Académique Documents
Professionnel Documents
Culture Documents
Dr. D.KEZIA
Associate Professor,
St. Martins Engineering college,
Dulapally, Near Kompally, Qutubullapur, Hyderabad,
Telangana -500014, India
Dr. T. SATHISH
Project-Scientist,
Andaman and Nicobar Center for Ocean Science and Technology
(ANCOST), National Institute of Ocean Technology (NIOT),
(Ministry of Earth Science, Govt. of India), Industrial Estate Road,
Dollygunj Port Blair - 744103, Andaman and Nicobar Islands, India
PREFACE
The term fermentation is derived from the Latin word for forever to boil thus
describing the appearance of the action of yeast on extracts of fruit or malted grain. The
boiling experience is due to the production of CO2 bubbles caused by the anaerobic
catabolism of the sugars present in the extract. Pasteur applied the term Fermentation to
those anaerobic reactions through which microorganisms obtained energy for growth in
the absence of oxygen. Today fermentation has much broader meaning. It applies to both
aerobic and anaerobic metabolic activities of the microorganisms where in specic
chemical changes are brought about by an organic in substrate.
A variety of substances such as alcohols, organic acids, amino acids, vitamins,
antibiotics, enzymes, single cell proteins, hormones etc., are produced through
fermentations by employing different microorganisms. The enzymatic yield obtained
from fermentation, cost of their production and downstream processing cost determines
the nal cost of the enzyme. To develop a viable industrial process, lowering production
cost and increasing enzyme productivity are very important. Selection of best
fermentation techniques and optimization of culture conditions contribute much in
achieving enzyme productivity. Currently, enzyme production by microorganisms can be
achieved using submerged fermentation or solid state fermentation.
This book is aimed at helping new comers to know about upstream and
downstream processing of enzymes and also it helps to understand the fermentation
techniques and avoid obvious mistakes and pitfalls we have all made. It has been written
for researchers, scientists, students, academicians coming from various disciplines of
Chemistry, Biology, Engineering and industry personnel as well. The content in this book
is quite readable and thorough in its presentation of the subject provides the readers a
broad coverage and insight into the area. To meet the above objectives this book covers
following topics: chapter-1 contains introduction, mechanism of action, sources and
classication of proteolytic enzymes. This chapter also deals with properties and
applications of alkaline proteases. Chapter 2-5 contains practical aspects on isolation,
screening and characterization of protease producing microorganisms, protease
production by submerged fermentation, optimization by Plackett-burman design,
Response surface methodology, solid-state fermentation, enzyme recovery and
purication procedures, characterization of puried enzyme, estimation of kinetic
parameters, evaluation of industrial applications. Chapter-6 deals with various
fermentation strategies to improve the yield. The present book has comprised of six
chapters presenting an overview of the research being carried out in laboratory in the eld
of fermentation technology.
Dr. D. KEZIA & Dr. T. SATHISH
CONTENTS
PREFACE ..................................................................................................... 03
TITLE
SR. NO
PAGE
NO.
CHAPTER-1
07
CHAPTER-2
18
CHAPTER-3
22
CHAPTER-4
35
CHAPTER-5
53
CHAPTER-6
67
REFERENCE
73
APPENDIX -I
83
1.0 INTRODUCTION
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. Alkaline proteases are so far exploited as industrial catalysts in various
industrial sectors. 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 alkaline proteases production resulted in continuous exercise for search of new microbial resources. Extreme environments are important sources for isolation of microorganisms for novel industrial
enzymes production (Kumar & Takagi, 1999). Enzyme cost is also the most critical factor
limiting wide use of alkaline proteases 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 production of the growth
substrate (Enshasy et al. 2008; Kirk et al. 2002). In this regard, SMF method is used to
optimize the parameters of enzyme production and SSF which uses cheap agricultural residues which have enormous potential in reducing enzyme production cost. So, studies on
alkaline proteases that are produced in SMF and SSF by alkaliphilic microorganisms are
scarce in literature as a result, it is of great importance to pursue such studies.
1.1 MECHANISM OF ACTION OF PROTEASES
The catalytic site of proteases is anked on one or both sides by specicity subsites, each
able to accommodate the side chain of a single amino acid residue from the substrate.
These sites are numbered from the catalytic site S1 through Sn toward the N terminus of
the structure and Sl' through Sn' toward the C terminus. The residues which they accommodate from the substrate are numbered Pl through Pn and P1' through Pn', respectively.
The structure of the active site of the protease therefore determines which substrate residues can bind to specic substrate binding sites of the protease, thereby determining substrate specicity of a protease (Fig. 1.1)
Fig. 1.1: Active sites of proteases. The catalytic site of proteases is indicated by * and
the scissile bond is indicated by + ; S1 through Sn and S1' through Sn' are the specicity subsites on the enzyme, while P1 through Pn and P1' through Pn' are the residues on the substrate accommodated by the subsites on the enzyme (From
http://journals.asm. org/misc/reprints.dtl).
7
12
PH
optimum/stability
9.5
Industrial applications
Detergents and heavy duty
laundry powders
10.0-12.5
Bacillus licheniformis
8.2
12.0-13.0
Dehairing/leather industry
Bacillus sp.
(Savinase/Durazym)
9.0-11.0
Detergent formulations
Detergent formulations
Catalyst for N-protected amino
acids
Bacillus rmus
8.0
Detergent industry
9.5
Bacillus licheniformis
(Alcalase)
8.2
Bacillus subtilis
8.5
pI
9.0
4.8
Reference
Huang et al. 2003
Hutadilok-Towatana et al.1999
Molecular weight
Method
Reference
(kDa)
32
SDS-PAGE Huang et al. 2003
28
25/40
30
28
32
33.5
25/40
30
Ferrero et al.1996
Takami et al.1989
Kumar, 2002
Huang et al. 2003
Rahman et al.1994
Ferrero et al.1996
Gupta & Beg, 2003
Bacillus sp., although they do affect the alkaline enzymes produced by Streptomyces sp.
(Kumar & Takagi, 1999).
1.5.6 Kinetic Parameters
To develop any enzyme-based process, knowledge of the kinetic parameters of the
enzyme under study is of utmost importance. To be precise, kinetic properties like Vmax,
Km, Kcat, and Ea knowledge are essential for designing enzyme reactors or quantifying
the applications of the enzyme under different conditions. This information also helps in
understanding the catalytic behavior related to enzyme-substrate and environment specicity. Various natural complex substrates like casein, azocasein, BSA, gelatin etc., and
synthetic substrates such as para nitroanilide esters are used for determining kinetic
parameters for proteases. The synthetic substrates are much more popular than complex
substrates for dening Km and Vmax as they are convenient (Kumar, 2002; Larcher et
al.1996; Kemel et al. 2011; Subbarao et al. 2009). For an alkaline protease from B.
mojavensis, the Km for casein decreased with corresponding increase in Vmax, as the
reaction temperature was raised from 45 to 60C (Beg et al. 2002). In contrast, the Km
and Vmax for an alkaline protease from Rhizopus oryzae increased with an increase in
temperature from 37C to 70C.
1.6 APPLICATIONS OF PROTEASES
In the present era for development of environmentally friendly technologies, proteases
are believed to have extensive applications in different sectors ranging from domestic to
leather processing, environmental pollution abatement to neutraceutical applications,
health care products to diagnostic kits developments and value added product production
to bioremediation processes.
1.6.1 Detergents
The detergent industry emerged as the single major consumer of alkaline protease since
1913 (Kalisz, 1988). At present, approximately 25% of the total worldwide sales of
enzymes (Kalisz, 1988) used in detergent industry. Proteases are stable in a broad range of
pH and temperatures. They are compatible with surfactants, perfumes and bleaches. Further they are stable and have good shelf life. They play a key role in stain degradation and
removal (Kumar, 2002; Joo et al. 2003; Banik & Prakash, 2004; Ferid Abidi, 2008;
Subbarao et al. 2009; Haddar et al. 2010; Ashis et al. 2008).
1.6.2 Leather Industry
The enzymatic dehairing process is gaining importance as an alternative to chemical
methods in present day concern on development of eco-friendly technologies as this process in reduction of toxicity in addition to improvement of the quality of the leather and
other chemicals (soda sulde) (Sivasubramanian et al. 2008; Ganesh et al, 2008; Vasudeo
et al. 2011; Mukhter & Ikram-Ul-Haq, 2008; Ramakrishna, 2010; Arunachalam &
Sarita,2009). The major building blocks of skin and hair are proteinaceous. Hence, proteases are used for selective hydrolysis of non-collagenous constituents of the skin and
for the removal of non-brin proteins such as albumins and globulins. The purpose of
soaking is to swell the hide.
1.6.3 Food Industry
The basic hydrolysis character of proteases was exploited to convert solid proteins from
meat, sh or legumes into liquid slurries or protein hydrolysates, to improve their avor,
14
texture, functionality and nutritional quality (Watanabe et al. 1995; Keivan et al. 2009) in
addition to production of high-value functional ingredients from proteins, bioactive peptides with various functionalities, reduce allergenicity of food proteins and protect food
quality in food industry (Gautheir & Pouliot, 2003). Proteases were extensively used to
improve the palatability of reformulated low-carbohydrate/ high-protein foods.
1.6.4 Dairy Industry
Major application of proteases in the dairy industry is in the manufacture of cheese. World
shortage of calf rennet due to the increased demand for cheese production intensied the
search for alternative microbial milk coagulants. Proteases produced by GRAS (genetically regarded as safe) microbes such as Mucor michei, Bacillus subtilis, and Endothia
parasitica which were gradually replacing chymosin in cheese making and ripening
(Schmidt ,1979).
1.6.5 Feed Industry
Proteins are essential dietary components and have a signicant effect on feed quality due
to their broad substrate specicity in hydrolysis of hemoglobin, casein, egg yolk, soy, gelatin, sh, and other proteins to lower molecular weight peptides and subsequent production of amino acids that are absorbed by the body. Many publications showed that proteases increase the digestibility of the proteins in soybean meal (Cheng et al.1995)
reported the use of alkaline proteases from (B. subtilis B72 and B. licheniformis PWD-1)
for hydrolysis of feather keratin for obtaining a protein concentrate for fodder production.
1.6.6 Pharmaceutical Industry
Wide diversity and specicity character of proteases is used in developing effective therapeutic agents including as contact-lens enzyme cleaners and enzymic debrides.
