brought about complete removal of hair within 24
h at room temperature and 12 h at 37°C. Since protease was produced from readily available complex
substrates and agro-industrial wastes, the three Bacillus species appear to have substantial potential for
application in various proteolytic processes. Thus, identification of these Bacillus isolates at a molecular
level and purification as well as detailed characterization of the types of proteases are recommended for
effective utilization in tannery industries.
Keyword: Bacillus spp., casein hydrolysis, dehairing, enzyme activity, protease,
xiv
1. INTRODUCTION
Proteases are hydrolytic enzymes found in every organism to undertake important physiological
functions. These include: cell division, regulating protein turnover, activation of zymogenic
performance, blood clotting, lysis of blood clot, processing and transport of secretory proteins
across membrane, nutrition, regulation of gene expression and virulence factors. Proteases differ
in their specific activities, substrate specificities, pH and temperature optima and stability, active
site, and catalytic mechanisms. All these features contributed in diversifying their classification
and practical applications in industries involving protein hydrolysis (Saeki et al., 2007).
The estimated value of worldwide use of industrial enzymes has increased from $1 billion in
1995 to $1.5 billion in 2000 (Kirk et al., 2002). As per the forecast, the global demand for
enzymes was estimated to rise 7% per annum through 2006 to $6 billion in 2011 (McCoy, 2000).
Proteases represent one of the major groups of industrial enzymes because of their widespread
use in detergents and dairy industries and industrial sales of protease are estimated at more than
$350 million annually (Kirk et al., 2002). Proteases account for the 60-65% of the global
industrial enzyme market and out of this 25% constitute alkaline proteases, 3% trypsin, 10%
renin and 21% other proteases (Bhosale et al., 1995; Rao et al., 1998). Proteases show a vast
diversity in their physico-chemical and catalytic properties and a lot of literature is available on
their biochemical and biotechnological aspects (Rao et al., 1998). The proteases of industrial
importance are obtained from animals, plants and microorganisms. The proteolytic enzymes
hydrolyze the peptide links of proteins and peptides to form smaller subunits of amino acids and
are produced both extracellularly as well as intracellularly (Gajju et al., 1996). The Proteases
play an important role in a wide range of industrial processes viz., baking, brewing, detergents,
leather processing, pharmaceuticals, meat tenderization, cosmetics and medical diagnosis
(Bhalla et al., 1999; Gupta et al., 2002; Najafi et al., 2005)
Proteases are essential constituents of all forms of life on earth. Microbial proteases are among
the most important, extensively studied groups since the development of enzymology.
2
Neutralophilic and alkaliphilic microbial alkaline proteases possess a considerable industrial
potential due to their biochemical diversity and stability at extreme pH environments,
respectively (Moon et al., 1994). However, the demanding industrial conditions for technological
applications and cost of protease production required continuous exercise for search of new
microbial resources. Enzyme cost is also the most critical factor limiting wide use of protease for
different applications. A large part of this cost is accounted for the production cost of the
enzyme. Therefore, reduction in the production cost of enzymes could greatly reduce the cost of
the enzyme. In submerged fermentation up to 40% of the total production cost of enzymes is due
to the cost of the growth substrate (Enshasy et al., 2008). In this regard, solid state fermentation
(SSF) which uses cheap agricultural residues have enormous potential in reducing enzyme
production cost. So, studies on protease that are produced in SSF by microorganisms are scarce
in literature. As a result, it is of great importance to pursue such studies. This type of
fermentation process also does not require high caliber equipment and energy for agitation to
provide oxygen.
Proteases catalyze the addition of water across amide (and ester) bonds to cleave using a reaction
involving nucleophilic attack on the carbonyl carbon of the scissile bond. They differ widely in
their properties such as substrate specificity, active site and catalytic mechanism and possess
different profiles for mechanical stress, chemical environment, pH and temperature for stability
and activity. Because of their broad substrate specificity, proteases have a wide range of
applications such as in leather processing, detergent formulations, baking, brewing, meat
tenderization, cheese manufacture, soy sauce production, protein hydrolysate, pharmaceutical
industries, waste treatment, silk industry, recovery of silver from waste photographic film, as
well as analytical tools in basic research and have high commercial value (Godfrey and West,
1996). Among the bulk of industrial enzymes, proteases from plant, animal, and microorganism
constitute around 60 % of the total worldwide enzyme sales (Kunamneni et al., 2003). Currently,
the largest share of the enzyme market has been held by detergent proteases which are active and
stable at alkaline pH.
They are also important from a physiological point of view, as they are involved in many cellular
processes like protein turn over and digestion as well as fungal morphogenesis, spore formation
3
and spore germination. Yet, there is a continued search for proteases having novel properties
with known and newer applications.
Proteases play crucial roles in different applications: in producing the food we eat, the clothes we
wear, the drugs we need, the detergents we use, even in producing fuel for our automobiles, etc.
Apart from use in various production processes towards greater efficiency, enzymes are also
important in reducing both energy consumption and combating environmental pollution.
Proteases are enzymes which catalyze the hydrolysis of protein molecules. They are so far
exploited as industrial catalysts in various industrial sectors, leather processing being the most
interesting and potential area for large scale application (Kumar and Bhalla, 2005).
Ethiopians are believed to have been practicing traditional leather processing since their ancient
civilizations. This local knowledge has been transferred through generations and is being widely
practiced these days to process leathers of cattle origin for making their shoes, clothes, beds,
cushions and many other items primarily among the rural communities. They use small ponds
usually on the sides of rivers and embed the leather for a period of time to remove hairs.
However, very little has been done to promote this work through the application of
biotechnology. At present there are few scientific reports available in Ethiopia on the potential
microbial isolates that can be used in the process of dehairing. Therefore, there is a need to
investigate the role and contribution of microorganisms during traditional leather processing. The
outcome of this study may be helpful to develop a protocol and apply this knowledge for modern
dehairing process in leather production at industrial level. Moreover, this study may help to
document information on our microbial wealth and their proteolytic enzymes that can potentially
be used for other applications using modern fermentation processes. To this effect, this research
was attempted to address the following objectives.
General objective:
 The general objective of the study was to isolate, characterize, and optimize production
of industrial protease through solid state fermentation from traditional leather processing
ponds and to evaluate its application for leather industry.
4
Specific objectives:
 To isolate potential protease producing bacteria from ponds used in leather
processing around Gondar town.
 To determine the effect of different culture conditions on protease production
from selected bacterial isolates under solid-state fermentation
 To characterize the produced protease in terms of various physico-chemical
parameters (Temperature and pH).
 To evaluate potential application of the proteases for dehairing in leather
processing.
5
2. LITERATURE REVIEW
2.1. Enzyme
An enzyme is a protein that catalyzes, or speeds up, a chemical reaction. Enzymes are essential
to sustain life because most chemical reactions in biological cells would occur too slowly, or
would lead to different products, without enzymes. A malfunction (mutation, overproduction,
underproduction or deletion) of a single critical enzyme can lead to a severe disease. Like all
catalysts, enzymes work by lowering the activation energy of a reaction, thus allowing the
reaction to proceed much faster. Enzymes may speed up reactions by a factor of many millions.
An enzyme, like any catalyst, remains unaltered by the completed reaction and can therefore
continue to function. Because enzymes, like all catalysts, do not affect the relative energy
between the products and reagents, they do not affect equilibrium of a reaction (Abdullah, 2006).
However, the advantage of enzymes compared to most other catalysts is their sterio-, regio- and
chemo selectivity and specificity. While all enzymes have a biological role, some enzymes are
used commercially for other purposes. Many household cleaners use enzymes to speed up
chemical reactions (Gioppo et al., 2009).
2.2. Proteases
Proteases (proteinases, peptidases or proteolytic enzymes) are enzymes that break peptide bonds
between amino acids of proteins. The process is called proteolytic cleavage, a common
mechanism of activation or inactivation of enzymes especially involved in blood coagulation or
digestion. Protease constitutes one of the most important groups of industrial enzymes
accounting for about 60% of the total worldwide enzyme sales. Protease is one of the digestive
enzymes that conduct proteolysis, by hydrolysis of the peptide bonds that link amino acids
together in the polypeptide chain forming the protein. Proteases belong to the class of enzymes
known as hydrolases which catalyse the reaction of hydrolysis of various bonds with the
participation of a water molecule (Muthu and christudhas, 2012).
6
2.3. Occurrence of Protease Enzyme
Proteases occur naturally in all organisms. These enzymes are involved in a multitude of
physiological reactions from simple digestion of food proteins to highly regulated cascades (e.g.
the blood-clotting cascade, the complement system, apoptosis pathways and the invertebrate
prophenoloxidase-activating cascade). Acid proteases secreted into the stomach (such as pepsin)
and serine proteases present in duodenum (trypsin and chymotrypsin) enable us to digest the
protein in food; proteases present in blood serum (thrombin, plasmin, Hageman factor, etc.) play
important role in blood-clotting, as well as lysis of the clots, and the correct action of the
immune system. Other proteases are present in leukocytes (elastase, cathepsin G) and play
several different roles in metabolic control. Proteases determine the lifetime of other proteins
playing important physiological role like hormones, antibodies or other enzymes, this is one of
the fastest "switching on" and "switching off" regulatory mechanisms in the physiology of an
organism (Kumari et al., 2012).
Bacteria also secrete proteases to hydrolyse (digest) the peptide bonds in proteins and therefore
break the proteins down into their constituent monomers. Bacterial and fungal proteases are
particularly important to the global carbon and nitrogen cycles in the recycling of proteins and
such activity tends to be regulated in by nutritional signals in these organisms. A secreted
bacterial protease may also act as an exotoxin and be an example of a virulence factor in
bacterial pathogenesis. Bacterial exotoxic proteases destroy extracellular structures. Protease
enzymes are also used extensively in the bread industry in bread improver (Sana et al., 2006).
2.4. Classifications of Proteases
Proteases (EC 3.4.21-24) are classified based on chemical nature of the active site, the reaction they
catalyze, and their structure and composition (Rao et al., 1998). The major classes are again
classified into sub classes based on pH, catalytic site on polypeptide, occurrence, and so on.
Based on the catalytic site on the substrate, proteases are mainly classified in to endoproteases
and exoproteases (Rao et al., 1998). Endoproteases preferably act at the inner region of the
polypeptide chain. By contrast, exoproteases preferentially act at the end of the polypeptide
chain. Exoproteases are further classified in to amino peptidases (those proteases which act at the
7
free N-terminus of the polypeptide substrate), and carboxypeptidases (those proteases which act
at the free C-terminal of the polypeptide chain (Rao et al., 1998).
Similarly, endoproteases are also classified based on the functional group present in active site
and pH optimum. The different classes of proteases based on their catalytic active site include:
Serine proteases: Serine proteases are proteases having a serine group (-OH) in their active site.
Cystein proteases:Cystein proteases are proteases having a thiol (-SH) group in their active site.
Metalloproteases: Metalloproteases are proteases requiring divalent metal ion for their catalytic
activity.
Aspartic protease: Aspartic proteases are proteases with aspartic residue at their catalytic active
site.Other rare proteases also contain other amino acid residues at their active site, such as
threonine and glutamic acid.
Based on their optimal pH requirements, proteases are also classified as:
Acid proteases:Acid proteases are proteases which are active in the pH ranges of 2-6 (Rao et al.,
1998) and are mainly of fungal in origin (Aguilar et al., 2008). Common examples in this
subclass include aspartic proteases of the pepsin family. Some of the metalloprotease and cystein
proteases are also categorized in as acidic proteases.
Neutral proteases: Neutral proteases are proteases which are active at neutral, weakly alkaline
or weakly acidic pH .Majority of the cystein proteases, metalloproteases, and some of the serine
proteases are classified under neutral proteases. They are mainly of plant in origin, except few
fungal and bacterial neutral proteases (Aguilar et al., 2008).
Alkaline proteases:Alkaline proteases are optimally active in the alkaline range (pH 8-13),
though they maintain some activity in the neutral pH range as well (Horikoshi, 1999). They are
obtained mainly from neutralophilic and alkaliphilic microorganisms such as Bacillus and
Streptomyces species. In most cases the active site consists of a serine residue, though some
alkaline proteases may have other amino acid residue in their active site (Rao et al., 1998).
8
2.5. Sources of Protease Enzyme
Proteases are widely distributed in animals, plants, fungi and bacteria. Several proteases from
different sources are currently in the market, but almost all are products of microbial origin.
