INNOVATIVE FERMENTATION

STRATEGIES FOR PROTEOLYTIC

ENZYMES PRODUCTION

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

INNOVATIVE FERMENTATION STRATEGIES FOR

PROTEOLYTIC ENZYMES PRODUCTION

Authors : Dr. D. KEZIA

Dr. T . SATHISH
ISBN : 978- 1-63040- 518-2
Publisher : SARA BOOK PUBLICATION
303, Maharana Pratap Complex
B/H.V. S. Hospital
Paldi, Ahmedabad - 380006.
Phone: +91 8866003636, 9904000288
First Edition : 2015
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Price : 300/-

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

INTRODUCTION TO PROTEOLYTIC ENZYMES

07

CHAPTER-2

SCREENING OF PROTEASE PRODUCING

MICROORGANISMS

18

CHAPTER-3

ENRICHMENT OF PROTEASE PRODUCTION BY

PROGRESSIVE OPTIMIZATION METHODS

22

CHAPTER-4

ECONOMICALLY VIABLE PROTEASE

PRODUCTION BY SOLID STATE FERMENTATION
USING AGRO INDUSTRIAL WASTE MATERIALS
AND OPTIMIZATION OF PRODUCTION
CONDITIONS

35

CHAPTER-5

PURIFICATION AND EVALUATION OF

INDUSTRIAL APPLICATION OF PROTEOLYTIC
ENZYMES

53

CHAPTER-6

FERMENTATION STUDIES FOR IMPROVED

PRODUCT FORMATION

67

REFERENCE

73

APPENDIX -I

83

CHAPTER-1: INTRODUCTION TO PROTEOLYTIC

ENZYMES

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

1.2 SOURCES OF PROTEASES

Since proteases are physiologically necessary for living organisms, they are ubiquitous,
found in a wide diversity of sources such as plants, animals and microorganisms (Rao,
1998; Ward, 1985). Fortunately, enzymes can be separated from living cells and perform
catalysis independent of their physiological environment. Commercial proteases are
derived from animal tissues, plant cells and microbial cells via fermentation.
1.2.1 Plant Proteases
The use of plants as a source of proteases is governed by several factors such as the availability of land for cultivation and the suitability of climatic conditions for growth. Moreover, production of proteases from plants is a time-consuming process. (-amylase,
papain, bromelain, urease, cin, polyphenol oxidase (tyrosinase), lipoxygenase, etc.), represent some of the well-known proteases of plant origin (Rao,1998). Plant-derived products are perceived as safe and natural ingredients for use in the food applications and
may offer unique benets and functionality.
1.2.2 Animal Proteases
The most familiar proteases of animal origin are pancreatic trypsin, chymotrypsin, pepsin
and rennin. Trypsin is the main intestinal digestive enzyme responsible for the hydrolysis
of food proteins (Rao, 1998). Chymotrypsin is found in animal pancreatic extract. Pure
chymotrypsin is an expensive enzyme and is used only for diagnostic and analytical
applications (Rao, 1998). Pepsin is an acidic protease that is found in the stomach of
almost all vertebrates (Rao, 1998). Pepsin was used in laundry detergents as early as
1913, but is now being replaced by a mixture of serine and metal microbial proteases that
appear to be less degradable by soaps, alkaline conditions and high temperatures
(Adinarayana et al. 2003). Rennet is a pepsin-like protease that is produced as an inactive
precursor in the stomach of all nursing mammals. It is used extensively in the dairy industry to produce a stable curd with good avor (Rao, 1998).
1.2.3 Microbial Proteases
The inability of the plant and animal proteases to meet current world demands has led to
an increased interest in microbial proteases (Rao, 1998). Proteases of bacteria, fungi and
viruses are increasingly studied due to its importance and subsequent applications in
industry and biotechnology. Commercial application of microbial proteases is attractive
due to the relative ease of large-scale production as compared to proteases from plants
and animals. Microbial proteases account for approximately 40% of the total worldwide
enzyme sales. Proteases from microbial sources are preferred to the enzymes from plant
and animal sources since they posses almost all the characteristics desired for their biotechnological applications (Rao, 1998). In general microbial proteases are extracellular
in nature and are directly secreted into the fermentation broth by the producer, thus simplifying downstream processing of the enzyme as compared to proteases obtained from
plants and animals (Gupta et al. 2002a). Microbial proteases, especially from Bacillus sp.
have traditionally held the predominant share of the industrial enzyme market of the
worldwide enzyme sales with major application in detergent formulations and leather
industries (Beg et al. 2003; Negi & Banerjee, 2006; Vasudeo et al. 2011).