Collagenolytic protease was orally administered as a pretreatment against for osteoporosis, used in the preparation of isolated rat liver cells and the scission of collagen-like peptides in fusion proteins (Barthomeuf et al. 1989) used alkaline protease to hydrolyze collagen to produce low molecular weight peptides of therapeutics use while used them for
brinolytic activity. Proteases also revealed their importance in dissolution of blood
clots, in treatment of sciatica, retained placenta (Eiler et al. 1993), adenovirus-mediated
cancer gene therapy (Kuriyama et al. 2000) and in therapy of thromboembolic diseases
(Myocardial infarction, embolisms and deep vein thrombosis). Protease with elastoterase
activity was used for the treatment of burns, purulent wounds, carbuncles, furuncles and
deep abscesses (Thangam & Rajkumar, 2002).
1.6.7 Peptide Synthesis
Since the rst report of (Bergman & Frankel-Conrat, 1937) on protease-catalyzed peptide
synthesis using the reverse-enzymatic reaction of hydrolysis, proteases were used for peptide synthesis (Clapes et al. 1997; Morihara et al. 1987). Proteases have been used successfully for the synthesis of dipeptides and tripeptide, regioselective sugar esterication
(Riva et al. 1988) and dia-stereoselective hydrolysis of peptide ester used alcalase for
kinetic resolution of N-protected amino acid esters in organic solvents, resolution of DLphenylalanine and DL-phenylglycine.
1.6.8 Photographic Industry
Alkaline proteases play a crucial role in the bioprocessing of X-ray or photographic lms
for silver recovery. These waste lms contain 1.52.0% silver by weight in their gelatin
15
layer, which can be used as a good source of silver for a variety of purposes. Alkaline proteases from B. coagulans PB-77 (Gajju et al. 1996), Bacillus sp. B18 (Fujiwara et al.
1991), B. subtilis (Fujiwara et al. 1991) played a crucial role in the bioprocessing of used
X-ray or photographic lms for silver recovery.
1.6.9 Textile Industry
Proteases are used in the silk industry for the degumming of silk, by splitting the albuminous proteins. Different proteases were used for degumming the silk (Freddi et al. 2003).
Proteases were used to wash down printing screens after use in order to remove the
proteinaceous glue used for thickening of printing paste (Freddi et al. 2003). They are also
used for softening of wool bers and to make 'shrinkproof' wool. A successful method
involving the partial hydrolysis of the scale tips with the protease was developed (Freddi
et al. 2003).
1.6.10 Alkaline Proteases in
Degradation of Proteinaceous Waste into Useful Biomass Recently, use of alkaline protease in the management of wastes from various food processing industries and household
activities has opened up a new era in the use of proteases in waste management. Fibrous
proteins such as horn, feather, nail and hair were converted into useful biomass, protein
concentrate or amino acids using proteases. Alkaline proteases can also be used to
solubilize sh meat in the production of animal glue and a fodder for animals from leather
industry waste, in hydrolysis of feather and horn (Atalo & Gashe, 1993), for the production of amino acids or peptides, for degrading waste keratinous material in household and
poultry refuse (Detoni et al. 2002; Brutt & Ichida, 1999; Mukhopadhyay & Chandra,
1992) used alkaline protease from B. subtilis for management of waste feathers from poultry slaughterhouses. Proteases along with other enzymes were directly added in the
digester to enhance the rate of biodegradation of polymeric substances which would
facilitate the conversion of monomeric constituents into methane and carbon dioxide.
1.6.11 Synthesis and Resolution of D, L-Amino Acids by Alkaline Proteases
Amino acids are important as a dietary supplement for both humans and domestic animals. Only the L-amino acids can be assimilated by living organisms. An extra- cellular
low molecular weight protease (6800 daltons) was puried to homogeneity from the culture ltrate of C. coronatus (NCIM 1328) and used in resolving the racemic mixture of
amino acids.
1.6.12 Medical Applications
Alkaline proteases are also used for developing products of medical importance exploited
the elastolytic activity of B. subtilis 316M for preparation of elastoterase, which was
applied for the treatment of burns, purulent wounds, carbuncles furuncles and deep
abscesses.
1.6.13 Other Applications
It is evident from above that alkaline proteases have a wide range of industrial applications. In addition to the applications already described, alkaline proteases are also used to
lesser extent in a large number of other elds, which may be technically interesting, but
are not commercial success in terms of microbial enzyme sales nevertheless they are the
upcoming areas of future enzyme industry. (Thangam & Rajkumar, 2002) reported that
some proteases can replace the Proteinase which is extensively used in DNA isolation
16
(Chiplonkar et al. 1985) have reported the use of alkaline protease from Conicliobolus sp.
as a substitute for trypsin in preparation of animal cell cultures. In another study, the effect
of culture supernatant of M. Purpureus CCRC 31499 on the growth rate of rape and amaranth seedlings was investigated (Liang et al. 2006).
The subsequent chapters in this book, experimental methods and the results obtained
from submerged fermentation studies, solid state fermentation, purication and characterization of proteases and application of proteases are written in detail.
17
2.0 INTRODUCTION
Bacteria of the genus Bacillus are known to produce a group of commercially important
enzymes including proteolytic enzymes. Alkaline proteases represent one of the largest
groups of industrial proteases useful in industrial process like in detergents, leather,
tanning, dairy, meat tenderization, baking, brewery, photographic industry etc. (Gupta et
al. 2002a). Naturally occurring and man-made alkaline inhabitants provide excellent
sources of microorganisms for research programmes in innovative microbiology. The
isolation and screening of microorganisms from different origins and the nature of
alkaline sources for alkaline proteases have been reported by various investigators
(Takami et al.1989). In the present investigation, a bacterial strain has been isolated from
soil sample collected from the Andhra University, Visakhapatnam, a potentially rich
source of alkalophiles, and a methodology has been standardized for the production and
partial characterization of the industrially important novel alkaline protease from
bacteria.
2.1 MATERIALS AND METHODS
2.1.1 Sample Collection
Samples were collected at different areas from garden soil in Visakhapatnam, A.P, India
and brought to laboratory the samples were stored at 4oC till further use.
2.1.2 Enrichment of Soil Microbial Organisms
1g of soil sample was suspended in 9.0 ml of sterile distilled water and mixed well for 1 h
at room temperature, 1.0 ml of suspension was inoculated in 50 ml of Yeast extractPeptone-Dextrose (YPD) medium consisting (gl-1) of glucose, 10.0; peptone, 7.5; yeast
extract, 7.5; K2HPO4, 0.50; MgSO4, 0.05 and CaCl2, 0.02, pH of the medium was adjusted
to 9.0 using 0.1N HCl or 0.1N NaOH solution. The culture was incubated at 37oC and at
200 rpm, After 24 h of incubation, the resultant culture was serially diluted using sterile
distilled water and 0.1 ml of this was spread on YPD medium agar-plates and incubated at
37oC for isolation of pure cultures. Each developed colony was picked up and maintained
on agar based YPD medium till further use
2.1.3 Screening for Proteolytic Activity
Proteolytic activity of isolated strains was screened by plating them on casein agar
medium consisting (gl-1) of casein -10.0, MgSO4- 0.05, CaCl2-0.02, FeSO4-0.01, yeast
extract -1.0 and agar- 20.0. These plates were incubated at 370C for 24 h. Bacteria
showing clear zones of caseinolysis on casein agar plates were identied as protease
producers. Based on hydrolysis zone, few colonies were selected and maintained on the
YPD medium slants at 40C. One of the colonies which were showing more caseionlysis
activity was designated as DKMNR and selected for further studies.
18
Fig. 2.1: Isolated bacterial strain DKMNR growing on the casein agar plate
showing a clear hydrolyzed zone.
2.2.3 Biochemical Characterization of isolate DKMNR
All the tests were performed at Institute of Microbial Technology (IMTECH)
Chandigarh, India. The colonies of strain DKMNR on nutrient agar plate were round,
wavy margins rough surface and opaque density. The cell growth is aerobic, gram
positive in nature and spore forming rod shaped bacteria. Figure 2.2 shows the scanning
electron microscopic pictures of the isolated bacteria DKMNR. The cells could survive
and grow in pH 5.0 to 11.0. The growth was studied in presence of different NaCl
concentrations; it was observed that DKMNR would grow even at 10% NaCl. Based on
the results, it was concluded that this isolate DKMNR may belong to Bacillus subtilis and
it was designated as Bacillus subtilis DKMNR.
Fig. 2.2: Scanning electron microscopic picture of the isolate DKMNR colony
20
Fig. 2.3: Neighbour joining phylogenetic tree constructed according to Kimura two
parameter model is showing phylogenetic relationship of Bacillus subtilis DKMNR
with the members of the genus Bacillus. Bootstrap values (>50) calculated from
1000 replications are indicated in the branch nodes and the accession numbers for
the reference sequences are given in the parenthesis. The bar represents 2
substitutions in 1000 nucleotides
21
Compound
Real
Starch
Maltose
Sucrose
Glucose
Fructose
Xylose
Galactose
Peptone
Casein
Yeast extract
Beef extract
Malt extract
Urea
Ammonium sulphate
Ammonium nitrate
Coded
X1
X2
X3
X4
X5
X6
X7
X8
X9
X10
X11
X12
X13
X14
X15
levels
0 (medium)
-1 (Low)
0.4
0.2
0.2
0.4
0.4
0.2
0.4
0.2
0.4
0.2
0.4
0.2
0.4
0.2
0.5
0.2
0.5
0.2
0.5
0.2
0.5
0.2
0.5
0.2
0.5
0.2
0.5
0.2
0.5
0.2
1(High)
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
23
The contribution of an ingredient towards the growth of the organism or yield of the
enzyme is determined based on the t-value (main effect) calculated from the experimental
result (Ramana Murthy, 1999). The nutrients are ranked based on their t-values. The
nutrient with highest t- value is considered to be the best and ranked one. The sign of the
effect indicates the level at which it is considered for further improvement. For example,
if a variable have the negative sign means the compound gives the best yield at the low
level and experiments should be carried out using further decreased concentration of the
compound. All experiments were carried out in triplicate and the average of protease productivity was taken as response (Y). The variables whose condence levels were higher
than 90% were considered to signicantly inuence on enzyme production.
Table 3.2: Planckett-Burman experimental design along with observed and predicted protease yield
Protease
S. No X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 Activity
(U/mL)
24
-1 -1 -1 -1 1
-1
-1
-1
-1
248
-1 -1 -1 -1 -1 -1
-1
-1
387
-1
-1 -1 -1
-1 -1
-1
-1
646
-1 -1 1
-1 -1 -1 -1
-1
-1
247
-1 -1
1 -1 1
-1
-1
-1
-1
-1
947
-1
1 -1 -1
-1 -1
-1
-1
-1
1055
-1
1 -1 -1 -1
-1
-1
-1
-1
267
1 -1 1
-1
-1
-1
-1
-1
-1
-1
450
-1 -1 -1 1
-1
-1
-1
-1
-1
555
10
-1 -1 1 -1 -1
-1
-1
-1
-1
610
11
-1
-1 1 -1
-1 -1
-1
-1
-1
850
12
-1 1
-1
-1
-1
-1
-1
-1
954
13
-1 -1
-1 -1 -1 -1
-1
-1
1104
14
-1
1 -1
-1 -1
-1
-1
-1
-1
1046
15
-1
1 -1 -1 -1
-1
-1
-1
-1
971
16
557
17
754
18
697
19
734
20
764
-1
(3.2)
Where xi is the coded value of the ith independent variable, Xi is the natural value of the ith
independent variable, X0 is the natural value of the ith independent variable at the center
point and Xi is the step change value.