Protease are produced by both neutralophilic and alkaliphilic microorganisms. These two groups
represent almost all sources of commercial alkaline proteases currently available in the market
(Moon et al., 1994).
2.5.1. Microbial proteases
Proteases are essential constituents of all forms of life on earth, including prokaryotic eukaryotic
organisms. The inability of the plant and animal proteases to meet the current world has led to an
increased interest in microbial proteases. The proteases available today in the market are derived
from microbial sources. This is due to their high productivity, limited cultivation space
requirement, easy genetic manipulation, broad biochemical diversity and desirable characteristics
that make them suitable for biotechnological applications (Genckal, 2004).
Proteases are ubiquitous and found in several microorganisms such as protozoa, bacteria, yeast
and fungi. Microbial proteases can be cultured in large quantities in a relatively short time by
established methods of fermentation and they also produce an abundant, regular supply of the
desired product. Microorganisms account for a two-third share of commercial protease
production in the world (Gupta et al., 2002; Vishwanatha, 2009).
Microbial proteases are among the most important hydrolytic enzymes and have been studied
extensively since the advent of enzymology. Microorganisms elaborate a large array of
proteases, which are intracellular and/or extracellular. Intracellular proteases are important for
various cellular and metabolic processes, such as sporulation and differentiation, protein
turnover, maturation of enzymes and hormones and maintenance of the cellular protein pool.
Extracellular proteases are important for the hydrolysis of proteins in cell-free environments and
enable the cell to absorb and utilize hydrolytic products. At the same time, these extracellular
proteases have also been commercially exploited to assist protein degradation in various
industrial processes (Gupta et al., 2002).
9
Protease production is an inherent capacity of all microorganisms; and a large number of
bacterial species are known to produce alkaline proteases of the serine-type, although very few
are recognized as commercial producers. Only those microbes that produce substantial amounts
of extracellular enzyme are of industrial importance. Microbial proteases account for
approximately 40% of the total worldwide enzyme sales as they possess almost all the
characteristics desired for biotechnological applications (Genckal, 2004; Kiran et al.,
2012).Although there are many microbial sources available for producing proteases, only a few
are recognized as commercial producers. Several products based on bacterial proteases have
been launched successfully in the market in past few years. Most of the commercial proteases are
of bacterial origin (Vigneshwaran et al., 2010). Though proteases are produced by variety of
bacteria
such
as
Bacillus,
Pseudomonas
aeruginosa,
Flavobacterium,
Clostridium,
Staphylococcus aureus, Achromo-bacter, Thermoactinomycesand species belonging to
Streptomyces, Bacillus is the major source and secretes a variety of soluble extracellular enzymes
which reflect the diversity of their habitats (Nirmal et al., 2011).
2.5.1.1.Proteases from neutralophiles
Neutralophiles are organisms that exhibit optimum growth at neutral pH range (Horikoshi,
1999). Their biochemical diversity significantly contributes in diversifying the protease
applications, and their market value (Moon et al., 1994). Bacillus subtilisandBacillus
licheniformisare the major and highly exploited neutralophilic organisms for alkaline protease
production. Members of the subtilisin super family of proteases that are used in almost all of the
technical protease application areas today are obtained from these groups (Maurer, 2004).
2.5.1.2.Proteases from alkaliphiles
Alkaliphiles are organisms isolated from extremely alkaline environments such as soda lake,
having their optimum growth pH above 9 (Horikoshi, 1999). Examples of alkaliphilic
microorganisms producing alkaline proteases include Bacillus firmus, Bacillus lentus, and
alkaliphilic Actinomycetes (Moon et al., 1994). Proteases from these sources are extremely
stable at high pH; as a result, they draw the attentions of many biotechnological companies and
researchers in the world (Patel et al., 2006).
10
2.6. Protease Production Methods
Selection of best fermentation technique and optimization of cultivation conditions offer
numerous advantages in reducing cost of enzyme production and increasing enzyme productivity
(Gizachew, 2009). Currently, enzyme production from microorganisms can be achieved using
SmF and SSF.
2.6.1. Submerged fermentation (SmF)
Submerged fermentation is the cultivation of microorganisms in liquid nutrient broth to produce
enzymes (Gangadharan et al., 2008). This involves growing carefully selected microorganisms in
closed vessels containing a rich broth of nutrient medium and a high concentration of oxygen. As
the microorganisms break down the nutrients, they release the desired enzymes into solution.
SmF has been traditionally used for the production of industrially important enzymes because of
the ease of control of different parameters such as pH, temperature, aeration, and moisture
content (Haddaoui et al., 1999). Despite of this importance, this enzyme production technique
has several drawbacks. Some of them are: (i) uses expensive synthetic media, (ii) consumes high
volumes of water and discards high volume of polluting effluents, (iii) requires high level of air
and (iv) offers low and diluted products that need further down streaming process and costs
(Gangadharan et al., 2008).
2.6.2. Solid state fermentation (SSF)
Solid-state fermentation (SSF) is defined as the growth of microorganisms on moist solid
substrates in the absence or low of free flowing water. In the SSF process, the solid substrate not
only supplies the nutrients to the cultures but also serves as an anchorage for the growth of
microbial cells (Kar et al., 2010; Perez et al., 2009). SSF systems appear promising due to the
natural potential and advantages they offer. Since SmF can be considered as a violation of their
natural habitat of the microorganisms, especially for fungi, SSF is by far better than SmF
(Singhania et al., 2010). Due to the advantages of SSF over SmF, recently SSF has been reported
9 to be the most appropriate process of enzyme production especially for developing countries
(Pandey, 2003; Serin et al., 2012). Furthermore, use of agricultural wastes has made SSF an
attractive method of enzyme production (Serin et al., 2012). The major factors that affect
11
microbial synthesis of enzymes in SSF system include selection of a suitable substrate and
microorganism, inoculums concentration, pH, temperature and moisture level (Barrios-Gonzalez
et al., 2009). Thus, optimization of fermentation conditions during fermentation time is essential
to increase productivity of the organisms and to extract enzymes that have better characteristics
(Pandey et al., 2000).
2.7. Protease Assay
The original protease assay uses casein as a substrate and involved TCA (trichloroacetic acid)
precipitation of the undigested substrate, followed by photometric quantification of the released
aromatic amino acids, using L-tyrosine as a standard. Due to their compact conformation, native
proteins are generally not very susceptible to degradation by proteases. Protein substrates for
proteases are most often hemoglobin or casein and must be completely soluble in buffer. Casein
precipitates below 6, so it is used at neutral to alkaline pH (Sumantha et al., 2006).
Peptide bonds are more exposed and liable to proteolytic attack when proteins are unfolded due
to denaturation. Diazotised protein allows measurement of solubilised peptide with a visible
range colorimeter. Hence, pH and temperature curves should be carried out to determine the
individual requirements of the system. Assay for proteases generally involves incubating the
enzyme with its substrate for a specific time period, arresting the reaction with TCA, and
measuring the absorbance of the solubilised peptide (Sumantha et al., 2006).
2.8. Factors Affecting Activity and Stability of Proteases
It is well established that extracellular protease production in microorganisms is greatly
influenced by media components and physicochemical factors. Therefore, various carbon and
nitrogen nutrient cost-effective substrates, divalent metal ions, environmental and fermentation
parameters such as pH, incubation temperature, time aeration, agitation speeds, inoculums ages
and density were found to affect the production of proteases (Oskouie et al., 2007; Qureshi et al.,
2011).
12
2.8.1. Effect of pH
The important characteristic of most alkalophilic microorganisms is their strong dependence on
the extracellular pH for cell growth and enzyme production. Initial pH of the medium required
for obtaining maximum production depends not only upon the bacterium but also upon the
ingredients of the medium. Maximum alkaline protease production has been reported from
different pH ranges (7-11) by different Bacillus species. The pH requirements vary from species
to species or even in different strains of the same species isolated from different habitats. Some
bacterial cultures in unbuffered media have been observed to exert a change in pH following the
growth and alkaline protease production (Nadeem, 2009). The advantage in the use of carbonate
in the medium for an alkaline protease has been well demonstrated. The culture pH also strongly
affects many enzymatic processes and transport of various components across the cell membrane
(Nirmal et al., 2011).
2.8.2. Effect of temperature and incubation period
The mechanism of temperature control of enzyme production is not well understood. However,
studies by Frankena et al. (1986) showed that a link existed between enzyme synthesis and
energy metabolism in bacilli, which was controlled by temperature and oxygen uptake.
Incubation period for maximum yield of enzyme varies among various species or even in the
same species isolated from various sources. Many workers have reported a broad incubation
period ranging from 36 to 96 h for the maximum yield of protease enzyme by Bacillus strains
(Nadeem, 2009).
2.8.3. Effect of substrate
The production of enzymes is highly influenced by carbon and nitrogen sources. A number of
carbon sources such as glucose, sucrose, fructose, glycerol etc. have been used as carbon sources
for protease production. Studies have also indicated that a high carbohydrate concentration
repressed enzyme production. Therefore, carbohydrates can be added either continuously or in
aliquots (fed batch) throughout the fermentation to supplement the exhausted component
(Gitishree and Prasad, 2010).
13
Organic nitrogen sources like soya bean meal, corn step liquor, soya oil, corn glutan etc as well
as inorganic nitrogen such as nitrates or ammonium salts, amino acids etc have been used for
protease production. Complex nitrogen source is preferred as it is slowly degraded in medium
resulting in the availability of low levels of amino acids/peptides in the medium which act as
inducers of protease production (Nadeem 2009; Nirmal et al., 2011).
Cells may require divalent cations for the production of proteases and their activity. Presence of
various metal ions in the growth medium affects the production of enzymes including
improvement of alkaline protease activity. Ca2+ stabilizes the structure of most of microbial
alkaline proteases and results in increased proteolytic activity of the enzyme. The other metal
ions that have been reported to affect proteolytic activity of bacterial alkaline proteases are Mg2+,
Fe3+, Zn2+, Mn2+, K+, Co2+and Cu2+ (Nadeem, 2009).
2.8.4. Effect of moisture content
Moisture is one of the most important parameters that influences the growth of the organism and
thereby enzyme production in SSF (Mrudula et al., 2011). Moisture is causes swelling of the
substrates, thereby facilitating better utilization of the substrate by microorganisms (Mrudula et
al., 2011). Low and high moisture levels of the substrate affect the growth of the microorganism
resulting in lowing enzyme production and stability. On the other hand, high moisture content
leads to reduction in substrate porosity, changes in the structure of substrate particles and
reduction of gas volume (Kunamneni et al., 2005).
2.8.5. Time course
Maximum production of enzyme is related to incubation time and become high when the cell
population entered into stationary phase of growth. In addition, the incubation time used for
achieving the maximum enzyme level is governed by the characteristic of the culture and is
based on growth rate and enzyme production (Serin et al., 2012). Thus, knowing maximum
protease production period could be beneficial in managing production cost associated with
incubation time.
14
2.8.6. Inoculum size
Inoculum size is an important factor for the production of enzyme. Lower inoculum size results
in a lower number of cells in the production medium while higher inoculums level leads to rapid
utilization of substrate in a short period. Hence, using of optimum inoculum size is important
requirement to utilize the substrate and to produce the desired products (Saxena and Singh,
2011).
2.9. Industrial Application of Proteases
Proteases have several industrial applications. These include: as processing aid in leather tanning
industries, as detergent additive, in protein hydrolysis, in pharmaceuticals production, and in
chemical synthesis (Gupta et al., 2002). According to Rao et al. (1998), Proteases account more
than 25% of the global enzyme market.
2.9.1. Application of proteases in leather industry
The global environment is gradually deteriorating because of the socio-economic activities of
humankind such as processing industries. Many industrial processes cause adverse changes in
the immediate environmental change and therefore being challenged by society. Of these, leather
industries and the increased amount of feathers generated by commercial poultry
processing may represent a pollution problem and needs adequate management (Shih, 1993).
Leather processing involves a series of unit operations. At each stage, various chemicals are used
and varieties of materials are expelled (Thanikaivelan et al., 2004). Depilation or dehairing of
hides and skins in leather industry is traditionally done with chemical methods using lime,
sodium sulfide, etc, which contributes to 80-90% of the total pollution load in the leather
industry and generates noxious gases as well as solid wastes, e.g. hydrogen sulfide and lime
(Thanikaivelan et al., 2004). Therefore, leather industry is one of the industries looking up to
enzymes to reduce the impact of tanning processes on the environment (Sundararajan et al.,
2010).