----------------------------
(3.3)
Where Y is the predicted response, 0 is intercept term, i is linear effect, ii is the squared
effect, and ij the interaction effect. The full quadratic equation for 6 factors is given by
model 3.4.
Y = 0 + 1x1 + 2x2 + 4x3 + 4x4 + 5x5 + 6x6 + 11x1*x1 + 12x1*x2 + 13x1*x3 + 14x1*x 4
+ 15x1*x5 + 16x1*x6 + 22x2*x2 + 23x2*x3 + 24x2*x4 + 25 x2*x5 + 26 x2*x6 + 33 x3*x3+
34 x3*x4+ 35 x3*x5 + 36 x3*x6+ 44 x4*x4+ 45 x4*x5+ 46 x4*x6+ 55 x5*x5+ 56 x5*x6+ 66
x6*x6 ----------------------------------------(3.4)
Table 3.3: Experimental range and levels of the independent variables
S. No
Levels
Coded
1
2
3
4
5
Variable
Real
Temperature (C)
step change
0
1
33
34
X1
-2
31
-1
32
pH
Agitation (rpm)
Size of inoculum (ml)
Glucose (%w/v)
X2
X3
X4
X5
8.0
160
1.0
0.5
8.5
180
1.5
1.0
9.0
200
2.0
1.5
Peptone (%w/v)
X6
0.5
0.75
1.0
2
35
9.5
220
2.5
2.0
10
240
3.0
2.5
0.5
20
0.5
0.5
1.25
1.5
0.25
For this study, 26-1 fractional factorial design with 12 star points and 6 replicates at the central points was employed to t the second order polynomial model, which indicated that
50 experiments were required for this procedure.
25
The design and results of FFCCD experiments for studying the effect of six independent
variables were presented along with the mean predicted and observed responses in Table
3.5. The regression equations obtained after the analysis of variance (ANOVA) gave the
level of protease production as a function of the initial values of glucose, peptone, pH,
temperature, agitation and volume of the inoculum.
3.2 RESULTS AND DISCUSSION
3.2.1 Screening of nutrients using Plackett-Burman design
In the present investigation, the signicance of 15 different carbon and nitrogen compounds on production of protease was screened at two levels (high and low values) by
applying the 20 experimental PlackettBurman design. Table 3.2 gives the experimental
plan along with the results. It was observed that the enzyme production was varied
between 247 - 1104 Uml-1. It indicates that the selected nutritional compounds show a signicant effect on production of the protease.
Based on experimental data, the Pareto chart effects was plotted for identifying the factors that are important in enzyme production in this bacterial strain. This chart shows the
factors main effect estimates on the horizontal axis. The selected factors main effects are
rank ordered according to their signicance. The chart also show a vertical line to indicate
the statistical signicance (P=0.05). If selected variable is signicant in the process, the
variable-bar crosses the vertical line or vice versa.
From the Pareto chart (Fig.3.1) carbon sources (glucose, sucrose, maltose and fructose)
and nitrogen sources (peptone, urea, casein and ammonium nitrate) are signicant. Table
3.4 indicates the ANOVA data from this Table it is observed that the peptone has the highest effect (-350.5) and followed by the glucose (300).
Fig. 3.1: Pareto chart showing the effect of medium components on protease
yield.
26
The observed lowest p-value (0.00024) and highest F-value (153.601) (F > P value) for
peptone indeed suggested that this is the most important nutritional source for the protease production. After the peptone, glucose shows the lowest p-value (0.000447) and highest F-value (112.528). From the Table 3.4, it was observed that peptone, urea, ammonium
nitrate, maltose and fructose were showing the highest effect at their lowest concentrations indicates that these compounds are required in small amounts. It is also evident from
the Table 3.2 where the highest activity was observed in the 6th, 13th and 14th runs where
peptone was found in the low concentrations. Where as in 1st and 7th runs peptone was
found in the higher concentrations at this run the lowest activity was observed.
It is evident from Fig. 3.2 that among selected variables peptone and glucose were seen as
outliers with positive mean values separated from other variables. The outlier's variables
have the more positive inuence on the protease production. This suggested that further
optimization of these variables improves the enzyme production in this B. subtilis
DKMNR.
Table 3.4: Effects and ANOVA for selected variables
S.
No
Effect
Coefci
ents
Mean/
692.150 692.150
Intercept
SS
df
MS
t-value p-value
54.7261 0.000001
X1
-35.250 -17.625
X2
X3
X4
X5
X6
-10.000
-5.000
400
400.0
X7
-43.000 -21.500
7396
7396.0
10
X8
11
X9
130.500
68121
12
X10
13
X11
26.000
13.000
2704
2704.0
0.8452
0.9194 0.409934
14
X12
19.500
9.750
1521
1521.0
0.4754
0.6895 0.528415
15
X13
16
17
X14
X15
18
Error
12797
19 Total SS
65.250
4970
4970.3
3199.2
1447329 19
27
Table 3.5: Experimental design along with observed and predicted protease
activity.
Size of
S. Temp.
Agitation
Glucose Peptone
pH
Protease activity (U/mL)
inoculum
No (C)
(rpm)
(% w/v) (% w/v)
(ml)