The enzymatic treatment destroys undesirable pigments, increases the skin area and thereby
clean hide is produced. Bating is traditionally an enzymatic process involving pancreatic
15
proteases. However, recently, the use of microbial alkaline proteases has become popular.
Alkaline proteases speed up the process of dehairing, because the alkaline conditions enable the
swelling of hair roots; and the subsequent attack of protease on the hair follicle protein allows
easy removal of the hair (Gupta, 2002a; Genckal, 2004; Ikram, 2008; Nadeem, 2009; Ray, 2012).
There are three series of unit operation that involve leather processing.
1) Pre-tanning operation stage is the stage that is used to clean hide or skin in leather industry.
The non-collagen part of hide or skin proteins such as albumin, globulin, mucoids, and fibrous
proteins such as elastin, keratin, and reticulin are removed during this stage (Sivasubramanian et
al., 2008). (2) The tanning operation stage is a step that is used to stabilise the skin or hide matrix
and (3) Post-tanning or finishing operations is the stage that is used to add aesthetic quality of
leather (Thanikaivelan et al., 2005).
In pre-tanning stage, hide or skin undergoes series of treatment stages. These include: raw skin
preservation, socking, liming, dehairing and deliming, bating, degreasing, and pickling. The
pretanning stage uses several chemicals such as sodium sulphide, sodium chloride, lime,
chlorinated compounds and others that contribute for the generation of 80-90% of the total
pollution load released by leather industry (Thanikiavelan et al., 2004). Of these, dehairing
accounts for one third of these total pollution generated by leather industry (Kamini, et al.,
1999). The use of alkaline protease in soaking, dehairing and tanning shown to greatly reduce the
amount of pollution generated.
Soaking is a key step which prepares the hide for subsequent operation step by cleaning and
softening of hides and skins with water. This process results in solubilisation and elimination of
salts and globular proteins contained within the fibrous structure (Thanikaivelan et al., 2004). It
uses cured skin and is usually carried out under alkaline condition. Water, antiseptics such as
sodium hypochlorite, sodium pentachlorophenate, formic acid, and so on are used in soaking.
Addition of chemicals like sodium sulphide or sodium tetrasulphide aids the soaking process. It
has been reported that the use of alkaline proteases aid the soaking stage by breaking soluble
proteins of the inside matrix, and release salts and hyluaronic acid. As a result, water uptake
facilitated at alkaline at an alkaline pH (Kamini et al., 1999).
16
Dehairing is also an important operation in tanneries conventionally practiced using lime and
sodium sulphide (Thanikaivelan et al., 2004). In this process, the skin/hide is painted with
sulphide which helps to reduce the disulphide bond that is responsible for attachment of hair
keratin in epidermis. This brings about complete removal of hair, but the hair root remained
within skin (Sivasubramanian et al., 2008). In this process, lime contributes to the dehairing
process by opening up the collagen fiber structure. The use of alkaline protease has proven
superior and efficient for selective removal of the non-collagen part of hide/skin (Kamini et al.,
1999).
Compared to conventional dehairing, the use of alkaline protease for dehairing has the following
advantages (Thanikaivelan et al., 2005; Mukhtar and Ul-Haq, 2008). (1). Significant reduction or
even complete elimination of the use of sodium sulphide. (2). their non-polluting effect and
biodegradability. (3). Activity under mild conditions. (4). Reduction in dehairing time. (5). their
specificity. (6). Recovery of hair of good quality and strength with a good saleable feature that
can be used to develop animal feed additives. (7). Creation of an ecologically conducive
atmosphere for the workers. Thus, the skin/hide easily handled by work men. (8). enzymatically
dehaired leathers have shown better strength properties and greater surface area. (9).
Simplification of pre-tanning processes by cutting down one step.
The advantages of enzymatic dehairing include hair-saving dehairing process, a reduction of
sulfide content in the effluent, recovery of hair which is of good quality and elimination of the
bate in the de-liming (Choudhary et al., 2004; Brandelli, 2008; Arunachalam and Saritha, 2009).
Enzyme produced by Bacillus subtilis S14 has a potential to complete elimination of the need for
toxic sodium sulfide during dehairing process in leather industry (Macedo et al., 2005).
Therefore, the ever increasing attention to the environmental impact of leather industry has
necessitated for the development of enzyme based processes as potent alternatives to pollution
causing chemicals.
Keratinolytic proteases lacking collagenolytic and having mild elastolytic activities are
increasingly being explored for dehairing process because these enzymes would help in the
selective breakdown of keratin tissue in the follicle, thereby pulling out intact hairs without
affecting the tensile strength of leather (Macedo et al., 2005). B. subtilis S14 produces a
17
keratinolytic protease (KerS14), which does not have any detectable effect on collagen that is a
crucial property for an enzyme intended to be used in skin dehairing (Macedo et al., 2008).
Therefore, introduction of alkaline proteases for leather processing have a potential to reduce
environmental pollution, decrease processing time and improve leather quality.
2.9.2. Food and feed industry
Proteases find huge potential in various food and feed industrial applications such as in dairy
industry (milk protein-casein and whey protein hydrolysis for use in cheese flavour
development), baking industry (treatment of flour in the manufacture of baked goods and
improvement of dough texture, flavour, and colour in cookies, etc.), brewing industry, soy
protein hydrolysis, soy sauce production, gelatine hydrolysis, meat protein recovery, fish protein
hydrolysis and meat tenderization)and improves digestibility of animal feeds (Gupta 2002a;
Sumanthaet al., 2006; Ikram, 2008; Nadeem, 2009).
2.9.3. Dairy industry
The major application of proteases in dairy industries is the manufacture of cheese. In cheese
making, the primary function of proteases is to hydrolyze the specific peptide bond to generate pk-casein and macro peptides. The most significant property of acidic proteases is the ability to
coagulate milk proteins (casein) to form curds from which cheese is prepared after the removal
of whey. By virtue of this property, microbial acidic proteases have largely replaced the calf
enzyme (rennet), facilitating the expansion of the cheese manufacture industry whose
development was hurdled by animal rights issues. Alkaline protease was also used for the
production of whey protein hydrolysate, using cheese whey in an industrial whey bioconversion
process. The proteases produced by GRAS (generally regarded as safe) microbes such as
Bacillus subtilis, Mucormichei, and Endothiaparasitica have been used in cheese production It is
also involved in lactose reduction and flavour modification in dairy applications (Gupta,2002a;
Sumanthaet al., 2006; Ikram, 2008 Nadeem 2009; Vishwanatha,2009; Ray, 2012).
2.9.4. Baking Industry
Proteolytic enzymes are used for processing strong gluten flours with high resistance and
elasticity and low extensibility. The dough obtained from strong gluten flours cannot expand
18
under pressure of the gas fermentation which shows that it has little capacity to retain the gas.
Dough elasticity is improved at low doses of protease and it is reduced at higher doses. A limited
action of proteases causes weakening of the gluten network, while a strong action destroys this
network, completely loses its elasticity and the dough becomes sticky Proteases degrade proteins
in flour and improves the plastics proprieties of the dough, which makes the dough easier to
handle during the technological process for baking biscuits, crackers and cookies (Sumanthaet
al., 2006; Nadeem 2009).
2.9.5. Brewing industry
Proteases are used in brewing industry for extracting sufficient proteins from malt and barley and
for obtaining the desired level of nitrogen nutrients. In the production of brewing wort protease
are used to solublize protein from barley adjuncts, thereby releasing peptides and amino acids
which can fulfill the requirement of the nitrogen supply. The proteolytic enzymes are used in
chill proofing, a treatment designed to prevent the formation of precipitates during cold storage.
In beer, hazes are formed due to the presence of proteinanceous substances which also
precipitate the polyphenols and oligosaccharides. Hydrolysis of the protein components prevents
aggregation of the insoluble complex and hence used as seasoning materials from the foods
containing various proteins, the degradation of the turbidity complex resulting from protein in
fruit juices and alcoholic liquors, and the improvement of quality of protein-rich foods
(Sumanthaet al., 2006).
2.9.6. Manufacture of soy products
Proteases have been used from ancient times to prepare soy sauce and other soy products that are
known to be rich sources of proteins. Proteolytic modification of soy proteins helps to improve
their functional properties such as high solubility, good protein yield, and low bitterness. The
hydrolysate is used in protein-fortified soft drinks and in the formulation of dietetic feeds. They
are also known to be highly digestible and nutritious, and affect a number of physiological
activities such as antioxidative activity, fibrinolytic activity, lowering of blood pressure, and
prevention of osteoporosis (Kim et al., 2011). Microorganisms including Bacillus, Aspergillus,
19
and Rhizopusspecies and proteases are commonly used as a starter for fermenting soybeans
(Genckal, 2004).
2.9.7. Removal of bitter components of protein hydrolysates
Hydrolysis of food proteins is widely employed for value addition through improvement of
nutritional characteristics, retarding deterioration, improvement of functional properties and
removal of toxic or inhibitory ingredients. Proteases are used to hydrolyse proteins from plants
and animals for the production of hydrolysates of well-defined peptide profiles of high
nutritional value. These protein hydrolysates play an important role in blood pressure regulation
and are used in infant food formulations specific therapeutic dietary products and the
fortification of fruit juices and soft drinks. In recent years there has been substantial interest in
developing enzymatic methods for the hydrolysis of soya protein, gelatin, casein, whey and other
proteins in order to prepare protein hydrolysates of high nutritional value (Singhal et al., 2012).
In developing commercial products from these proteins, emphasis is placed on achieving a
consistent product in high yields, having desirable flavour, nutritional and/or functional
properties (Genckal, 2004; Ikram, 2008). One of the attributes that reduces the consumer
acceptance of protein hydrolysates is the bitterness caused by the presence of low molecular
weight (>10 kDa) peptides containing pro, leu, tyr, phe, ala etc in specific combinations. The
peptidases that can cleave these AA are valuable in debittering protein hydrolysates (Rao et al.,
1998).
2.9.8. Feed processing
Enzymes have been used for decades to improve the utilization of cattle, swine and poultry diets.
Protease enzymes may improve the digestion of cereal grains, because starch digestion is
partially a function of the protein- starch matrix within the seed. Treating steam flaked sorghum
with an enzyme mixture improved weight gain and feed efficiency. Keratinolytic activity of
alkaline protease has also been exploited in the production of proteinaceous fodder from waste
feathers or keratin-containing materials. Feather meal is relatively inexpensive and is shown to
be superior to soybean meal in terms of total cysteine, valine and threonine content, and the
Hydrolyzed meal can replace soybean meal at 7% dietary level. The crude enzyme can also serve
20
as a nutraceutical product, leading to significant improvement in broiler performance. Papain and
bromelain have been used to improve the nutritional value of feeds (Genckal, 2004; Ikram, 2008;
Nadeem 2009).
2.9.9. Detergent industry
Enzymes have long been of interest to the detergent industry for their ability to aid in the
removal of proteinaceous stains and to deliver unique benefits that cannot otherwise be obtained
with conventional detergent technologies. Applications of detergent proteases have grown
substantially and the largest application is in household laundry detergent formulations. The
increased reliance of detergent manufacturers on enzyme technology is because of consumerrecognizable cleaning benefits, the addition of completely new performance benefits, fabric
restoration and an increased performance/cost ratio, because of the availability of more efficient
enzymes and the industry trend toward reduced pricing (Shamkant and Raghunath, 2013).
In addition, enzyme suppliers and detergent manufacturers are actively pursuing the development
of new enzyme activities that address the consumer-expressed need for improved cleaning, fabric
care and antimicrobial benefits. Apart from their use in laundry detergents, proteases are also
popular in the formulation of household dishwashing detergents and both industrial and
institutional cleaning detergents (Gupta, 2002a; Genckal, 2004; Ikram, 2008; Nadeem, 2009;
Ray, 2012).
2.9.10. Medical and pharmaceutical industry
Alkaline proteases are used for developing products of medical importance. These include
elastolytic activity for the preparation of elastoterase, which was applied for the treatment
ofburns, purulent wounds, carbuncles, furuncles and deep abscesses, asthrombolytic agent
having fibrinolytic activity (Gupta, 2002a ; Genckal, 2004; Ikram,, 2008; Nadeem, 2009; Ray,
2012).