Observed Predicted Error
1
32
8.5
180
1.5
1.0
0.8
1007
1011.445 -4.445
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
31
8.5
8.5
8.5
8.5
8.5
8.5
8.5
9.5
9.5
9.5
9.5
9.5
9.5
9.5
9.5
8.5
8.5
8.5
8.5
8.5
8.5
8.5
8.5
9.5
9.5
9.5
9.5
9.5
9.5
9.5
9.5
9.0
180
180
180
220
220
220
220
180
180
180
180
220
220
220
220
180
180
180
180
220
220
220
220
180
180
180
180
220
220
220
220
200
1.5
2.5
2.5
1.5
1.5
2.5
2.5
1.5
1.5
2.5
2.5
1.5
1.5
2.5
2.5
1.5
1.5
2.5
2.5
1.5
1.5
2.5
2.5
1.5
1.5
2.5
2.5
1.5
1.5
2.5
2.5
2.0
2.0
1.0
2.0
1.0
2.0
1.0
2.0
1.0
2.0
1.0
2.0
1.0
2.0
1.0
2.0
1.0
2.0
1.0
2.0
1.0
2.0
1.0
2.0
1.0
2.0
1.0
2.0
1.0
2.0
1.0
2.0
1.5
1.3
1.3
0.8
1.3
0.8
0.8
1.3
1.3
0.8
0.8
1.3
0.8
1.3
1.3
0.8
1.3
0.8
0.8
1.3
0.8
1.3
1.3
0.8
0.8
1.3
1.3
0.8
1.3
0.8
0.8
1.3
1.0
1129
687
1262
1373
1497
1350
1353
997
1035
887
1079
1221
1705
995
876
699
694
1396
1107
1037
1143
1284
1126
674
965
1104
857
1138
1142
934
1159
1656
1113.045
928.345
1216.745
1341.945
1530.345
1319.645
1328.745
966.545
1067.445
855.245
1051.345
1217.345
1756.945
983.245
929.145
656.845
716.745
1355.045
1121.645
1075.645
1185.745
1262.545
1167.445
709.245
1006.345
1081.645
899.045
1194.245
911.645
960.945
1165.545
1595.720
15.955
-241.34
45.255
31.055
-33.345
30.355
24.255
30.455
-32.445
31.755
27.655
3.655
-51.945
11.755
-53.145
42.155
-22.745
40.955
-14.645
-38.645
-42.745
21.455
-41.445
-35.245
-41.345
22.355
-42.045
-56.245
230.355
-26.945
-6.545
60.280
29
Size of
S. Temp.
Agitatio
Glucose Peptone
pH
Protease activity (U/mL)
inoculum
No (C)
n (rpm)
(% w/v) (% w/v)
(ml)
Observed Predicted Error
34
35
9.0
200
2.0
1.5
1.0
1311
1327.320 -16.320
35
33
8.0
200
2.0
1.5
1.0
1483
1411.020 71.980
36
33
10.0
200
2.0
1.5
1.0
1186
1214.020 -28.020
37
33
9.0
160
2.0
1.5
1.0
1145
1078.120 66.880
38
33
9.0
240
2.0
1.5
1.0
1502
1524.920 -22.920
39
33
9.0
200
1.0
1.5
1.0
1276
1295.220 -19.220
40
33
9.0
200
3.0
1.5
1.0
1379
1315.820 63.180
41
33
9.0
200
2.0
0.5
1.0
1205
1158.520 46.480
42
33
9.0
200
2.0
3.0
1.0
1312
1314.520 -2.520
43
33
9.0
200
2.0
1.5
0.3
1120
1167.920 -47.920
44
33
9.0
200
2.0
1.5
1.5
1410
1318.120 91.880
45
33
9.0
200
2.0
1.5
1.0
1712
1737.320 -25.320
46
33
9.0
200
2.0
1.5
1.0
1708
1737.320 -29.320
47
33
9.0
200
2.0
1.5
1.0
1745
1737.320 7.680
48
33
9.0
200
2.0
1.5
1.0
1680
1737.320 -57.320
49
33
9.0
200
2.0
1.5
1.0
1754
1737.320 16.680
50
33
9.0
200
2.0
1.5
1.0
1737
1737.320 -0.320
The above results were analyzed using statistical programme and the coefcient of determination (R2) was calculated as 0.9575 for alkaline protease production by this bacterial
strain indicating that the statistical model can explain 95.75% of variability in the
response and only 4.25% of the total variations were not explained by the model. The R2
value should always be between 0 and 1. If the R2 is closer to 1.0, the stronger the model
and the better it predicts the response. The adjusted R2 value corrects the R2 value for the
sample size and for the number of terms in the model. The value of adjusted determination coefcient (Adj R2 = 0.9053) was also very high suggesting a higher signicance of
the model used for analyzing the data (Cochran & Cox, 1957). In this enzyme production
study the adjusted R2 value (0.9053) was lesser than the R2 value (0.9575). This is
because, if there are many terms in the model and the sample size is not very large, the
adjusted R2 may be noticeably smaller than the R2. At the same time, a relatively lower
value of the coefcient of variation (CV= 7.4 %) indicated a better precision and reliability of the experiments carried out by (Cochran & Cox, 1957)
30
31
Effect
SS
df
MS
Mean/
Intercept
1737.32
1737.32
X1
-67.1
-134.2
180096 1
180096
21.6821 0.00012
X1*X1
-68.95
-137.9
152131 1
152131
18.3153 0.00031
X2
-49.25
-98.5
97023
X2*X2
-106.2
-212.4
360910 1
360910
43.4505
1E-06
X3
111.7
223.4
499076 1
499076
60.0844
X3*X3
-108.95
-217.9
379843 1
379843
45.7299
1E-06
X4
5.15
10.3
1060.9
0.12772 0.72421
X4*X4
-107.95
-215.9
372902 1
372903
44.8942
X5
39
78
60840
60840
7.32461 0.01289
X5*X5
-125.2
-250.4
501601 1
501601
60.3885
X6
37.55
75.1
X6*X6
-123.58
-247.15
X1*X2
10.938
21.875
3828
X1*X3
-25.625
-51.25
21013
X1*X4
92.188
X1*X5
-46.625
-93.25
69565
X1*X6
17.375
34.75
9660
X2*X3
-19.063
-38.125
11628
X2*X4
-61.625
-123.25
121525 1
p-value
X2*X5
12.188
24.375
X2*X6
1061
56400
1E-06
0
488665 1
184.375 271953 1
488665
58.8311
3828.1
0.46087
0.5043
271953
9660.5
32.7408
9E-06
1.16304 0.29252
121525
14.6305 0.00092
4753.1
0.57224
65.938
131.875 139128 1
139128
16.7498 0.00048
X3*X4
-73.688
-147.38
173755
20.9186 0.00015
X3*X5
-0.25
-0.5
0.00024 0.98776
X3*X6
31.625
63.25
32005
X4*X5
-30.688
-61.375
30135
X4*X6
X5*X6
-23.813
43.125
-47.625
86.25
18145
59513
Error
Total SS
4753
173755 1
1
182737 22 8306.2
4299893 49
F-value
0.4574
It is observed that except linear term of inoculum size all linear and quadratic terms of all
variables were signicant. But, the interaction terms of temperature with pH, agitation
speed and peptone concentration were insignicant, similarly the interactions of pH with
agitation speed and glucose concentration were also insignicant. Even the interactions
of agitation speed with concentrations of glucose and peptone were insignicant and in
the same way the interactions of inoculum size with concentrations of glucose and
peptone were also insignicant remaining all other interactions were signicant (Table
3.6). The model F-value of 18.36, and values of probability > F (<0.05) indicated that the
model terms were signicant.
The regression model developed was represented in the form of 2D contour plots. The
yields of alkaline protease for different concentrations of variables could also be predicted from the respective contour plots as shown in Figures 3.4 (a-f). (Cochran & Cox,
1957). It was evident from the contour curves that the alkaline protease production was
highly and interactively inuenced by all selected fermentation parameters Fig.3.4 (a-f).
Figure 3.4a shows the interaction of temperature and pH. From the Fig 3.4a it is observed
that the contours are elliptical in nature and slightly inclined towards the temperature indicates that the temperature is much more important than medium pH. The Fig. 3.4c shows
that the pH was also inuencing the protease production when interacted with other factors. The organism is able to produce the protease at pH higher than 8 and below 10, but
still higher than this it has a negative inuence on enzyme production.
An interesting enzyme productivity status was noticed with the interactive state of carbon
and nitrogen source supplemented environment. The enzyme production was inhibited
by higher concentrations of these two nutrients (in the selected range) and more enzyme
yield was observed when both of them near to the central point and much variation were
observed in case of peptone (Fig. 3.4f). Moreover, the Fig. 3.4f also suggested that for
achieving maximum enzyme production, glucose and peptone concentrations should be
nearer to central point's concentrations. However higher enzyme production was also
observed in controlling the ratio of casein and starch. In general it was noticed that higher
concentration of any carbon source results in reduction of protease production due to catabolic repression of glucose. In alkalophillic actinomyces sp. supplementation of more
than 0.5% of carbon source resulted in decreased protease production (> 60%). However,
enhanced protease production was reported with corn steep liquor (Malathi &
Chakraborty, 1991) as nitrogen sources. Comparatively, in this isolate higher concentration of carbon source in presence of low nitrogen concentration reduced protease production.
A numerical method (Myers & Montgomery, 1995) was used to solve the regression equation 3.5. The optimal values of the six test variables in coded units were observed to be x1=
-1.84, x2 = 0.1647, x3 = 1.26, x4 = -1.402, x5 = 0.7623 and x6 = 0.4958. The predicted value
of Y (protease activity) at the above conditions is 1885.7 Uml-1. The real values of the six
variables were obtained by substituting the respective coded values in equation 3.2.
The optimum conditions of selected fermentation parameters were predicted using RSM
and the maximum predicted protease production (1885.7 Uml-1) could be achieved with
the medium consisting of glucose 1.88 %wv1; peptone 1.124 %wv1; pH-9.08; temperature 31.16 C; agitation speed 225.2 rpm and inoculum size 1.29 ml in 250 ml ask for 24
h culture having absorbance of 1.0 at 600 nm. The validation experimental protease pro33
duction data revealed 1885.7 Uml-1 under optimized conditions. The experimental value
of the protease production was almost equal if we consider 95% of the condence limits
for the prediction of Y value at optimized conditions with shake ask results.
34
The present study was aimed to exploit the locally available, inexpensive agro-substrate
for alkaline protease production using B. subtilis DKMNR under solid-state fermentation
and optimization of the various parameters for improvement of the yield.
4.1MATERIALS AND METHODS
4.1.1 Collection and processing of the substrates
Various agro industrial materials such as rice bran, wheat bran, coconut oil cake, Zuzubi
oil cake, peanut press cake, rice husk, green gram husk, red gram husk, bengal gram husk,
black gram husk, soya bean meal, corn cobs, corn stover, corn leaves, sweet sorghum
pulp, sugarcane baggase and sugarcane leaves are obtained from the local agricultural
market. Whereas apple pomac, pineapple waste, orange waste, banana peels and orange
peels were obtained from the local fruit market and juice shops. The potato peels, mashed
potatoes, processed tea powder and processed coffee waste were collected from the
home. All the materials were dried at 800C for 2 hours and the leaf materials and fruit pulp
were powdered. All the materials were sieved to avoid the ne powder, which causes the
clumps formation upon addition of water, and decrease the substrate availability to the
microorganism.
4.1.2 Solid-state fermentation
Selected substrate materials were used as a solid medium for protease production. Five
grams each of the substrate was taken separately in 250 ml Erlenmeyer asks and moisturized with 5 ml of distilled water. These asks were sterilized and inoculated with 2 ml
of inoculum solution. The asks were mixed thoroughly and incubated at 320C in an incubator for 48 hours.
4.1.3 Mixture Design
Mixture design was chosen to study the effect of mixed substrates on protease production.
The best three substrates, which could yield highest protease at individual level were chosen. A simplex lattice design was employed to optimize the substrate mixture. All the
experiments were conducted according to the (Sathish et al. 2008).
In a mixture design, two models have to be taken into account, one for each mixture being
considered. In this case, a product model can be used with the two groups of components
x and z. This model is represented by Eq. (4.1):
y = f(x)*g(z) + e
. (4.1)
In Eq. (4.1) y is the response, the functions f(x) and g (z) are separated polynomial models
that represent the two mixtures, and e is a zero-mean random variable with variance independent of x and z.
The polynomial models used in Eq. (4.1) was modied some terms from the complete
polynomial expression in order to eliminate the constraint originated in the correlated
variables. Eq. (4.2) shows the canonical form of the quadratic model:
. (4.2)
36
Geometrically, in Eq. (4.2) the parameter i represents the expected response to the pure
mixture xi=1, xj=0, ji. The rst term in the Eq. (4.2) represents the response when blending is strictly additive and there are no interactions between the components of the mixture. The quadratic term ijxixj represents the excess response over the linear model due to
the interaction between two components, and this effect is often called synergism or
antagonism.
4.1.4 Enzyme extraction
The enzyme was extracted according to the method described by (Prakasham , 2006). Fermented medium was mixed thoroughly with 50 mM glycineNaOH buffer, pH 11 for 30
min and the extract was separated by squeezing through a cloth. This process was
repeated three times and extracts were pooled together and then centrifuged. The
supernatant was used as enzyme source for protease assay.
4.1.5 Optimization of the various process parameters
In order to increase the protease production in the solid-state fermentation various process parameters such as pH (5 to 12), moisture content (20 to 200 % w/w1), inoculum size
(0.5 to 3.5) and temperature (26 to 400C) were optimized.
4.1.5.1 Effect of carbon and nitrogen additives on protease production
The optimum SSF medium was supplemented with different carbon and nitrogen sources
initially at 0.5g. Further the best suitable additional carbon and nitrogen source was studied at various concentrations in order to improve the protease production.
4.2 RESULTS AND DISCUSSION
4.2.1Evaluation of Different Agro-Industrial Material for Alkaline Protease Production
The selection of an ideal agro-biotech waste for economic enzyme production in solidstate fermentation process depends upon several factors, mainly related with cost and
availability of the substrate material. Thus it involves screening of several agro-industrial
residues. Agro industrial materials such as rice bran (RB), wheat bran (WB), coconut oil
cake (COC), zuzubi oil cake (ZOC), peanut press cake (PPC), rice husk (RH), green gram
husk (GH), red gram husk (RH), bengal gram husk (BGH), black gram husk (BLH), soya
bean meal (SM), corn cobs (CC), corn stover (CS), corn leaves (CL), sweet sorghum pulp
(SSP), sugarcane baggase (SB) and sugarcane leaves (SL) (Fig.4.1), household waste
materials like apple pomac (AP), pine apple waste (PW), orange waste (OW), banana
peels (BP), orange peels (OP), potato peels (PP), mashed potatoes (MP), processed tea
powder (PTP) and processed coffee waste (PCW) Figure.4.1 were used as solid support/substrate matrices for production of enzyme by B. subtilis DKMNR. The data indicated that protease production pattern varied with the type of agro-waste. This phenomenon might be attributed to the dual role of solid materials on supply of nutrients to the
growing microbial culture and providing anchorage for the growing cells.