21
2.9.11. Photographic industry
Alkaline proteases play a crucial role in the bioprocessing of used X-ray or photographic films
for silver recovery. Conventionally, this silver is recovered by burning the films, which causes
undesirable environmental pollution. Furthermore, base film made of polyester cannot be
recovered using this method. Since the silver is bound to gelatin, it is possible to extract silver
from the protein layer by proteolytic treatments. Proteolytic hydrolysis of gelatin not only helps
in extracting silver, but also the polyester film base can be recycled (Gupta, 2002a; Genckal,
2004; Nadeem, 2009; Ray, 2012).
2.9.12. Chemical industry
Proteases have been used successfully for the synthesis of dipeptides and tripeptide,
regioselective sugar esterification and dia-stereoselective hydrolysis of peptide esters. Enzymatic
peptide synthesis offers several advantages over chemical methods, e.g. reactions can be
performed stereospecifically and reactants do not require side-chain protection, increased
solubility of non-polar substrates, or shifting thermodynamic equilibria to favor synthesis over
hydrolysis. Enzymatically synthesized small peptides (usually di or tripetides) are being used
successfully for human and animal nutrition and also as pharmaceuticals and agrochemicals.
Some relevant examples are the synthesis of the leading non-caloric sweetener aspartame, the
lysine sweet peptide, kyotorphin, angiotensin, enkephalin and dynorphin and some nutritional
dipeptides and tripeptides (Keivan et al., 2009).
Protease are also used for the production of biodegradable films, coatings and glues from
keratinous waste products like hair, feathers, skin, fur, animal hooves, horns etc. for compostable
packaging, agricultural films or edible film applications. Keratin structure is chemically modified
and hydrolyzed to produce stable dispersions for such applications (Gupta, 2002a; Genckal,
2004).
2.9.13. Textile industry
One of the least explored areas for the use of proteases is the silk industry and only a few patents
have been filed describing the use of proteases for the degumming of silk. Sericin, which is about
22
25% of the total weight of raw silk, covers the periphery of the raw silk fibers, thus providing the
rough texture of the silk fibers. This sericin is conventionally removed from the inner core of
fibroin by conducting shrink-proofing and twist-setting for the silk yarns, using starch. The
process is generally expensive and therefore an alternative method suggested is the use of
enzyme preparations, such as protease, for degumming the silk prior to dyeing (Gupta, 2002a;
Genckal, 2004; Nadeem, 2009; Ray, 2012).
2.9.14. Management of industrial and household wastes
The global environment is gradually deteriorating because of the socio-economic activities of
humankind such as processing industries. Many industrial processes cause adverse changes in
the immediate environmental change and therefore being challenged by society. Of these, leather
industries and the increased amount of feathers generated by commercial poultry processing may
represent a pollution problem and needs adequate management. In this regard, the use of alkaline
protease in the management of wastes from various industries and household activities opened
up a new era in the use of proteases in waste management. Proteases solubilize proteinaceous
waste and thus help lower the biological oxygen demand of aquatic systems. Alkaline protease
from B. subtilis was used for the management of waste feathers from poultry slaughter houses
(Gupta, 2002a; Genckal, 2004; Ikram, 2008; Nadeem, 2009; Ray, 2012).
2.10. Status of Research and Potential Applications of Proteases in Ethiopia
In Ethiopia, isolation of protease was conducted by Gessesse (1997), and Gessesse and Gashe
(1997) and Gessesse et al., (2003).These reports describe the isolation of Bacillus species AR
009 and B. pseudofirmusAL-89 from an alkaline soda lake in the Ethiopian Rift Valley Area.
The use of nug meal as a low-cost substrate for the production of alkaline protease was also
described with the intent of practical applications. Some studies suggested the potential
applications for the enzymatic and/or microbiological hydrolysis of feather to be used as animal
feed supplement (Genckal, 2004). In recent years, limited efforts are also being made to improve
the management of tannery wastes in the local industries using proteases (Gessesse et al., 2011).
It is therefore crucial to continue the efforts of searching for more potent isolates in the
environment as there are plenty of potential applications in the food industries, animal feed
23
processing and management of wastes of various industrial activities and municipal household
garbage wastes.
24
3. MATERIALS AND METHODS
3.1. Description of the Study Area
The study was conducted at the University of Gondar, Department of Biotechnology, Molecular
Biology Laboratory, Gondar town, which is found in North West Ethiopia, 730 km away from
the capital city of Ethiopia, Addis Ababa. Geographically, the town is located at 12° 36’ North
latitude and 37° 28’ East longitude with an elevation of 2133 meters above sea level. Average
maximum and minimum temperature is 29°C (in March and May) and 10°C (in January and
December), respectively. The mean relative humidity for an average year is recorded as 55.7
% and on monthly basis it ranges from 40 % in January to 79 % in July (Central Statistics
Office, 2011). Ethiopians are believed to have been practicing traditional leather processing
since their ancient civilizations. Local people around Gondar town are also using this activity to
make their beds, shoes and cushions by embedding the skin in those local ponds to remove the
hair from the skin.
3.2. Research Design
The design of the research involved screening for protease producing bacterial species from
randomly selected water and soil samples and experimentally determining both optimal growth
and reaction conditions for maximum production of protease. All experiments for protease
production were conducted in triplicate with Erlenmeyer flasks of 250 ml capacity and their
average values taken. The experimental design used for the optimization of cultivation
conditions was conducted following the OVAT (One Variable At a Time) method (Abdel-Fattah
et al., 2013).
3.3. Sample Collection and Isolation of Protease Producing Isolates
Samples were collected from traditional leather processing ponds/wastes (water and soil from
stagnant pond used for dehairing) and was kept in sterile tubes in refrigerator, at 4°C until used
for further investigation. Isolation of protease producing microorganisms were carried out after
enrichment using solid state fermentation medium containing (g/g) wheat bran, 10; K2HPO4,
25
0.1; MgSO4, 7H2O, 0.02; CaCl2, 0.01; and casein, 1.0 in a 250ml Using Erlenmeyer flask,
moistening agent (distilled water) were added in such a way to give final bran to moisture ratio
of 1:3, thoroughly mixed, and autoclaved at 121 ºC for 15 minutes. Then, each flask was
inoculated with 10 % (v/w) aliquots of mud suspensions as inoculums and incubated at 37 °C for
5 days (Ghasemi et al., 2011).
From thoroughly mixed enriched fermented solid substrate of each sample, one gram was taken
and suspended into 30 ml glass tube containing five ml of sterilized distilled water. Then the
glass tube was placed on a 121 rpm shaker for 30 minutes at room temperature. This suspension
was serially diluted (10-1to 10
-12
) and spread on agar plates. Individual colonies were isolated
and screened for protease production (Rasooli et al., 2008).
3.4. Screening for Protease Production
Screening of isolates for protease production was carried out using casein-yeast extract peptone
(CYP) agar medium containing (g/l): casein, 10; bacteriological peptone, 5; yeast extract, 1;
K2HPO4, 1; MgSO4.7H2O, 0.2; CaCl2, 0.1; and agar, 15. (Amare Gessesse et al., 2003). After
inoculation, the plates were incubated at 37 °C for 48 hours. Formation of halo zone around the
colonies, resulting from casein hydrolysis was taken as positive for proteolytic activity. These
colonies were isolated and streaked repeatedly in fresh plates until single uniform colonies were
obtained (Amare Gessesse et al., 2003).
To select an isolate which gives protease with high activity, a loop full of culture from agar plate
was taken and inoculated into 30 ml glass tube containing 5 ml of protease production medium
and incubated overnight at 121 rpm at room temperature. Then, 5 % (v/v) of the 16 hr inoculum
was inoculated into 50 ml of same medium kept in 250 ml Erlenmeyer flask and incubated with
rotary shaking (121 rpm) at room temperature for 5 days. Five ml of the fermented broth was
taken and centrifuged at 6000 rpm for 5 minutes and the cell free supernatant was used as
enzyme source (Alkando et al., 2011).
26
3.5. Preparation of Substrates for Solid State Fermentation
Agricultural residues namely wheat bran and rice bran were collected from local market in
Gondar town. Before using these substrates for fermentation, the unnecessary parts of the
substrates such as adhered surface dust particles were first separated and removed by washing
with distilled water. After that, the remaining useful materials were sun-dried and ground to
small size in a mixer grinder and finally used as a substrate for the production of protease under
SSF (Soni et al., 2003).
3.6. Solid State Fermentation
SSF medium containing (g/g): wheat bran, 10; K 2HPO4, 0.1; MgSO4. 7H2O, 0.02; CaCl2 0.01;
and casein, 1.0 was prepared in a 250 ml Erlenmeyer flask and the solid substrate to moistening
agent ratio was adjusted to 1:3, unless stated otherwise. After autoclaving, sterile sodium
carbonate was added to give a final concentration of 10% (w/w), inoculated and incubated at
37°C for 5 days. From the fermented substrate, alkaline protease was harvested by soaking the
fermented solid with ten volumes of distilled water per gram solid substrate (wheat bran), in
shaking (121rpm) condition for 30 minutes at room temperature (Soni et al., 2003). At the end of
the extraction, the suspension was hand squeezed through a double layered muslin cloth and the
particulate materials clarified by centrifugation at 10,000 rpm for 5 minutes.
3.7. Determination of the Protease Activity of Selected Isolates
3.7.1. Protease assay
Protease activity was determined using casein as a substrate as described by Hema and Shiny
(2012). The reaction mixture contained a total volume of 2 ml which in turn was composed of 1
ml of 1% casein in 50 mM sodium phosphate buffer (pH 7) and 1 ml enzyme solution. After 20
min of incubation at 37°C, the reaction was terminated by adding 2 ml of 10% trichloroacetic
acid (TCA) and again incubated at 37°C for 20 min. After separation of the un-reacted casein
precipitate by centrifugation at 10000 rpm for 15 min, 0.5 ml of clear supernatant was mixed
with 2.5 ml of 0.5M Na2CO3 and 0.5 ml of 1N Folin-Ciocalteau’s phenol reagent. After
incubation for 20 min at 37°C, absorbance was measured at 660 nm against a reagent blank. One
27
unit of protease activity is defined as the amount of enzyme that releases 1 µg amino acid
equivalent to tyrosine per minute under the standard assay conditions (Sevic and Demirkan,
2011).
Units/ml = μ mole of tyrosine x reaction vol
Sample vol x reaction time x vol assay
(Source:Folin and Ciocalteu, 1929).
3.7.2. Tyrosine calibration curve
As a reference to protease enzyme activity, tyrosine standard curve was generated using an
appropriate amount of tyrosine diluted in water. The suitably diluted samples (0.1 – 1.5 mg/ml)
were treated similar to the experimental enzyme catalyzed reaction mixture and then were
measured using a spectrophotometer at a wavelength of 660 nm (Hema and Shiny, 2012).
3.8. Phenotypic Characterization of the Bacterial Isolates
3.8.1. Macroscopic characterization of isolates
Colony characteristics such as margin, elevation, opacity, pigment and shape were investigated
microscopically and by direct observation of the 24 hours old colony on the nutrient agar plate
(Duncan, 2005).
3.8.2. Microscopic characterization of isolates
Gram staining and endo-spore staining features as well as motility of microorganisms were
studied microscopically.
3.8.2.1.Gram staining
This was carried out by using standard techniques with a step-wise application of Crystal violet
solution, iodine solution, ethanol (95%) and Safranin solution as described in Harley and
Prescott (2002).
28
3.8.2.2.Motility test
Bacterial motility was observed directly from examination of the culture tubes containing semisolid media of nutrient agar containing casein substrate following incubation. Growth in semisolid medium was spread out from the line of inoculation for motile organisms. Highly motile
organisms were providing growth throughout the tube. Growth of non motile organisms only
occurs along the stab line (Murray et al., 2007).
3.8.2.3.Endospore staining
Endospore staining was carried out by preparing heat fixed smears from a 24 h old bacterial
culture on clean microscopic slides. The slides were then covered with Malachite green and
placed in a beaker that had been kept in a boiling water bath for 3 to 5 minutes to allow the dye
to penetrate the endospore. After counterstaining the vegetative cells with Safranin solution, the
bacteria were observed using a standard microscope (Harley and Prescott, 2002).
3.8.3. Biochemical characterization of isolates
A loop-full of sample from an overnight culture was streaked on to nutrient agar plate and
incubated for 24 hrs at 37°C. From the resulting agar culture, a loop-full of culture was again
added to media containing different biochemicals and incubated at appropriate temperature for
24 hours. Presence or absence of changes in the media was recorded as positive and negative,
respectively, and the results were interpreted as per the information provided by Bergey’s
Manual of Determinative Bacteriology (Holt et al., 1994) used for identification of bacterial
isolates.
3.8.3.1.Catalase test
A thick emulsion was prepared on a clean slide for each bacterial isolate. Three drops of 3%
hydrogen peroxide were added on each of the slides. Formation of bubbles was recorded as
positive result (Adetunji et al., 2012).
29
3.8.3.2.Starch hydrolysis
This test was carried out by dividing starch agar plate into three equal sectors using a marker.
After labeling the sectors with the isolates’s code, the test organisms (isolates) were spot
inoculated and incubated for 24 h (Harley and Prescott, 2002). Zone of hydrolysis of starch was
detected as a brownish clear zone in a blue black background after flooding the starch agar plate
with iodine solution. The presence of zone of hydrolysis on the plate indicated the ability of the
test organism to metabolize starch.
3.8.3.3.Urea hydrolysis
Urease test was carried out by preparing urea broth containing phenol red as pH indicator. After
inoculating the broth with the test isolate and incubating the culture for 24 h, color change of the
broth from red to pink was observed and recorded as a positive result for urease test (Srinivas et
al., 2013).
3.8.3.4.Carbohydrate fermentation tests
Carbohydrate fermentation patterns of microorganisms were studied using media containing
different carbohydrates as carbon source and phenol red as the pH indicator. The experiment
involved both control and experimental groups. The fermentation of carbohydrates such as
glucose, lactose, galactose, D-xylose, mannitol and cellulose was noticed by observing color
change from red to yellow after 24 hours of incubation (Harley and Prescott, 2002).
3.8.3.5.Gas production using triple sugar iron test (TSI)
Gas production was detected using TSI agar slants which were prepared from a mixture of agar,
a pH-sensitive dye (phenol red), 1% lactose, 1% sucrose, 0.1% glucose, sodium thiosulfate and
ferrous sulfate (Harley and Prescott, 2002; Sharma, 2007). The bacterial isolate to be studied was
inoculated both by streaking on slant and stabbing the butt. After incubating the inoculated TSI
agar slant tubes for 24 hours, presence of H2S, color change on the slant and in the butt were
observed and interpreted according to Sharma (2007). Production of H2S was indicated by the
blackening of the TSI medium.
30
3.9. Seed Culture Medium
For enzyme production, bacterial cells from a 24 h aged culture were inoculated into 100 ml
Erlenmeyer flasks containing 50 ml of sterile inoculation medium containing glucose, CaCl2,
K2HPO4 and MgSO4 and casein as substrate. The cultures were grown at 37oC for 24 h. After
incubation for 24 h, 2% (v/v) of the culture was used to inoculate the production medium
(Ghaemi et al., 2007).
3.10. Optimization of the Growth Conditions for Production of Protease
3.10.1. Effect of time on the production of protease
To determine the optimum period for maximum production of protease, the culture in the
medium containing wheat bran, peptone, yeast extracts, casein, CaCl2, K2HPO4 and MgSO4 was
incubated at 37°C for 24-72 hrs and the protease activity was determined at 12 h intervals. Thus
2 ml of culture broth was collected after each interval and protease activity was determined as
described above in section 3.7.1.
3.10.2. Effect of temperature on the production of protease
The optimum temperature for protease production was determined by incubating inoculated
production media at different temperatures (i.e. 25, 30, 37, 40, 45 and 50°C), for as long as the
protease activity began to drop from the maximum. Protease activity was then determined for
each culture every 12 hours till a decline in activity was observed by taking a cell free
supernatant and testing using the method of Sevinc and Demirkan (2011).
3.10.3. Effect of pH on the production of protease
The effect of pH on the production of protease was investigated by adjusting the pH of the
production media to5.0, 6.0, 7.0, 8.0, and 9.0 and incubating at 37°C for as long as the protease
activity began to drop from the maximum. Adjustment of pH was done using 1N NaOH and
31
0.1N HCl solutions. Following this, protease activity was determined for each culture every 12
hours till a decline in activity was observed using the method of Sevinc and Demirkan (2011).
3.10.4. Effect of carbon source on the production of protease
Glucose, rice bran, wheat bran, and sucrose were used as carbon sources. The cultures were
incubated at 37°C for as long as the protease activity began to drop from the maximum (Akcan,
2012). Protease activity for each culture was then determined every 12 hours using the method of
Sevinc and Demirkan (2011).
3.10.5. Effect of nitrogen source on the production of protease
Two different sources of nitrogen, viz. organic nitrogen and inorganic nitrogen were tested for
their potentials to enhance protease production. The production medium was initially
supplemented with different organic nitrogen sources such as yeast extract, peptone, casein, each
at 1% (w/v) and inorganic nitrogen sources such as, (NH4)2SO4, and NH4Cl at 1% (w/v) were
tested after incubating culture at 37oC for as long as the protease activity began to drop from the
maximum (Akcan, 2012).The effect was studied by determining the protease activity for each
culture every 12 hours till a decline in activity was noted as in sections 3.7.1.
3.10.6. Effect of NaCl concentration on the production of protease
NaCl was added at various concentrations, i.e. 0.0, 0.2, 0.4, 0.6 and 0.8M, into the protease
production medium and assay for crude enzyme (protease) activity was carried out by incubating
each culture for as long as the protease activity began to drop from the maximum(Agrawa et
al.,2012). Effect of concentration of NaCl was studied by determining the protease activity for
each culture every 12 hours till a decline in activity was noted as in sections 3.7.1.
3.10.7. Effect of moisture level on protease production
The effect of moisture level on protease production of the selected bacterial isolates (1%
inoculum) was determined by adding moistening medium to wheat bran at level of 1:2, 1:3, 1:4
and 1:5 (w/v). SSF medium was incubated at 37°C and the crude enzymes were harvested to
32
determine protease activity every 12 hours until the activity began to show a decline. The
procedures used to determine the activity of the crude enzyme were similar to those shown in
sections 3.7.1.
3.10.8. Effect of inoculum size on protease production
The effect of inoculums size on protease production of the selected bacterial isolates was
assessed by inoculating the SSF medium (a medium composed of a1:3 wheat bran to moistening
agent ratio) with inoculum size of 5%, 10%, 15% and 20%. Protease activity was determined for
each culture using the same procedures indicated in sections 3.7.1.
3.11. Characterization of Protease
3.11.1. Effect of pH on the activity and stability of protease
Crude enzyme obtained from each isolate was incubated for 20 minutes at pH 5, 6, 7, 8, 9, 10
and 11 with phosphate buffer (pH 7.0). The effect of pH on the protease activity was studied by
determining the remaining activity following the standard protease assay procedures described
above. The effect on the stability was studied by pre-incubating for 12 hours and determining the
remaining activity following the standard protease assay procedures described above in section
3.7.1 (Oliveira et al., 2010).
3.11.2. Effect of temperature on the activity and stability of protease
This experiment was performed by incubating protease for 20 min at different temperatures viz.:
30, 40, 50, 60, 70, 80 and 90ºC. The effect of temperature on the protease activity was studied by
determining the remaining activity following the standard protease assay procedures described
above. Whereas, the effect of temperature on the protease stability was studied by pre-incubating
the crude enzyme for 12 hours and determining the remaining activity following the protease
assay procedures described above from section 3.7.1.
33
3.12. Partial Purification of Crude Enzyme
Partially purified enzymes were obtained by ammonium sulfate precipitation and dialysis using
membrane tube (Saxena and Singh, 2011). Ammonium sulfate powder was added slowly to the
crude enzymes until 80% saturation was reached and the crude enzymes were allowed to
precipitate for 60 min with gentle mixing at room temperature. The precipitates were recovered
by centrifugation at 12,000 rpm for 20 min at room temperature. The precipitates recovered from
ammonium sulfate precipitation were dissolved in 0.1 M phosphate buffer (pH 7) for 4 h. Using
membrane dialysis tube, the precipitates obtained from ammonium sulfate precipitation were
dialyzed overnight against the same buffer and re-centrifuged. Finally, the supernatants were
used as partially purified enzymes for further study. Enzyme activity was determined at each step
by the assay method described above.
3.13. Data Analysis
The enzymatic activity assay was performed in triplicate with three independent replicates.
Statistical evaluation for significant differences between mean values was done using one-way
ANOVA at 95% level (P ≤ 0.05) with the help of SPSS (version 16.0) statistical software and all
graphical and tabular data were generated using Microsoft Excel 2007.
34
4. RESULTS AND DISCUSSION
4.1. Isolation, Screening and Selection of Protease Producing Bacteria
4.1.1. Isolation and screening of protease producing bacteria
Based on colony morphology, a total of 147 colonies were isolated from the three different
sample sources. Out of the total isolated colonies, 85 colonies (57.8%) were positive for protease
production. As it can be seen from Table 1, the isolates showed a great variation in the size of the
clear zone of hydrolysis they produced on milk agar plates ranging from the least 1 mm to the
largest 20 mm. This indicates the possible capability of the isolates to produce potential protease,
which could be used for different industrial applications. In addition, the capability of the isolates
to hydrolyze milk agar is in good agreement with the earlier reported 1-20mm diameter of clear
zone formed on casein agar (Akpomie et al., 2012; Ogbonnaya and Odiase, 2012; Verma et al.,
2011).
4.1.2. Selection of the best protease producing bacteria
From the total of 85 positive isolates, 15 isolates with relatively large clear zone of hydrolysis
were selected for further investigation. The selection of potent bacteria was done by comparing
the isolates with each other in terms of both their diameter of clear zone of hydrolysis and their
protease activities (Table1). The results showed that the isolates with higher clear zone of
hydrolysis also gave higher protease activities (Table1). This step resulted in selection of three
potentially potent isolates, i.e. Ds-7 from Dashen soil, Ew-9 from Enfraz water and Sw-11 from
Seveha water.
35
Table 1. Screening of the 15 protease producing isolates
Sample sources
Positive isolates
Zone
of Protease
activity
clearance (mm)
after 48hrs (U/ml)
Ds-6
11
14.6
Ds-7
14
24.1
Dashen Ds-8
08
9.2
Ds-9
12
17..7
Ds-10
10
12.5
Ew-5
11
9.7
Water sample from
Ew-7
13
12.3
Enfraz
Ew-8
09
7.0
Ew-9
14
14.7
Ew-11
12
11.5
Sw-7
09
9.5
Sw-8
10
10.6
Water sample from
Sw-9
13
12.1
Seveha
Sw-10
11
11.0
Sw-11
15
14.0
Soil samples collected
From
Brewery
36
Ds-7
Sw-11
Ew-9
Figure 1. Zone of hydrolysis of casein by the three selected isolates
4.2. Phenotypic Characterization of the Bacterial Isolates
The isolates Ds-7, Ew-9 and Sw-11 were identified as spore-forming bacterial species that
belong to the genus Bacillus based on the information obtained from Bergey’s Manual of
Classification of Determinative Bacteriology (Table 2.). Although members of the genus
Clostridiumare also spore-forming bacteria, the 3 isolates of this study do not belong to this
genus as they are catalase positive and capable of growing under aerobic condition in the
ordinary incubator.
37
Table 2. Morphological and biochemical characteristics of the selected bacterial isolates
Bacterial Isolates
Parameters
Ds-7
Ew-9
Sw-11
Cell shape
Long, Rod
Long, Rod
Short, Rod
Cell arrangement
Chain
Pair
Pair
Colonial pigmentation
White
Red
White
Gram staining
+ve
+ve
+ve
Endospore staining
+ve
+ve
+ve
Motility test
+ve
+ve
+ve
Catalase test
+ve
+ve
+ve
Indole production
-ve
-ve
-ve
H2S production
-ve
-ve
-ve
Starch hydrolysis
+ve
+ve
+ve
Gelatin hydrolysis
+ve
+ve
+ve
Casein hydrolysis
+ve
+ve
+ve
Gas production
-ve
-ve
-ve
Urea hydrolysis
-ve
-ve
-ve
Glucose fermentation
+ve
+ve
+ve
Sucrose Fermentation
+ve
+ve
+ve
Key: +ve=Positive, -ve= Negative
4.3. Effect of Culture Conditions on Protease Production under SSF
In this section, the results of the effect of time, growth temperature, medium pH, moisture level,
carbon source, nitrogen source, NaCl concentration and inoculum size on protease production of
the selected isolates are described and discussed.