Maximum protease production (914 Ugds1) was observed with green gram husk while
minimum protease production (145 Ugds1) was noticed with rice husk as substrate/support material. Soya bean meal and bengal gram husk were found to be best substrates for protease production next to the green gram husk. This data further supported
that, the composition of the substrate was one of the important parameters for evaluation
of extracellular microbial enzymes production. The results were in accordance with the
37
observations made with alkalophilic and thermophilic Bacillus species JB-99 (Johnvesly
et al. 2002), and others reported about bacterial strains (Prakasham, 2006; Gessesse,
1997). However, the results varied in the production values suggesting that the present
investigated bacterial strain was different in its metabolic and biochemical aspects to that
of Bacillus species JB-99 (Johnvesly et al. 2002). Evaluation of protease production values of the strain with different organisms reported in the literature did not indicate that,
this strain could be a potential organism after optimization and scale up studies (Table
4.1). To obtain the maximum protease production the fermentation media is modied by
mixing the different agro-industrial wastes. Three different wastes such as soybean meal,
bengal gram husk and green gram husk were selected as they exhibited the maximum protease activity when used as individual substrates.
Fig. 4.1: Protease production by isolated Bacillus subtilis DKMNR using market
and house hold waste.
Table 4.1: Protease production by different microorganisms
38
Microorganism
Protease production
(Ugds1 material)
References
Bacillus sp ecies
35,000
Prakasham ,2006
Pigeon pea
12,430
Bacillus species
Wheat bran
429
Bacillus species
Lentil husk
168
Rhizopus oryzae
Wheat bran
358
Rhizopus oryzae
Wheat bran
58.7
Coded
Real
1
2
3
4
5
1
0
0
0.5
0.5
5
0
0
2.5
2.5
0
1
0
0.5
0
0
5
0
2.5
0
0
0
1
0
0.5
0
0
5
0
2.5
685.00
904.00
823.00
966.00
998.00
685.74
910.38
808.83
969.17
986.26
-0.74
-6.38
14.16
-3.17
11.73
6
7
8
9
0
0.6667
0.1667
0.1667
0
3.4
0.8
0.8
0.5
0.1667
0.6667
0.1667
2.5
0.8
3.4
0.8
0.5
0.1667
0.1667
0.6667
2.5
0.8
0.8
3.4
967.00
1045.00
915.00
1019.00
949.62
1023.91
923.74
1010.83
17.37
21.08
-8.74
8.16
10
0.3333
1.6
0.3333
1.7
0.3333 1.7
917.00
970.47
-53.47
SM= Soybean Meal; GH = Green gram husk; BGH = Bengal gam husk
Data from the experimental design was further analyzed by employing a multiple linear
regression using protease yield as the response. Sequential F-tests, on the linear to full
cubic solutions were performed for appropriate model selection (based on highest Fstatistics signicance) suitable for protease production. ANOVA results of the all four
models are presented in Table 4.3. The quadratic model showed a high F value (22.24)
and low p value (0.00588). The analysis of R2 value revealed that the special cubic and
cubic model have higher t (R2 special cubic = 0.9593 & R2 Cubic = 0.9840) than the quadratic model (R2quadratic = 0.9588).
S. No
Model
Linear
Quadratic
3
4
40
SS
R2
Adjusted R2
0.328304 0.2725
0.0647
0.9074
73184.79 13711.06
49.89
1609.46 1239.64
1.3114
0.8780
0.8560
SM
GH
BGH
SM*GH
SM*BGH
Coefcients
685.7483
910.3847
808.8392
684.4141
809.3232
t-Value
22.10519
29.34638
26.07304
4.78690
5.66054
p-Value
0.000025
0.000008
0.000013
0.008731
0.004801
GH*BGH
506.5960
3.54321
0.023944
The task of optimizing mixtures of different substrates for protease production can be predicted using a triangular surface response methodology as triaxial diagrams are graphical
representation of a combination of raw materials, rather than a predictive illustration of
the product yield. Figure 4.2 depicts the triangular graphs showing the level curves of protease yield obtained from (Eq.4.7) as a function of the substrate type. From the graph it is
observed that the highest yield was nearer to the GH having equal distance to the GH
SM and GH-BGH axis. The SM- BGH axis demonstrated the least protease production.
41
Further, a numerical method given by (Myers and Montgomery, 1995) was used to solve
the regression Eq. 4.7 to optimize the substrate mixture ratio. The results indicated that
the optimum mixture for higher protease production is 44.4% of GH, 25% SM and 30.6%
BGH by dry weight. For this substrate combination the predicted protease yield was
1029.864 Ugds1. Our results validated these fermentation conditions with a protease production rate of 1045 Ugds1. A similar optimization approach was performed by Sathish et
al. (2008), for L-glutaminase production in solid state fermentation in cutinase production in submerged fermentation.
suggested that, the enzyme production was inuenced by the pH of the medium. Maximum protease production (1246 Ugds1) was observed at pH 9.0 (Fig. 4.3). The synthesis
of the enzyme increased with increase of the pH of the medium towards alkaline range
from neutrality up to 9.0 and was less constant in the pH range 9.012.0 by B. subtilis
DKMNR. The enzyme production pattern suggested that, the isolated bacterial strain was
alkalophilic in nature and produces maximum quantity of enzyme at alkaline pH conditions.
Further evaluation of enzyme data in the studied pH range indicated a linear increase in
the biocatalyst production up to pH 9.0. The observed variation in protease production
under solid state fermentation with mixed substrate attributed to higher enzyme production in the pH range of 9.0 to 10.0. In the literature the authors reported that protease from
solid state cultures of Aspergillus parasiticus showed optimum activity at pH 8.0 and 80
% less activity at pH 5.0. (Johnvesly et al. 2002) working on alkaline thermo stable protease found that, the enzyme produced by thermo alkalophilic Bacillus species JB-99
showed catalytic activity in a broad pH range (6.0 to 12.0) with 11.0 as optimum pH. The
data generated in the present investigation suggested that, the inuence of pH on alkaline
protease produced by isolated B. subtilis DKMNR might be related with synthesis level
because, the extraction of the enzyme after solid state fermentation was performed using
alkaline pH buffer. The adapted extraction procedure eliminated the inhibition of protease at cellular transport and at activity level and the observed growth associated nature of
this enzyme production in this bacterial strain.
43
44
45
46
Fig. 4.6: Effect of incubation temperature on the production of protease by isolated B. subtilis DKMNR.
4.2.7 Role of different carbon sources on protease production
The selection of an ideal substrate for enzyme production in solid-state fermentation process is one of the critical factors to be considered (Subbarao et al. 2009; Sathish and
Prakasham, 2010). This is because, some of the nutrients may be available in sub-optimal
concentrations, or even absent in the substrate and therefore no substrate may be suitable.
In such cases, it would be necessary to supplement substrate externally with decit nutrients to have optimal yields. Several carbon sources such as galactose, arabinose, fructose,
maltose, soluble starch, glucose, xylose, mannose and ribose were selected and supplemented to the solid medium at 1 % level in order to enhance the microbial growth.
The data suggested that, supplementation of external carbon source inuenced alkaline
protease production in this bacterial strain and all the selected carbon sources showed positive impact on cellular metabolism leading to the production of the enzyme (Fig.4.7).
The results indicated that the selected media (mixture of soybean meal, bengal gram husk
and green gram husk) were not the ideal substrate for alkaline protease production by this
isolated Bacillus subtilis DKMNR because of the deciency in carbon source. Improvement of cell growth and subsequent metabolite synthesis, in several microorganisms, was
noticed upon supplementation of external carbon sources (Malathi & Chakraborty,
1991). However, enhancement in enzyme production levels varied with the type of carbon source (Fig. 4.7). Glucose supplemented conditions supported maximum production
with an increase of 112% over control (no external carbon source supplementation).
Protease production was observed to be 12% increase over control condition, with glucose as external carbon source. This data suggested that, glucose was not a repressor of
protease enzyme in the bacterial strain under investigation unlike the observed catabolic
47
repression by glucose in B. subtilis and B. licheniformis (Frankena et al. 1986; Kole et al.
1998).
One interesting phenomenon in this investigation was maltose, which is a disaccharide
with two monomers of glucose units supported protease production better from this bacterial species when compared to carbon source (Fig.4.7). This might be attributed to the
fact that, maltose as such might enter inside the cell before conversion into glucose units.
In fact, maltose metabolism in bacterial cells begins at maltose-6 phosphate and subsequently gets converted to glucose-6 phosphate and is metabolized further. This apparently eliminates the glucose associated physiological changes in the medium such as lowering of solution pH especially the medium is alkaline in nature. Such glucose-mediated
reduction in protease production associated with lowering of pH was noticed by Zamost
et al. (1990), while working on production and characterization of a thermostable protease by an asporogenous mutant of B. stearothermophilus. This was further evidenced
from the fact that reduced alkaline protease production in lower pH medium environment.
In order to know optimum requirement of carbon source for better alkaline protease production by the bacterial strain is under investigation in solid-state fermentation conditions, the enzyme production pattern was investigated by supplementation of different
concentrations of glucose (0 to 2%) (Fig. 4.8). The data revealed that alkaline protease
production in this bacterial strain was regulated by availability of glucose in the medium
and maximum production (1971 Ugds1) occurred with 1.25% glucose concentration
under experimental conditions. Approximately 15% improvement of enzyme yield was
noticed under 1.25% glucose supplemented conditions when compared to 0.5 %. Further
increase in glucose concentration adversely affected protease production in B. subtilis
DKMNR under solid-state fermentation condition. The results obtained were in accordance with reported alkaline protease production in the presence of different sugars
(Adinarayana et al. 2003).
Fig. 4.7: Effect of different carbon sources on the production of protease by isolated B. subtilis DKMNR.
48
Fig. 4.8: Inuence of glucose concentration on the production of protease by isolated B. subtilis DKMNR.
4.2.8 Role of different nitrogen sources on protease production
Nitrogen source is one of the essential requirements for healthy microbial growth and is
required to produce several cellular organic compounds such as amino acids, nucleic
acids, proteins and cell wall components. Though most of the microorganisms metabolize inorganic and organic nitrogen sources, the preference varies with the genetic nature
of microbe and type of product produced (Johnvesly et al, 2002). It was reported that alkaline protease comprised 15.6 per cent nitrogen and its production was regulated by the
availability of nitrogen in the medium (Pandey et al. 2000; Kole et al. 1998). Therefore,
inuence of different nitrogen sources on alkaline protease yield by isolated B. subtilis
DKMNR was investigated by supplementing 0.5 % selected nitrogen compound to solid
medium under optimal fermentation environment and measuring enzyme production.
Experimental data revealed that complex nitrogen sources yield maximum alkaline protease production in the bacterial strain. The data indicated the importance of nitrogen
requirement for production of the protease by the Figure 4.9. A media with the mixture of
SM, BGH and GH was insufcient nutrient medium for protease production. So, different
nitrogen sources on protease production by B. subtilis DKMNR under solid state fermentation conditions with the above media was investigated. Casein as nitrogen source
showed maximum inuence by enhancing the enzyme production to that of other organic
and inorganic nitrogen sources. The increase in protease yield was observed to be approximately 2-fold over control. Similar observations were noticed in the case of protease production by different microbial species (Johnvesly et al. 2002).