38
4.3.1. Effect of time course on protease production
In the present study, the optimum time for protease production from the three isolates was found
to be 48 hrs with protease activities of 3.5 U/ml, 2.7 U/ml, and 4.2 U/ml, for Ds-7, Ew-9 and Sw11, respectively (Fig. 2). Though three of the isolates gave maximum protease at 48 hrs but
isolate Sw-11 gave highest protease and followed by Ew-9 and Ds-7 this variation might be due
to the inherent features of the isolates. After 48 hours of incubation time, there was neither
further increase nor a pronounced drop in protease production. This might be due to the decrease
in microbial growth associated with the depletion of available nutrient, loss of moisture content,
production of toxic metabolites and autolysis caused by the protease produced (Sumantha et al.,
2006).
Protease Activity (U/ml)
12
10
8
Sw-11
6
Ew-9
4
Ds-7
2
0
24
36
48
60
72
Incubation time (hr)
Figure 2 . Effect of incubation time on protease production
These results are in accordance with observations made by Durhams (1987), Gessesse (1997)
and Qadar et al. (2009), where maximum enzyme production was observed during growth of the
culture at the late exponential phase and early stationary phase of the growth and thereafter the
number of viable cells decreased due to depletion of readily available carbon sources and other
nutrients.
39
4.3.2. Effect of temperature on the production of protease
The optimum temperature for production of proteases by isolates Ew-9 and Sw-11 was found to
be 37°C, which resulted in protease activities of 10.1 U/ml and 9.0 U/ml, respectively. Whereas
for isolate Ds-7, maximum protease production (9.3 U/ml) was obtained at 40°C. Isolate Ew-9
gave highest protease as compared with the others and followed by isolate Ds-7 and Sw-11
respectively. This might be due to the fact these isolates prefer a mesophilic growth conditions.
However, a considerable decrease in protease activity was observed with further increase in
temperature beyond the maximum in all three isolates (Fig.3). It might be due the fact that at
high temperature, the growth of the bacteria was hindered.
Protease Activity (U/ml)
12
10
8
6
Ds-7
4
Ew-9
2
Sw-11
0
25
30
37
40
45
50
Incubation Temperature (°C)
Figure 3. The effect of incubation temperature on protease production.
According to the report of Abdel Nasser et al. (2007) high temperature may inactivate the
expression of the gene responsible for the synthesis of protease enzyme. At relatively low
temperature (< 25°C), protease production was very low probably due to slow growth of the
bacterial isolates at low temperature.
Several reports indicate that maximum protease production was achieved at 35-40°C for certain
Bacillus spp. (Qadar et al., 2009; Kumara et al., 2012; Josephine et al., 2012). On the basis of the
40
temperature requirement for maximum protease production, it can be gathered that isolates Ds-7,
Ew-9 and Sw-11 belong to the mesophilic protease producers.
4.3.3. Effect of initial pH on the production of protease
The optimum pH for protease production for the three isolates was 7.0 although the enzyme was
active in the pH range of 7- 11 (Fig. 4).At pH 7, the protease activities for Ds-7, Ew-9 and Sw-11
were 13.5 U/ml, 12.5 U/ml and 10.2 U/ml, respectively. As it is shown from the graph isolate
Ds-7 gave highest protease and followed by isolates Ew-9 and Sw-11. This variation might be
due to the fact these isolates were collected from different ponds and they prefer a neutral pH.
However, from the survey of literature it can be seen that the optimum pH range for protease
production is generally between 7 and 9 (Al-Shehri and Mostafa, 2004; Qadar et al., 2009;
Sevinc and Demirkan, 2011; Josephine et al., 2012).
Protease Activity (U/ml)
16
14
12
10
8
Ds-7
6
Ew-9
4
Sw-11
2
0
5
6
7
8
9
Initial pH of the media
Figure 4. The effect of initial pH of the media on protease production
Further increase in initial pH values resulted in the decrement of protease production. This might
be because the isolates had preference for neutral pH to optimally grow in the medium
(Gangadharan et al., 2006).Normally, Bacillus spp. prefer neutral or slightly alkaline or a range
between 6.8 and 7.2 pH for protease production at the initial stage of fermentation (Benjamin et
al., 2013). For bacteria isolated from mesophilic environments, reports from earlier studies
41
revealed that an optimum pH for protease production was pH 7 (Meenakshi et al., 2009; Ashwini
et al., 2011).
4.3.4. Effect of different carbon sources on the production of protease
Among the various carbon sources used in this study, the easily available complex carbon
sources like wheat bran and rice bran were found to be the best for protease production by the
selected isolates. Wheat bran showed maximum enzyme production which was even better than
that produced on glucose for isolates Ds-7 (20.0 U/ml) and Ew-9 (12.9 U/ml), whereas glucose
was better for isolate Sw-11 (12.8 U/ml) (Fig 5). All of the isolates gave different protease
production in relation to different carbon sources, this variation might be due the inherent nature
of the isolates.
Protease Activity (U/ml)
25
y = -0.55x2 - 0.37x + 20.25
R² = 0.8475
y = 0.725x2 - 4.615x + 16.375
R² = 0.6703
20
y = -1.025x2 + 4.755x + 5.525
R² = 0.3057
15
Ds-7
Ew-9
10
Sw-11
5
0
Wheat bran
Rice bran
Glucose
Sucrose
Figure 5. The effect of different carbon sources on protease production
Microbial growth medium for enzyme production at industrial scale takes about 30-40% of the
production cost (Enshasy et al., 2008). By using wheat bran alone, appreciable amount of
protease can be produced with reduced cost. The production of large amount of protease from
complex carbon sources suggests the presence of enough nutrients in wheat bran that promote
enzyme production and support very little growth of the isolates.
42
4.3.5. Effect of nitrogen source on the production of protease
Effect of various nitrogen sources (organic and inorganic nitrogen sources) on protease
production of the three selected isolates (i.e. Ds-7, Ew-9 and Sw-11) was also examined. It was
observed that the growth medium containing casein yielded highest activity in isolates Ds-7 &
Ew (i.e.33.5 U/ml & 37.6 U/ml respectively). Whereas yeast extract was highest for isolate Sw11 corresponding with 43 U/ml. This was followed by peptone, yeast extract, ammonium
Protease Activity (U/ml)
sulphate and ammonium chloride (Fig.6).
50
45
40
35
30
25
20
15
10
5
0
Ds-7
Ew-9
Sw-11
Peptone
Yeast Extract
Casein
(NH4)2 SO4
NH4Cl
Figure 6. Effect of different nitrogen sources on protease production
As shown in the above figure, organic nitrogen sources (casein, yeast extract and peptone)
enhance protease production better than inorganic nitrogen sources (ammonium sulphate and
ammonium chloride). This maximum protease production by casein, peptone and yeast extract
might be due to the presence of high nutritional amino acids in these organic nitrogen sources.
By contrast, least production of protease was observed in SSF medium supplemented with
ammonium sulphate and ammonium chloride, respectively. These findings were generally in
agreement with the results reported by Shyam et al (2013). The low level protease production
might be due to the inability of the bacterial isolates to utilize these nitrogen sources or due to the
inhibitory effect of the inorganic N sources. In connection with this, Niadu and Devi, (2005) also
reported the repressing ability of inorganic nitrogen sources in Bacillus isolate.
43
4.3.6. Effect of inoculum size on protease production
The size of inoculum plays an important role in the production of high protease (Saxena and
Singh, 2011; Shyam et al., 2013). In the present study, 10% was found to be an optimum
inoculum size for protease production for all isolates (i.e. 12.5 U/ml, 11.3 U/ml and 13.6 U/ml
for isolates Ds-7, Ew-9 and Sw-11, respectively). All of the isolates show different activity at
inoculum concentration of 10% this variation is might be due to the fact that the isolated are
collected from different sources.
Protease Activity (U/ml)
40
35
30
25
20
Sw-11
15
Ew-9
10
Ds-7
5
0
5
10
15
20
Inoculum Size in (%)
Figure 7. The effect of inoculum size on protease production
In this study, inoculum size higher or lower than 10% has been shown to decrease protease
production. The decrease in protease yield at lower inoculum size might be due to the longer
time required by the bacterial isolates to grow to an optimum number to utilize the substrate and
form the desired product. On the other hand, the low protease production at higher inoculum size
(>20%) might be due to the stressful conditions created by the microbial cells such as depletion
of nutrients, pH fluctuation, change in availability of oxygen and competition for limited
resources (Kumar et al.,2010; Shyam et al.,2013).
44
4.3.7. Effect of moisture content on protease production
The effect of moisture level on enzyme production was determined by growing the bacterial
isolates on wheat bran supplemented with moistening agent (distilled water) at different ratios
(w/v). In all isolates, maximum protease activity was shown at moisture content 1:3 (i.e. 9.5
U/ml, 11.9 U/ml and 10.5 U/ml for isolates Ds-7, Ew-9 and Sw-11 respectively). Even if the
optimum moisture level for the isolates were at 1:3, but they gave different activity at 1:3 this
might be due to the inherent nature of the isolates.
Protease Activity (U/ml)
14
12
10
8
Ds-7
6
Ew-9
4
Sw-11
2
0
1:20
1:30
1:40
1:50
Wheat bran to Moisturing agent ratio (w/v)
Figure 8. Effect of moisture level on protease production
Among several factors that are essential for microbial growth and enzyme production under
solid-state fermentation, moisture level is one of the most critical factors (Pandey et al., 2000;
Mrudula et al., 2011). In the present study, in all isolates, high enzyme activity was obtained
when the substrate to moisture ratio maintained at 1:3. In all isolates, any further increase or
decrease of moisture ratio from the optimum (1:3) resulted in a slight decline of enzyme
production. This slight reduction of enzyme yields at low moisture level might be due to
clumping of solid particles, reduction in solubility of the nutrients of the substrate, low degree of
swelling and higher water tension (Mrudula et al., 2011).
The low enzyme activity at high moisture level (at 1:5) might be due to decreased oxygen
availability and steric hindrance of the growth of the isolates by reduction in porosity of the
wheat bran (Mrudula et al., 2011).
45
Different studies showed difference in optimum moisture content needed for production of
protease. Saxena and Singh (2011) reported that 1:3 moisture content as an optimum moisture
ratio for enzyme production from Bacillus species, which was in agreement with the present
study. On the other hand, Salwa et al. (2012) reported that the optimum moisture levels required
for enzyme production by Bacillus cereus and Bacillus species were 1:2 and 1:2.5, respectively.
These reports demonstrated slightly lower moisture ratio for maximum enzyme production
compared to the result obtained in the present study. This might be due to the difference in the
nature of the solid substrates used for fermentation.
4.3.8. Effect of NaCl concentration on the production of protease
Various NaCl concentrations (i.e. 0, 0.2, 0.4, 0.6, 0.8M) were used to determine the optimum
level required for the production of protease by the three selected isolates (i.e. Ds-7, Ew-9 and
Sw-11).It was observed that the growth medium containing 0.2M NaCl yielded the maximum
protease production in isolates Ds-7 and Sw-11 corresponding with 6.8 U/ml and 4.9 U/ml
respectively. Whereas, for isolate Ew-7 0.4M of NaCl was resulted in maximum protease activity
(i.e 4.9 U/ml). The present study considerably well agreement with the study conducted by
(Huang et al., 2003) who reported on halophilic and alkaliphilic bacterial isolates at 4M NaCl.
8
Protease Activity (U/ml)
7
6
5
Ds-7
4
Ew-9
3
Sw-11
2
1
0
0.00M
0.2M
0.4M
Conc. of NaCI
0.6M
0.8M
46
Figure 9. Effect of NaCl concentration on the production of protease
4.4. Characterization of Protease
4.4.1. Effect of pH on the activities of proteases of the selected isolates
The effect of pH on the activity of protease was studied by incubating the growth medium at pH
values ranging from 5 to 11 and at a temperature of 37°C for 20 min. The highest protease
activity from Sw-11andEw-9 was shown at pH 9.0 whereas the highest value from Ds-7 was
recorded at pH 8 (Fig.10).
Protease Activity (U/ml)
14
12
10
8
Sw-11
6
Ew-9
4
Ds-7
2
0
5
6
7
8
9
10
11
pH
Figure 10. Effect of pH on the activity of protease
This finding suggests that the enzymes would be useful in processes that require a wide pH range
from slightly acidic to alkaline medium. The results of the present study were in line with
activity of protease produced from Bacillus cereus at wide range of pH and maximum activity at
pH 7.0 (Mrudula et al., 2011).
47
Moreover, it was in agreement with the activity of the earlier reported protease produced from
Bacillus species, which was in the range of pH 6-8 with an optimum activity at pH range of 7-8
(Salwa et al., 2012). Also, relative lowest protease activity of isolates Ds-7 (1.0 U/ml) and Sw-11
(1.1 U/ml) were observed at pH 5 while lowest protease activity of isolate Ew-9 (1.7 U/ml) was
observed at pH 6. This might be due to deformation of structural and functional groups present in
the active site of the enzymes due to reaction with hydrogen or hydroxide ion.
4.4.2. Effect of pH on the stability of proteases of the selected isolates
The effect of pH on enzyme stability was examined by incubating the reaction mixture at pH
values ranging from 5.0 to 11.0 and a temperature 37°C for 12 hours with casein in sodium
phosphate buffer. The results showed that the stability of protease was higher at pH values
ranging from 8.0 to 10.0 than at lower pH values exhibiting maximum stability at pH 9.0 in Ds-7
and Ew-9; and at pH 8 in S-11 (Fig. 11).
Protease Activity (U/ml)
14
12
10
8
Sw-11
6
Ew-9
4
Ds-7
2
0
5
6
7
8
9
10
11
pH
Figure 11. Effect of pH on stability of protease
These findings suggest that the proteases of the three isolates belonged to the alkaline protease
class. In agreement with this, the optimum pH for stability of alkaline proteases from Bacillus spp.
has been previously reported in various studies as lying between 9.0 and 11.0 (Deng et al.,
2010).
48
4.4.3. Effect of temperature on the activities of proteases of the selected isolates
Effect of temperature on the activity of protease was studied, by incubating the culture filtrate
with the substrate at temperatures ranging from 30 to 90ºC and at optimum pH for 20 min. The
highest protease activity for isolates Ds-7 andSw-11 was recorded at 50ºC, whereas for Ew-9 it
was 40oC (Fig.13). Some earlier reports had also indicated varying optimum temperatures in the
range of 50-80°C (Al-Sheri and Mostafa, 2004; Deng et al., 2010).
Protease Activity (U/ml)
120
100
80
60
Sw-11
40
Ew-9
Ds-7
20
0
30
40
50
60
70
80
90
Temperature (ºC)
Figure 12. Effect of incubation temperature on the activity of proteases
The results of this study clearly indicate that the optimum temperature of proteolytic activity
exceeds the optimum temperature of enzyme production as already reported by Al-Shehri and
Mostafa (2004).
4.4.4. Effect of temperature on the stability of proteases of the selected isolates
The effect of temperature on the stability of proteases was also measured by pre-incubating them
at the optimum pH for 12 h. As shown in Fig. 13, the enzyme is active at temperatures between
30 and 80°C, with a highest stability obtained when held at 50°C for Ds-7 and Sw-9 for 12 h.
However, the protease of Ew-7 showed maximum stability at 40oC with a similar incubation
time.
Protease Activity (U/ml)
49
45
40
35
30
25
20
15
10
5
0
Ds-7
Ew-9
Sw-11
30
40
50
60
70
80
90
Temperature (°C)
Figure 13. . Effect of temperature on the stability of proteases
According to reports in stability of enzymes (Al-Shehri and Mostafa, 2004), the protease activity
was relatively stable at temperatures ranging from 50-65°C and 85.2% of the activity was
retained after incubation at 60°C. The stability of protease enzyme could be due to the
organisms’ genetic adaptability to carry out their biological activities at higher temperatures (AlShehri and Mostafa, 2004).
4.5. Partial Purification of Crude Enzymes for Hair Removal
The crude enzymes produced from the selected bacterial isolates were partially purified by
ammonium sulfate precipitation at 80% saturation level and dialysis using phosphate buffer.
After precipitating the crude enzymes of isolates Ds-7, Ew-9 and Sw-11 by adding ammonium
sulfate, purity of the enzymes were increased by 2.4, 2.6 and 2.1 folds, respectively. Moreover,
after dialysis, purity of the enzymes of the isolates Ds-7, Ew-9, and Sw-11 were increased by
2.8, 2.9, and 2.5 folds, respectively (Table 3).
50
Table 3. Partial purification of Protease produced from selected bacterial isolates
Bacterial Isolates
Ds-7
Ew-9
Sw-11
Purification steps
Crude
(NH3)2SO4
Precipitation
Dialysis
Crude
(NH3)2SO4
Precipitation
Dialysis
Crude
(NH3)2SO4
Precipitation
Dialysis
Total volume (ml)
50
20
Enzyme Activity
(U/ml)
5.8
13.8
Final Purification
(folds)
1.0
2.4
9
50
21
16.5
10.2
26.5
2.8
1.0
2.6
8
50
18
30.0
6.5
13.5
2.9
1.0
2.1
11
16.5
2.5
4.5.1. Enzymatic cow hide dehairing
To evaluate the potential use of this enzyme as a hide depilating agent in leather industries, pair
of cow hide was taken and added to 250 ml flask containing enzyme, its pH adjusted to 7.5 and
placed on shaker (121 rpm) for 24 hr. One at a time from each pair taken and hair removal trail
was done. As shown in Fig. 14 complete dehairing of the enzyme treated skin was achieved in 12
hr, at room temperature, with 12.8 U/ml cow hide resulting pelt (hide) of natural pore (grain) on
dehaired surface.
51
(a)
(c)
(b)
(d
Figure 1 Results of cow hide dehairing experiment: (a) & (b) raw hide before dehairing, and (c) and (d) are enzyme treated cow
hide dehairing with fingers.
Results of enzymatic cow hide dehairing showed successful use of this enzyme as a dehairing
agent. Complete dehairing of hide was achieved at 12 hr. Because of specificity to hydrolyse
non-collagen protein part at hair roots in hide, proteases are very important in shortening hide
dehairing time and in production of high quality full gain leather having natural hair pores on the
surface (Sivasubramanian et al., 2008). Cow hide usually treated with dehairing chemicals in a
drum for 24 hr (Thanikaivelan et al., 2004). Shortening of deharing time has been also reported,
20 hr for Aspergilus flavus protease by Malathi and Chakraborty, (1991), and 9 hr for keratinases
of Bacillus subtilis S14 by Macedo et al., (2005). Thus, protease has a potential to substitute
environmentally objectionable dehairing chemicals for hide/skin dehairing in leather industries
and for production of quality leather products (Xu et al., 2009).
52
5. SUMMARY, CONCLUSION AND RECOMMENDATION
5.1. Summary and Conclusion
The objective of this study was to isolate potentially potent protease producing bacteria (Bacillus
spp.) from three different sample sources (water from Seveha and Enfraz and soil from Dashin)
and to optimize their cultivation condition for maximum protease production. Proteases are one
of the most important groups of industrial enzymes with considerable application in the animal
feed processing, leather industry, medical activity, beverage industry and others sectors.
From a total of 147 pure bacterial colonies, 85 (57.8%) were found as protease positive, out of
the 85 protease positives, following selection criteria on the basis of their clear zone diameter on
milk agar plate, Ds-7 soil sample collected around Dashin brewery, Ew-9 water sample collected
from Enfraz and Sw-11 water sample collected from Seveha. Based on the results of different
morphological, physiological and biochemical tests done, these isolates were found to be
members of the genus Bacillus spp.
The effect of different physical and chemical parameters; incubation period (24, 36, 48, 60
and72); temperature (25, 30, 37, 40, 45, and 50°C); initial pH of media (5, 6, 7, 8 and 9);
different carbon sources (wheat bran, rice bran, glucose and sucrose); organic and inorganic
nitrogen sources (casein, peptone, yeast extract, ammonium sulphate and ammonium chloride);
inoculums size (5%, 10%, 15% and20% v/v); NaCl concentration (0.00, 0.2, 0.4, 0.6, and 0.8M)
and moisture level (1:2, 1:3, 1:4 and 1:5 v/w) on protease production by these isolates were
studied. The potential of the crude enzyme harvested from these isolates were also evaluated for
hair removal from a piece of skin.
Time courses of protease production in all isolates indicates that the production increases as time
increases up to the optimum time of incubation and decline after wards. The maximum protease
was harvested after 48 hrs in all isolates. The effect of different temperatures show as the
incubation temperature increases production increases up to the optimum temperature, but
53
beyond the optimum production decreased. Isolates Ew-9 and Sw-11 produce maximum protease
at 37°C, where as 40°C was optimum for isolate Ds-7. All isolates produced maximum protease
at pH 7 when compared to some slight acidic and alkaline pH.
Isolate Ds-7 and Ew-9 gave maximum protease in medium supplemented with wheat bran where
as isolate Sw-11 gave high protease in the present of glucose. The effect of nitrogen sources
indicated that in all isolates organic nitrogen sources resulted in maximum protease production
as compared to inorganic nitrogen sources. In isolate Ds-7 and Ew-9 maximum protease was
obtained in a medium containing casein, where as yeast extract gives maximum protease in
isolate Sw-11.
The production curve of effect of different size of inoculums on protease production revealed
that protease production increased when the percent of inoculums increased up to the optimum
and decreased beyond the optimum size. In all isolates maximum protease were harvested in
10% v/v inoculums. The effect of moisture level on protease production indicated that protease
production increased with increased bran to moistening agent till optimum decreased beyond the
optimum and all isolates give maximum protease at 1:3 v/w bran to moisture ratio. On the other
hand, production of protease is also influenced by the concentration of NaCl on the growth
media. The optimum NaCl concentration was found to be 0.2 M for isolates Ds-7 and Sw-11 but
0.4M was optimum for isolate Ew-9.
Although many potent isolates are on market for enzyme production, scientists prefer studying
new isolates because they could be alternative for commercial use in many aspects. Many studies
showed that researches will continue to isolate alternative strains for production of enzymes as
well as proteases. The isolated new source of protease producing bacteria, from the soil and
water samples that are collected from traditional leather processing ponds might be an alternative
source for the potential industrial applications.
54
5.2. Recommendations
Based on the findings of this study, the following recommendations were made:
1. The isolates that were selected in the study are only three, increasing the number of isolates
could also increase the chance of obtaining bacteria with interesting futures that could be
selected to future large scale applications.
2. Identification of the three Bacillus isolates needs to be fully characterized at a molecular level.
3. To determine the maximum hydrolyzing potential of the enzymes further purification of the
protease should be done.
4. The methods for large scale production and purification of the proteases from each isolate
needs to be developed.
5. Test of the enzymes dehairing capability at tannery experimental level.
55
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65
7. APPENDICES
Appendix A: Standard curves
Tyrosine Standard Curve
To prepare the standard curve 0.5M of Na2CO3, 50mM of sodium phosphate buffer, pH 7.00,
diluted 1N Folin reagent and 10 mg/ml of Tyrosine stock solution were used. A required amount
of buffer and Tyrosine were added in each test tube except blank. Then 2.5 ml of 0.5M Na2CO3
was added in each test tube including blank. After 500μl of 1N Folin reagent was added in each
test tube including blank, the solution was mixed immediately and kept for 30min at room
temperature. Finally, the optical density (OD) was measured at 660nm using spectrophotometer
and the standard curve was plotted (Fig. 1).
Based on the above procedures and experimental results the following standard curve was
obtained. So, to determine the protease activity of the 3isolates, the following calibration curve
was used with the regression coefficient of R2=0.996.