49
Fig. 4.9: Effect of different nitrogen sources on the production of protease by isolated B. subtilis DKMNR.
50
51
Fig. 4.11: Effect of incubation time on the production of alkaline protease by isolated B. subtilis DKMNR.
52
5.0 INTRODUCTION
Recent developments indicated that proteases with high activity prole at alkaline pH and
high temperatures have potential applications in variety of elds including enzymatic
debridement and the natural healing process in the skin ulcerations (Gupta et al. 2002a;
Anwar & Saleemuddin, 1998). They have also gained importance in view of their cleaving of peptide bonds in aqueous environments and synthesize them in non-aqueous conditions.
Physical, biochemical, thermal, molecular and catalytic properties of proteases
vary with the producing organism (Ghorbel et al. 2003). In general, most of the industrial
proteases face some limitations (Joo et al. 2003) and their use highly depends on their stability during isolation, purication and storage in addition to their robustness against solvents, surfactants and oxidants (Sivasubramanian et al. 2008) Hence, in depth knowledge
on protease kinetics of fermentation and catalytic level of protease production by newly
isolated strain is one of the pre-requisites for evaluation of its potential for biotechnological application (Davis ,1964) . In the present studies a strain of Bacillus subtilis DKMNR
was assayed for its ability to digest casein.
5.1 MATERIALS AND METHODS:
5.1.1Protein estimation
The protein content was determined by the Lowry method by using BSA as standard.
5.1.2 Enzyme recovery and purication procedure
5.1.2.1Ammonium sulphate precipitation
The crude broth obtained after fermentation was centrifuged at 5000 X g for 10 min to
remove the cell biomass. Solid ammonium sulphate was added slowly to the culture
supernatant to get 60% saturation, stirred for 60 min and left for overnight at 4oC. The precipitate was harvested by centrifugation at 10,000 X g for 10 min, dissolved in 50 mM
glycine-NaOH buffer and dialyzed against same buffer overnight (4oC). The dialyzed
sample was then assayed for protease activity and protein content.
5.1.2.2 Sephadex G-100 Chromatography
Dialyzed enzyme was loaded on to a column of sephadex G-100 (1.5 x 90 cm) previously
equilibrated with 50 mM glycine-NaOH buffer (pH 11) and then eluted at a ow rate of 10
ml h-1 with the same buffer containing sodium chloride gradient from 0.1 to 1M. The
absorbance of fractions was checked at 280 nm. Fractions were assayed for protease
activity with casein as substrate. Protease active fractions were pooled and concentrated
for further characterization.
53
54
55
Total
protein
(mg)
864
234
39
Fig. 5.2: Effect of substrate concentration on the protease activity from 0.4 to 1.6%.
5.5.2.2 Effect of pH on the protease activity and stability
In general, species of Bacillus are reported to secrete mostly two types of extracellular proteases, a neutral or metallo-protease exhibit optimum activity at pH 7.0 and an alkaline
protease having pH optima between 9.0 and 11.0 (Ghorbel et al. 2003). The enzyme produced by Bacillus subtilis DKMNR exhibited its optimum activity at pH 11.0 indicating
the enzyme to be alkaline protease group. Any further variation in pH of reaction mixture
resulted reduced catalytic activity (Fig.5.3). This activity variation was more and drastic
with increase of reaction mixture pH towards alkalinity. More than 50% reduction was
noticed with the change of 0.5 pH unit from 11.0 to 11.5 and further increase of 0.5 pH
unit resulted in 90% inhibition (Fig.5.3).
A progressive reduction of enzyme activity was observed with change of reaction mixture
pH towards acidic side indicating its robust nature in pH range of 5 to 11.0. The protease
was active more than 70 % in the range of pH 5.5-7.0 (Figure 5.3). Even though it is active
from pH 5.0 it shows only 50-70 % of its activity below pH 7.0 with respect to its activity
at pH 11.0 indicating its alkaline nature Nilegaonkar et al. (2007) reported protease with
broad pH range from 6.0 to 12.0 having optimum activity at pH 10.5 to 11.5. Protease of
Bacillus subtilis DKMNR was radically reduced with the change of pH in either side of
pH optima indicating the more activity enzyme.
57
59
Fig. 5.6: Effect of temperature on protease enzyme stability from 0 to 210 min.
5.5.2.6 Effect of metal ions on puried alkaline protease activity
To understand the role of different metal ions on the regulation of protease activity, the
enzyme activity was monitored in the presence of 10 mM concentration of selected metal
in reaction mixture. The effect of different metal ions on the activity of enzyme is shown
in the Figure 5.7.
Fig. 5.7: Effect of different metal ions on puried protease enzyme activity.
60
Presence of PbCl2 did not show any inhibition of alkaline protease activity indicating
enzyme from Bacillus subtilis DKMNR is not a metalloprotease. Na2+, Ca2+, Mg2+ and
Mn2+ ions positively regulated the enzyme activity while other tested metal ions did not
show much inuence except Ba2+ and Fe2+ compared to control. The activity regulation
was depended on the nature of ion. This was evident from the observations that 21, 12, 8
and 3 % activity improvement was noticed with the supplementation of Ca2+, Na2+, Mn2+
and Mg2+ ions, respectively, on the other hand > 60 % inhibition was observed in presence
of Ba2+ and Fe2+ ions. The Ca2+ ion stimulated the enzyme activity indicating more of calcium ions for enzyme optimal activity and may be attributed to calcium ion involvement
in stabilization of the enzyme molecular structure. In fact, calcium ions are known as
inducers and stabilizers of many enzymes and protect them from conformational
changes. Such type of metal dependent variation in proteases activity has also been
observed with serine proteases.
5.5.2.7 Effect of Inhibitors on protease activity
North, (1982) has classied proteases based on their sensitivity to various inhibitors. To
identify the class to which the alkaline protease produced by Bacillus subtilis DKMNR
belongs to, the enzyme activity was assayed in presence of different inhibitors and results
are presented in Table 5.2.
Table 5.2: Residual activity of puried alkaline protease at different
concentrations of inhibitors
Inhibitors
1 mM
Control
100
100
-mercaptoethanol
PMSF
Ido acetic acid
EDTA
PCMB
DFP
94
9
98
96
93
24
85
0
95
96
91
5
Presence of 1mM and 5mM of EDTA (Metallo protease inhibitor), Idoactetic acid (IAA,)
and p-chloromercuribenzoate (pCMB) which were inhibitors of cysteine protease
showed little or no effect on the alkaline protease of Bacillus subtilis DKMNR protease
suggesting this protease does not belongs to the metallo protease and cysteine protease
(Table 5.2). The aspartic protease inhibitor, -mercaptoethnol, was also ineffective as it
showed only 6 and 15% inhibition of proteolytic activity in presence of 1 and 5mM concentration of -mercaptoethnol. However, serine protease inhibitor PMSF completely
inhibited the enzyme activity even at very low concentration and the inhibition pattern is
typical of serine proteases.
5.5.2.8 Effect of surfactants and oxidizing agents on the alkaline protease activity
Table 5.3 depicts the inuence of various surfactants and reducing agents on alkaline protease of Bacillus subtilis DKMNR. The puried enzyme has shown stability in presence
of all studied compounds (Table 5.3). In fact the non-ionic detergents, Triton X-100 and
61
Control
100
Triton X-100
121
Tween-20
SDS
H2O2
106
84
97
Fig. 5.9: Arrhenius plot for protease enzyme of Bacillus subtilis DKMNR.
5.5.4 Evaluation of Industrial applications
5.5.4.1 Detergent stability
The compatibility studies of the puried enzyme with detergents revealed that the activity
of the enzyme decreased slightly with increasing the incubation time. A 3- 7% decrease in
protease activity was evidenced with increase of incubation time from 1 to 2h indicating
its compatibility with most of the branded detergents except Rin indicating its suitability
for formulation of commercial detergents (Table 5.4).
63
2h
Henko
90
83
Surf
75
72
Surf excel
84
76
Ariel
80
69
Rin
84
46
64
blood stain removal. Therefore applications of this enzyme in terms dehairing character
was investigating using goat skin without application of sodium sulde.
The enzyme precipitate (1% w/w in 10% water) dehaired the (6cm x 6cm) piece within 510 hrs at temperature of 30- 37 0C. Enzyme treated goat hide piece was white smooth
(Fig. 5.11b) as compared to untreated control (Fig. 5.11a) enzymatic process loosens
hair and epidermis because of degradation of specic proteins , glycoprotein's and
proteoglycans in the basal membrane, the present results are similar to those of our earlier studies on dehairing of goat hide by an alkaline protease from Aspergillus tamarri
dehaired the good skin at pH 9-11 temperature 30-37 0C with 1% enzyme concentration
and incubation period of 18-24h (Dayanandan, 2003).
Fig. 5.11: A) Control water treated goat hide piece B) Enzyme treated C) Control:
Enzyme untreated and DKMNR protease treated pieces.
The data depicted that there was no apparent damage to the collagen bers in dehaired
pelts. Protease produced from B.Subtilis has advantage in dehairing process as this
enzyme effectively un haired the goat skin with in 12h compared to literature reports
where alkaline proteases from B. circulans, B. cereus, A. tamarii dehaired the goat skin in
18, 21 and 24h, respectively (Nilegaonkar et al. 2007; Sivasubramanian et al. 2008;
Dayanandan, 2003). Indicating its potential application in leather industry for economizing the process.
66
6.0 INTRODUCTION
The term fermentation is derived from the Latin word forever to boil thus describing
the appearance of the action of the yeast on extracts of fruit or malted grain. The boiling
experience is due to the production of CO2 bubbles caused by the anaerobic catabolism of
the sugars present in the extract.
Pasteur applied the term fermentation to those anaerobic reactions through which
microorganisms obtained energy for growth in the absence of oxygen. Today fermentation has much broader meaning. It applies to both aerobic and anaerobic metabolic activities of the microorganisms where in specic chemical changes are brought about by an
organic in substrate.
A variety of substances such as alcohols, organic acids, amino acids, vitamins, antibiotics, enzymes, single cell proteins, hormones etc. are produced through fermentations by
employing different microorganisms. The enzymatic yield obtained from fermentation,
cost of their production, and downstream processing cost determines the nal cost of the
enzyme. To develop a viable industrial process, lowering production cost, and increasing
enzyme productivity are very important. Selection of best fermentation technique and
optimization of culture conditions contribute much in achieving enzyme productivity
(Barrios-Gonzalez et al. 2005). Currently, enzyme production by microorganisms can be
achieved using submerged fermentation or solid state fermentation.