Absorbance(660nm)
2.5
y = 0.0019x + 0.0327
R² = 0.996
2
1.5
1
0.5
0
0
200
400
600
Tyrosine(μg/ml)
800
1000
1200
66
Figure.1. Tyrosine standard curve for determination of protease activity
Appendix B: Results of protease activity at various physicochemical conditions
Appendix Table 1. Effect of physicochemical parameters on protease production
Parameter
Effect of Time (hr) on protease production
Ds-7
Ew-9
Sw-11
OD 660 nm
Protease
OD 660
Protease
OD 660 nm
Activity
nm
Activity
U/ml
U/ml
24 h
0.022
1.2
0.017
0.9
0.025
36 h
0.042
2.2
0.019
1.0
0.034
48 h
0.068
3.5
0.052
2.7
0.080
60 h
0.044
2.3
0.027
1.4
0.062
72h
0.032
1.7
0.020
1.0
0.045
о
Effect of Temperature( C) on protease production
25°C
0.117
6.1
0.096
5.0
0.088
30°C
0.122
6.4
0.136
7.1
0.097
37°C
0.148
7.7
0.192
10.1
0.172
40°C
0.179
9.3
0.184
9.6
0.129
45°C
0.132
6.9
0.124
6.5
0.116
50°C
0.098
5.1
0.094
4.9
0.102
Effect of pH on protease production
pH 5
0.152
7.9
0.141
7.4
0.115
pH 6
0.192
10.1
0.158
8.2
0.167
pH 7
0.264
13.8
0.240
12.5
0.196
pH 8
0.184
9.6
0.124
6.5
0.182
pH 9
0.132
6.9
0.098
5.1
0.174
Effect of Carbon source on protease production
Wheat bran
0.364
20.0
0.248
12.9
0.192
Rice bran
0.290
15.3
0.168
8.8
0.167
Glucose
0.310
16.2
0.198
10.3
0.246
Sucrose
0.178
9.3
0.174
9.1
0.142
Effect of Nitrogen source on protease production
Peptone
0.548
28.6
0.620
32.4
0.504
Yeast Extract
0.424
22.1
0.524
27.3
0.824
Casein
0.642
33.5
0.720
37.6
0.645
(NH4)2 SO4
0.262
13.7
0.462
24.1
0.426
NH4Cl
0.146
7.6
0.320
16.7
0.324
Protease
Activity
U/ml
1.3
1.8
4.2
3.2
2.3
4.6
5.1
9.0
6.7
6.1
5.3
6.0
8.7
10.2
9.5
9.1
10.0
8.7
12.8
7.4
26.3
43.0
33.7
22.2
16.9
Effect of Moisture Level on protease Production
1:2
0.124
6.5
0.176
9.2
0.140
7.3
67
1:3
1:4
1:5
0.182
0.108
0.096
9.5
5.6
5.0
0.228
0.172
0.162
11.9
8.9
8.5
Effect of Inoculum Size on Protease Production in (%)
5%
0.186
9.7
0.183
9.5
10%
0.242
12.5
0.216
11.3
15%
0.174
9.1
0.163
8.5
20%
0.140
7.3
0.150
7.8
Effect of NaCl concentration on protease production
0.00 M
0.074
3.9
0.036
1.9
0.2 M
0.128
6.8
0.042
2.2
0.4 M
0.098
5.1
0.094
4.9
0.6 M
0.028
1.5
0.024
1.3
0.8M
0.022
1.0
0.020
1.1
0.202
0.122
0.112
10.5
6.4
5.8
0.166
0.260
0.176
0.142
8.7
13.6
9.2
7.4
0.042
0.094
0.064
0.082
0.024
2.2
4.9
3.3
4.3
1.3
Appendix Table 2.Effects of physicochemicalparametersonprotease activity and stability
Parameter
pH5
pH 6
pH7
pH8
pH9
pH10
pH11
pH 5
pH 6
pH 7
pH 8
pH 9
pH 10
pH11
30°C
40°C
50°C
60°C
70°C
80°C
Effect of pH on protease activity
Ds-7
Ew-9
OD 660 nm Protease OD 660
Protease
activity
nm
activity
U/ml
U/ml
0.020
1.0
0.035
1.8
0.046
2.4
0.032
1.7
0.052
2.7
0.064
3.3
0.062
3.2
0.058
3.0
0.124
6.5
0.098
5.1
0.064
3.3
0.048
2.5
0.028
1.5
0.042
2.2
Effect of pH on protease stability
0.022
1.2
0.026
1.4
0.035
1.8
0.032
1.7
0.048
2.5
0.054
2.8
0.072
3.8
0.048
2.5
0.074
3.7
0.090
4.7
0.056
2.9
0.075
3.9
0.048
2.5
0.060
3.1
Effect of Temperature on protease activity
0.580
30.3
0.362
18.9
0.740
38.6
0.420
21.9
0.802
41.8
0.392
20.5
39.7
0.760
0.384
20.0
0.740
38.6
0.282
14.7
0.620
32.3
0.202
10.5
Sw-11
OD 660 Protease
nm
activity
U/ml
0.022
1.1
0.026
1.4
0.038
2.0
0.086
4.5
0.042
2.2
0.032
1.7
0.034
1.8
0.024
0.027
0.038
0.084
0.078
0.080
0.062
1.3
1.4
2.0
4.4
4.1
4.2
3.2
0.768
0.640
0.840
0.602
0.404
0.332
40.1
33.4
43.8
31.4
21.1
17.3
68
90°C
0.520
27.1
0.122
6.4
Effect of Temperature on protease stability
0.480
25.0
0.380
19.8
0.590
30.8
0.420
21.9
0.620
32.3
0.385
20.1
0.580
30.3
0.362
18.9
0.470
24.5
0.276
14.4
0.375
19.6
0.204
10.6
0.246
12.8
0.126
6.6
30°C
40°C
50°C
60°C
70°C
80°C
90°C
0.221
11.5
0.442
0.540
0.820
0.640
0.780
0.720
0.640
23.1
28.2
42.8
33.4
40.7
37.6
33.4
Appendix c: Results of stastical analysis of protease at different physiochemical parameters
(ANOVA Result).
(1) Effect of Time on Protease Production
ANOVA
Sum of Squares
Actat24h
Actat36h
Actat60h
Actat72h
Mean Square
Between Groups
.202
2
.101
Within Groups
.087
6
.014
Total
.289
8
9.662
2
4.831
.220
6
.037
Total
9.882
8
Between Groups
2.869
2
1.434
Within Groups
2.247
6
.374
Total
5.116
8
Between Groups
4.687
2
2.343
Within Groups
1.653
6
.276
Total
6.340
8
Between Groups
2.319
2
1.160
Within Groups
2.284
6
.381
Total
4.603
8
Between Groups
Within Groups
Actat48h
df
F
Sig.
7.000
.027
131.758
.000
3.831
.005
8.504
.018
3.046
.022
(2) Effect of Temperature on Protease Production
ANOVA
Sum of Squares
Act25oC
Between Groups
Mean Square
2
1.810
.120
6
.020
Total
3.740
8
Between Groups
6.629
2
3.314
.107
6
.018
6.736
8
Within Groups
Act30oC
df
3.620
Within Groups
Total
F
Sig.
90.500
.000
186.438
.000
69
Act37oC
Between Groups
10.007
2
5.003
.059
Within Groups
Act40oC
.353
6
Total
10.360
8
Between Groups
14.329
2
7.164
.058
Within Groups
Total
Act45oC
Between Groups
.347
6
14.676
8
1.119
2
.560
.290
6
.048
Within Groups
Total
Act50oC
1.409
8
Between Groups
.082
2
.041
Within Groups
.047
6
.008
Total
.129
8
84.962
.000
124.000
.000
11.594
.009
5.286
.047
(3) Effect of pH on protease production
ANOVA
Sum of Squares
ActpH5
actpH6
5.820
2
2.910
Within Groups
2.100
6
.350
Total
7.920
8
Between Groups
5.447
2
2.723
.133
6
.022
Total
Between Groups
Within Groups
actpH8
5.580
8
34.389
2
17.194
.426
2.553
6
Total
36.942
8
Between Groups
14.329
2
7.164
.058
Within Groups
ActpH9
Mean Square
Between Groups
Within Groups
actpH7
df
.347
6
Total
14.676
8
Between Groups
24.036
2
12.018
.033
6
.006
24.069
8
Within Groups
Total
F
Sig.
8.314
.019
122.550
.000
40.405
.000
124.000
.000
2.163E3
.000
(4) Effect of carbon source on protease production
Descriptive
95% Confidence Interval for
Mean
N
ActatWhitebran
ActatRicebran
Mean
Std.
Deviation
Std.
Error
Lower
Bound
Upper Bound
Minimum
Maximum
DS7
3 16.6000
7.00048 3.50024
5.4607
27.7393
6.10
20.20
Ew9
3 15.6000
1.95192 1.12694
10.7512
20.4488
13.60
17.50
SW11
3 16.3000
.43589
14.4245
18.1755
15.60
17.10
.75498
Total
16.2100
4.18369 1.32300
13.2172
19.2028
6.10
20.20
DS7
3 10.3250
2.99708 1.49854
Ew9
3
8.7667
SW11
3
Total
10
5.5560
15.0940
6.40
13.60
.51316
.29627
7.4919
10.0414
8.20
9.20
9.2000
.52915
.30551
7.8855
10.5145
8.80
9.80
9.5200
1.90426
.60218
8.1578
10.8822
6.40
13.60
70
ActatGlucose
ActatSucrose
DS7
4 11.7250
2.99486 1.49743
6.9595
16.4905
7.70
14.20
Ew9
3 12.5667
2.00083 1.15518
7.5963
17.5370
10.60
14.60
SW11
3 10.0333
.25166
.14530
9.4082
10.6585
9.80
10.30
Total
10 11.4700
2.23858
.70790
9.8686
13.0714
7.70
14.60
DS7
4 22.3250
8.68346 4.34173
Ew9
3
8.7333
3
7.0667
SW11
Total
10 13.6700
8.5077
36.1423
9.30
26.70
.15275
.08819
8.3539
9.1128
8.60
8.90
.70238
.40552
5.3219
8.8115
6.40
7.80
9.01111 2.84956
7.2238
20.1162
6.40
26.70
ANOVA
Sum of Squares
Act at
Mean Square
F
185.136
2
92.568
5.073
6
.846
190.209
8
90.736
2
45.368
.733
6
.122
Total
91.469
8
Act at
Between Groups
74.927
2
37.463
glucose
Within Groups
3.953
6
.659
78.880
8
whitebran
Between Groups
df
Within Groups
Total
Act at
Between Groups
ricebran
Within Groups
Total
Act at
Between Groups
8.420
2
4.210
sucrose
Within Groups
1.180
6
.197
Total
9.600
8
Sig.
109.476
.000
371.191
.000
56.858
.000
21.407
.002
(5) Effect of nitrogen sources
ANOVA
Sum of Squares
Actpepton
Between Groups
2
23.314
4.267
6
.711
50.896
8
710.340
2
355.170
.200
6
.033
710.540
8
Between Groups
39.980
2
19.990
Within Groups
17.360
6
2.893
Total
57.340
8
189.042
2
Total
Between Groups
Within Groups
Total
Actcasien
ActNHSO4
Mean Square
46.629
Within Groups
Actyeastexract
df
Between Groups
94.521
F
Sig.
32.786
.001
1.066E4
.000
6.909
.028
122.050
.000
71
Within Groups
ActNH4Cl
4.647
6
Total
193.689
8
Between Groups
124.429
2
62.214
.360
6
.060
124.789
8
Within Groups
Total
.774
1.037E3
.000
(6) Effect of inoculum size
ANOVA
Sum of Squares
Act5%
Between Groups
2
.840
.600
6
.100
Total
2.280
8
Between Groups
9.620
2
4.810
Within Groups
1.460
6
.243
11.080
8
.860
2
.430
.600
6
.100
1.460
8
Between Groups
.402
2
.201
Within Groups
.907
6
.151
1.309
8
Total
Actc15% Between Groups
Within Groups
Total
Act20%
Mean Square
1.680
Within Groups
Act10%
df
Total
F
Sig.
8.400
.018
19.767
.002
4.300
.009
1.331
.032
(7) Effect ofmoisture level
ANOVA
Sum of Squares
Act1to2
Between Groups
2
5.250
4.500
6
.750
15.000
8
Between Groups
8.720
2
4.360
Within Groups
3.000
6
.500
Total
11.720
8
Between Groups
25.929
2
12.964
2.887
6
.481
28.816
8
Total
Actc1to4
Mean Square
10.500
Within Groups
Act1to3
df
Within Groups
Total
F
Sig.
7.000
.027
8.720
.017
26.947
.001
72
Act1to5
Between Groups
Within Groups
Total
20.180
2
10.090
2.520
6
.420
22.700
8
24.024
.001
(8) Effect ofNaCl Concentration
ANOVA
Sum of Squares
Act0.00M
Between Groups
2
3.640
.060
6
.010
Total
7.340
8
Between Groups
6.242
2
3.121
.147
6
.024
6.389
8
12.860
2
6.430
.180
6
.030
13.040
8
Between Groups
.116
2
.058
Within Groups
.167
6
.028
Total
.282
8
Between Groups
.222
2
.111
Within Groups
.807
6
.134
1.029
8
Within Groups
Total
Actc0.4M
Between Groups
Within Groups
Total
Act0.6M
Act0.8m
Mean Square
7.280
Within Groups
Act0.2M
df
Total
F
Sig.
364.000
.000
127.682
.000
214.333
.000
2.080
.006
.826
.002
73