6.1 Submerged Fermentation (SmF)
Submerged fermentation is dened as the cultivation of microorganisms in liquid nutrient
broth. The growth of the microorganisms at a large scale involve use of closed large vessel
(up to 100 cubic meters in volume) containing a rich nutrient broth and enough amount of
oxygen (Enshasy et al. 2008). Fermentation parameters such as medium composition,
pH, and aeration are important variables that affect the production of enzyme in SmF
(Maurer, 2004; Kennedy & Krouse, 1999).
SmF has its own advantages and drawbacks. Serious pollution problems, low product concentration and high production cost are the major draw backs associated with the use of
SmF. However, presence of high water content that is favorable for most bacterial growth,
homogeneity of the fermentation system, ease in process parameters measurement and
presence of well developed industrial equipment are some of the advantages of submerged fermentation (Holker and Lenz, 2005). At present, more than 90 % of the commercial microbial enzymes, including alkaline proteases, are produced using submerged
fermentation (Aguilar et al. 2008).
67
68
pH
B. cereus
BG1
B. clausii I 52
37
200
48
10.6
37
700
48
B.
licheniformis
36
220
40
B.
licheniformis
MIR29
7.5
45
300
24
Reference
Yeast
Glucose Frikha et
extract
al. 2005
Soya bean Liquid Joo et al.
meal
malotose
2002
Soyabean Corn our Tang et
cake meal
al. 2004
Ammonium
sulphate
Casein
Ferrero et
al. 1996
69
70
Microorganism
pH
B.pseudorm
us
37
100
66
Yeast
extract
Casein Gupta et
acid
al. 2002a
hydrolysat
e
Bacillus sp.
10
37
180
96
Yeast
extract
Bacillus sp.
50
200
72
Casein
Gupta et
al. 1999
Bacillus sp.
NCDC- 180
11.0,
12
50, 55
28,29
B. polymixa
9.011.0
30
100
72
B.
licheniformis
and
B. coagulans
30
NS
48
Soya bean
meal ,
Wheat
bran
Asokan &
Jayanthi,
2010
Bacillus Sp.
55
NS
96
Beef
extract
Wheat
bran
Naidu &
Devi,
2010
B.
licheniformis
NH1
37
200
24
Viscera
sardinella
Hulled
grain of
wheat
Hadj-Ali
et al.
2010.
Pseudomonas
aeruginosa
MCM B- 327
30
150
36
Peptone
Lactose
Vasudeo
et al. 2011
B.
thuringiensis
cc7
8.5
29
150
24
Casein
Glucose
Chudasa
ma et
al. 2010.
Bacillus sp.
RGR-14
37
200
42
Soya bean
meal
starch
Oberoi et
al. 2001
Vibrio
uvialis
VM10
28
100
36
Casein
40
Reference
Kumar et
al. 1999
Maltose Venugopa
l&
Saramma,
2006
71
Design
Software
Yield
improvement
Reference
2.5-fold
Banerjee et
al.1999
Vermelho et al.
1996
Puri et al. 2002
PlakettBurman
Not specied
3.0-fold
DesignExpert
(Statease)
2.6-fold
Bacillus sp.
Taguchi methodology
Qualiteck
55 %
Prakasham et
al.2006
B. Mojavensis
A21
Central composite
14 fold
Haddar et al.
2010
B. subtilis C4
Plackett-Burman
&Central composite
Box- Behenken
2.6 fold
Romsomsa et al.
2010
Design
Expert
(Stat- Ease)
3.2 fold
B. subtilis P13
The major drawback of the statistical approach is that there are no precise guidelines for
the sequence of experiments to be conducted and the level combinations of different independent variables for each experiment. The system of laying out the conditions of experiments involving multiple factors was rst proposed by Sir R.A. Fisher in 1920s, popularly termed as fractional design of experiments (McLean and Anderson, 1984). A full
fractional design identies all the possible combinations for a given set of factors (Sayyad
et al. 2007). Since most industrial experiments usually demand a signicant number of
factors, a full factorial design results in performing a large number of experiments. To
reduce the number of experiments to a practical level, only a small set from all the possibilities is selected. The method of selecting a limited number of experiments which generates the most information is known as a partial fractional design.
6.5 OTHER STRATEGIES
Several different approaches such as cellular metabolic regulation, use of different fermentation strategies (submerged, solid state and slurry) and selection of low cost suitable
nutrient components and their interaction during fermentation were observed as components to enhance the productivity with minimum nutrient input for several microbial
strains (Prakasham et al. 2006; Sreenivasrao et al. 2004). Literature reports denoted that
optimization of process related parameters (fermentation conditions, nutrient components and biosystem-mediated) led to enhance the productivity with free cell fermentations (Sreenivasrao et al. 2004; Prakasham et al. 2006).
72
REFERENCE
Adinarayana, K., Ellaiah, P., & Prasad, S.D. (2003). Purication and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtillis PE11. AAPS Pharma. SciTech., 4, 1-9.
Agarwal, D., Pankaj, P., Tushar, B., & Shridhar, P. (2004). Alkaline Protease Production
by a soil isolate of Beauveriafeina under SSF condition: parameter optimization and
application to soy protein hydrolysis. Process Biochemistry, 40, 1131-1136.
Aguilar, C.N., Gutierrez-Sancez, G., Rado-Barragan, P.A., Rodriguez-Herrera, R., Martinez-Hernandez, J.L., & Contreras-Esquivel, J.C. (2008). Perspectives of solid state fermentation for production of food enzymes. Am.J.Biochem. & Biotech., 4,354-366.
Aikat, K., & Bhattacharyya, B.C. (2000). Protease extraction in solid state fermentation
of wheat bran by a local strain of Rhizopusoryzae and growth studies by the soft gel technique. Process Biochem., 35,907914.
Akhnazarova, S., & Kafarov, V. (1982). Experiment optimization in chemistry and chemical engineering. Moscow Russia: Mir Publications.
Anwar, A., & Saleemuddin, M. (1998). Alkaline proteases: a review. Bioresource Technology, 64, 175.
Arunachalam, C., & sarita, k. (2009). Protease enzyme: an ecofriendly alternative for
leather industry. Ind .J.SCI. Technol., 2, 29-32.
Ashis, K., Mukherjee, Hemanta Adhikari., & Sudhir Rai, K. (2008).Production of alkaline protease by a thermophilic Bacilus Subtilis under Solid-State fermentation (SSF) condition using Imparata Cyrinafrica grass and potato peel as low cost medium: Characterization and application of enzyme in detergent formation. Biochemical Engineering Journal, 39, 353-361.
Asokan, S., & Jayanthi, C.I. (2010). Alkaline protease production by bacillus
licheniformis and Bacillus coagulans. Journal of Cell and Tissue Research ,10, 21192123.
Atalo, K., & Gashe, B.A. (1993). Protease production by a thermophillic Bacillus sp (P001A) which degrades various kinds of brous proteins. Biotechnol.Lett.,15, 1151-1156.
Banerjee, U.C., Sani, R.K., Azmi, W., & Soni, R. (1999). Thermostable alkaline protease
from Bacillus brevis and its characterization as a laundry detergent additive. Process Biochemistry, 35,213-218.
73
Banik, R.M., & Prakash, M. (2004). Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Microbiol. Res.,159,135-140.
Barrett, A.J., Rawlings, N.D., & Woessner, J.F. (1998).Handbook of Proteolytic
Enzymes. San Diego: Academic Press.
Barrios-Gonzalez, J., Fernandez, F.J., Tomasini, A., & Mejia, A. (2005). Secondary
metabolites production by solid state fermentation. Malys. J. microbial.,1,1-6.
Barthomeuf, C., Pourrat, H., & Pourrat, A. (1989).Properties of a new alkaline proteinase
from Aspergillus niger. Chem. Pharm Bull., 37, 1333-1336.
Beg, Q.K., Sahai, V., & Gupta, R. (2003). Statistical media optimization and alkaline protease production from Bacillus mojavensis in a bioreactor, Process Biochemistry, 39,
2003-2009.
Beg, Q.K., Saxena, R.K., & Gupta, R. (2002). Kinetic constants determination for an alkaline protease from Bacillus mojavensis using response surface methodology. Biotechnol.
Bioeng., 78, 289295.
Browner, M.F., Smith, W.W., & Castelhano, A.L. (1995). Matrilysin inhibitor complexes:
common themes among metalloproteinases. Biochem., 34, 6601- 6610.
Brutt, E.H., & Ichida, J.M. (1999). Keratinase produced by Bacillus licheniformis. US
patent,5,877,000.
Cerny, G. (1978). Studies on the aminopeptidase test for the distinction of gram negative
from gram-positive bacteria. Eur.J.Appl.Microbiol.Biotechnol., 5,113-122.
Chaplin, M., & Bucke, C. (1990). The large-scale use of enzymes in solution. In M. Chaplin & C. Bucke [Eds.] , Enzyme Technology: Cambridge, England: Cambridge University Press
Cheng, S.W., Hu, H.M., Shen, S.W., Takagi, H., Asano, M.T., & sai, Y.C. (1995). Production and characterization of keratinase of a feather degrading Bacillus licheniformis.
Biosci. Biotechnol. Biochem., 59, 2239-2243.
Chiplonkar, J.M., Gangodkar, S.V., Wagh, U.V., Ghadge, G.D., Rele, M.V., & Srinivas,
M.C. (1985). Applications of alkaline protease from Conidiobolus in animal cell culture.
Biotechnol.Lett., 7,665-668.
Chudasama, C.J., Jani, S.A., Jajda, A.M., & patel, H.N. (2010). Optimization and production of alkaline protease from Bacillus Thuringiensis c7. Journal of cell and tissue
research, 10, 2257-2262.
Clapes, P., Pera, E., &Torres, J.L. (1997).Peptide bond formation by the industrial protease, neutrase, in organic media. Biotechnol. Lett., 19, 1023 1026.
74
Cochran, W.G., & Cox, G.M. (1957).Experimental design (2nd Ed.). New York, USA:
John Wiley and Sons.
Cowan, D.A. (1994). Industrial Enzymes, In Biotechnology-The science and the business. In V. Moses & R.E. Cape [Eds.], (pp.326-328). Switzerland, Harwood Academic
Publishers.
Davis, P.J. (1964). Disc electrophoresis II. Methods and application to human serum protein. Ann. NY. Acad Sci., 121,404447.
Dayanandan, A. (2003). Application of an alkaline protease in leather processing: an
ecofriendly approach. J.clean product, 11,533-536.
Detoni, C.H., Richter, M.F., Chagas, J.R., Henriques, J.A.P., & Termignoni, C. (2002).
Purication and characterization of an alkaline serine endopeptidase from a feather
degrading Xanthomonas maltophila strain. Can. J. Microbiol., 48,342-348.
Eiler A., Linder, P., Smirnoff, P., Newton, S., & Harpaz, S. (1993). Comparative study of
photolytic enzymes in the digestive tracts of the European sea bass and hybrid striped
bass reared in freshwater. Comp.Biochem.Physiol., 106A, 627-634.
Enshasy, E.H., Abuol-Enein, A., Helmy, S., & Azaly, E. (2008). Optimization of the
industrial production of alkaline protease by Bacillus licheniformis in different production scales. Aust.J.Bas.Appl.Sci., 2,583-593.
Ferid, A., Ferid, L., & Marzoukinejib, M. (2008). Production of alkaline proteases by
Botrytis Cinera using economic raw materials Assay as biodetergent. Process Biochemistry, 43, 202- 208.
Ferrero, M.A., Castro, G.R., Abate, C.M., Baigori, M.D., & Sineriz, F.
(1996).Thermostable alkaline protease of Bacillus licheniformis MIR29: isolation, production and characterization. Appl. Microbiol Biotechnol., 45,327-332.
Frankena, J., Koningstein, G.M., VanVerseveld, H.W., & Stouthamer, A.H. (1986). Effect
of different limitations in chemostat cultures on growth and production of extracellular
protease by Bacillus licheniformis. Appl. Microbiol., 24,106-112.
Freddi, G., Mossotti, R., & Innocenti, R. (2003). Degumming of silk fabric with several
proteases.106,101-112.
Frikha, G.B., Kamoun, A.S., Fakhfakh, N., Haddar, A., Manni, L., & Narsi, M. (2005).
Production and purication of calcium dependent protease from Bacillus cereus BGI. J
Ind Microbiol Biotechnol., 32,186-94.
Fujiwara, N., Tsumiya, T., Katada, T., Hosobuchi, T., & Yamamoto, K. (1989). Continuous recovery of silver from used X-ray lms using a proteolytic enzyme. Process
Biochem., 24,155156.
75
77
Kaur, S., Vohra, R.M., Kapoor, M., Beg, Q.K., & Hoondal, G.S. (2001). Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp.
P2.World Journal of Microbiology and Biotechnology, 17,125 -130.
Keivan, B.M., Giti, E., & Iraj, N. (2009). Production of alkaline protease by Bacillus
cereus and Bacillus polymixa in new industrial culture mediums and its immobilization.
African journal of Microbiology Research, 3,491-497.
Kemel, J., Olfa, G-H., Hanen, B.A., Laila, M., Rym, A., & Moncef, N. (2011).Alkaline
protease from Bacillus licheniformis Mp1: purication, characterization and potential
application as a detergent additive and for shrimp waste deproteinization. Process Biochemistry, 46, 1248-1256.
Kennedy, M., & Krouse, D. (1999). Strategies for improving fermentation medium performance. Ind. Microbial Biotechnol., 23,456-475.
Kirk, O., Borchert, T.V., & Fuglsang, C.C. (2002). Industrial enzyme applications. Curr.
Opinions Biotechnol., 13,345-351.
Kole, M.M., Draper, I., & Gerson, D.F. (1998). Production of protease by Bacillus subtilis
using simultaneous control of glucose and ammonium concentrations. J. Chem. Technol.
Biotechnol.,41,197-206.
Kumar, C.G. (2002). Purication and characterization of a thermostable alkaline protease
from alkalophilic Bacillus pumilus. Letters. Applied Microbiology, 34, 13-17.
Kumar, C.G., & Takagi, H. (1999). Microbial alkaline proteases from a bioindustrial viewpoint. Biotechnol. Adv., 17,561594.
Kumar, C.G., Tiwari, M.P., & Jany, K.D. (1999). Novel alkaline serine proteases from
alkalophilic Bacillus sp. purication and some properties. Process Biochemistry, 34,
441-449.
Kuriyama, N., Kuriyama, H., Julin, C.M., Lamborn, K., & Israel, M.A. (2000). Pretreatment with protease is a useful experimental strategy for enhancing adenovirus mediated
cancer gene therapy. Hum Gene. Ther., 11, 2219-2230.
Larcher, G., Cimon, B., Symoens, F., Tronchin, G., Chabasse, D., & Bouchara J.P. (1996).
A 33 kDa serine proteinase from Scedosporium apiospermum. Biochem. J., 315,119-126.
Liang, T.W., Lin, J.J., Yen, Y.H., & Wang, S.L. (2006). Purication and characterization
of a protease extracellularly produced by Monasxus purpureus CCRC 31499 in a shrimp
and crab shell powder medium. Enz. Microbial Biotechnol., 38, 74-80.
Mahalakshmi, Y., Sathish, T., Subba Rao, C.H., & Prakasham, R.S. (2010).Corn husk as a
novel substrate for enhanced production of rifamycin-B by Isolated Nocardia sp. RSP 3.
Process Biochem., 45, 4753.
78
Mahalaxmi, Y., Sathish, T., & Prakasham, R.S. (2009). Development of balanced
medium composition for improved rifamycin B production by isolated Amycolatopsis sp.
RSP-3. Lett. Applie. Microbiol., 49,533538.
Malathi, S., & Chakraborty, R. (1991).Production of alkaline protease by a new
Aspergillus avus isolate under solid state fermentation conditions for use as a depilation
agent. Appl. Environ. Microbiol., 57,712-716.
Maurer, K. (2004). Detergent proteases. Cur. Opin. Biotechnol., 15,330334.
McLean, R.A., & Anderson, V.L. (1984). Applied Fractional Factorial Designs. Marcel
Dekker, New York
Mitchell, D.A., & Lonsane, B.K. (1993). Solid substrate cultivation. Elsevier, London,
U.K.
Moon, S.Y., Oh, T.K., & Rho, H.M. (1994). Purication and characterization of an extra
cellular alkaline protease from Bacillus subtilis RM 615. Korean Biochem. J., 27, 323329.
Morihara, K., Oka, T., & Tsuzuki, H. (1967). Alkaline proteolytic enzyme of
Streptomyces fradiae: selection, isolation and preliminary characterization, Biochem.
Biophys.Acta., 139,382-397.
Mukhopadhyay, R.P., & Chandra, A.L. (1992). Application of a Streptomyces in the
removal of waste keratinous materials: Indus Biotechnology (pp.595-597). New Delhi,
India: Oxford & IBH publishing Co.Pvt.Ltd.
Mukhter, H., & Ikramul, H. (2008). Production of alkaline protease by Bacillus subtilis
and its application as a depilating agent in leather preprocessing. Pak.j. Bot., 40, 16731679.
Myers, R.H., Montgomery, D.C. (1995). Response surface methodology: Process and
Product optimization using designed experiments, (1st Edition), Wiley- Interscience,
USA.
Naidu, K.S.B., & Devi, K.L. (2010). Optimization of thermostable alkaline protease production from species of Bacillus using rice bran. African journal of Biotechnology,4,724726.
Negi, S., & Banerjee, R. (2006). Optimization of amylase and protease production from
Aspergillus awamari in single bioreactor through EVOP factorial design technique. Food
Technol. Biotechnol., 44,257-261.
Nilegaonkar, S.S., Zambare, W.P., Kanekar, P.P., Dhakephalkar, P.K., & Sarnaik, S.S.
(2007). Production and partial characterization of dehairing protease from Bacillus
cereus MCM B-326. Bioresource Techno., 98, 1238-1245.
79
North, M.J. (1982). Comparative biochemistry of the proteinases of eukaryotic microorganisms. Microbiol. Rev., 46,308340
Oberoi, R., Beg, Q.K., Puri, S., Saxena, R.K., & Gupta R. (2001). Characterization and
wash performance analysis of an SDS-stable alkaline protease from a Bacillus sp. World
Journal of Microbiology and Biotechnology,17,493-497.
Pandey, A., Soccol, C.R., Nigam, P., Brand, D., Mohan, R., & Roussos, S. (2000). Biotechnological potential of coffee pulp and coffee husk for bioprocesses. Biochem. Eng.
J., 6,153-162.
Pillai, P., Sweta, M., & Archana, G. (2011). Statistical optimization of production and tannery applications of a keratinolytic serine protease from Bacillus subtilis p13. Process
Biochemistry, 46, 1110-1117.
Plackett, R.L., & Burman, J.P. (1946). The design of optimum multi factorial experiments. Biometrika., 37,305325.
Prakasham, R.S., Subbarao, C.H., & Sarma, P.N. (2006). Green gram husk: an inexpensive substrate for alkaline protease production by Bacillus sp. in solid-state fermentation.
Bioresource Technol.,97,14491454.
Puri, S., Beg, Q.K., & Gupta, R. (2002). Optimization of alkaline protease production
from Bacillus sp. using response surface methodology. Curr. Microbiol., 44,286290.
Rahman, R.N.Z.A., Razak, C.N., Ampon, K., Basri, M., Yunus, W.M.Z.W., & Salleh A.B.
(1994). Purication and characterization of a heat-stable alkaline protease from Bacillus
stearothermophilus F1. Applied Microbiology and Biotechnology, 40,822-827.
Ramakrishna, D.P.N. (2010).Purication and properties of an extracellular alkaline protease produced by Bacillus subtilis (MTCC NO. 10110). Int. J.Biotechnol. Biochem., 6,
1-6.
Ramana, M., Mohan, E.V.S., & Sadhukhan, A.K. (1999). Cyclosporine-A production by
Tolypocladiumin atum using solid waste fermentation. Process Biochem., 34,263-80.
Ramesh, M.V., & Lonsane, B.K. (1990).Critical importance of moisture content in alphaamylase production by Bacillus licheniformis M27 in solid state fermentation. Appl.
Microbiol. Biotechnol., 33,501505.
Rao, M.B. (1998). Molecular and biotechnological aspects of microbial proteases.
Microbial Mol. Biol. Rev., 62,597-635.
Rawlings, N.D., &Barrett A.J. (1993). Evolutionary families of peptidases. Biochem.J.,
290,205-218.
Riva, S., Chopineau, J., Kieboom, A.P.G., & Klibanov, A.M. (1988). Protease-catalyzed
regioselective esterication of sugars and related compounds in anhydrous
dimethylformamide. J .Am. Chem Soc., 110,584589.
80
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APPENDIX -I
ABBREVIATIONS
ANNs
ANOVA
Analysis of Variance
BSA
CCD
DFP
EDTA
FFCCD
GA
Genetic Algorithms
GRAS
HCN
Hydrogen Cyanide
IMTECH
kDa
Kilo Daltons
pCMB
PMSF
RPM
RSM
SDS-PAGE
SmF
Submerged Fermentation
SSF
TLCK
TPCK
YPD
83