1.3 CLASSIFICATION OF PROTEASES

A number of microorganisms produce one or more types of protease enzymes with different pH optima for activity. Proteases are broadly classied as endo- or exoenzymes on
the basis of their site of action on protein substrates. They are also classied based on the
functional group at the active site, mechanism of action, pH optima, substrate specicity,
catalytic mechanism, 3-D structure and similarity in action to well characterized enzymes
like trypsin, chymotrypsin and elastase (Rawlings & Barret, 1993). Based on their amino
acid sequences, proteases are classied into different families and further classied into
clans to accommodate sets of peptidases that have diverged from a common ancestor
(Rawlings & Barrett, 1993). Exopeptidases act only near the ends of polypeptide chains,
further classied as amino-or carboxypeptidases based on their site of action at the N or C
terminus respectively. Aminopeptidases liberate a single amino acid residue, a dipeptide
(dipeptidyl peptidase) or a tripeptide (tripeptidyl peptidase). The substrate specicities of
the enzymes from bacteria and fungi are distinctly different and can be differentiated
based on the proles of the products of hydrolysis (Cerny,1978). Aminopeptidases may
be classied as aminopeptidase N or aminopeptidase A, depending on their preference for
neutral (uncharged) or acidic side chains, respectively. Most of the aminopeptidases fall
under metalloenzymes. Carboxypeptidases may be divided into three major groups,
serine carboxypeptidases, metallocarboxypeptidases and cysteine carboxypeptidases,
based on the nature of the amino acid residues at the active site of the enzymes. These
enzymes can also hydrolyze the peptides in which the peptidyl group is replaced by a
pteroyl moiety or by acyl groups. Other exopeptidases include dipeptidases, which cleave
a dipeptide and omega peptidases which release modied residues from N- or C- termini.
Endopeptidases are characterized by their preferential action at the peptide bonds in the
inner regions of the polypeptide chain away from the N or C termini. Based on the pH of
their optimal activity, they are also referred to as acidic, neutral, and alkaline proteases.
More conventionally, proteases are classied into four important groups according to the
Nomenclature Committee of the International Union of Biochemistry and Molecular
Biology, (International Union of Biochemistry, 1992) like serine proteases (EC 3.4.21),
cysteine proteases (EC 3.4.22), aspartate proteases (EC 3.4.23) and metallo proteases
(EC 3.4.24) (Kalisz, 1988; Rao,1998).
1.3.1 Serine proteases
Serine proteases are characterized by the presence of a serine group in active site. This
type of proteases hydrolyzes either esters or peptide bonds utilizing mechanisms of covalent catalysis and preferential binding in the transition state. They play an important role
in many processes, e.g. digestion of dietary protein, blood clotting cascade, and in several
pathways of differentiation and development. Based on their structural similarities, they
are grouped into 20 families, which are further subdivided into about six clans with common ancestors (Barett et al.1998). They are recognized by their irreversible inhibition by
3, 4-dichloroisocoumarin, Di-isopropyl uorophosphate (DFP), Phenyl methyl sulfonyl
uoride (PMSF) and Tosyl-Llysine chloromethyl ketone (TLCK). Serine proteases are
generally active at neutral and alkaline pH, with an optimum between pH 7 and 11. They
have broad substrate specicities including esterolytic and amidase activity. Their molecular masses range between 18 and 35 kDa. Their isoelectric points are generally between
pH 4 and 6. Serine alkaline proteases that are active at highly alkaline pH represents the
largest subgroup of serine proteases. Trypsin, chymotrypsin are the well-studied proteases of this sub-group.
9

1.3.2 Aspartic proteases

Aspartic proteases are commonly known as acid proteases and characterized by the
requirement of aspartic acid residues for their catalytic activity. The active-site aspartic
acid residue is situated within the motif Asp-Xaa-Gly in which Xaa can be Ser or Thr.
Most aspartic proteases show maximal activity at low pH (pH 3 to 4) and have isoelectric
points in the range of pH 3 to 4.5. Their molecular masses are in the range of 30 to 45kDa.
The members of the pepsin family have a bilobal structure with the active-site cleft
located between the lobes (Barrett et al. 1998). The aspartic acid proteases are inhibited
by pepstatin, leupeptin in the presence of copper ions. Microbial acid proteases exhibit
specicity against aromatic or bulky amino acid residues on both sides of the peptide
bond that is similar to pepsin. They are broadly divided into pepsin-like enzymes and
rennin like enzymes.
1.3.3 Cysteine proteases
The activity of all cysteine proteases depends on catalytic activity consisting of cysteine
and histidine. The order of Cys and His residues differ among the 20 families (Barrett et
al. 1998). Generally cysteine proteases are active in the presence of reducing agents such
as HCN or cysteine. Based on their side chain specicity, they are broadly divided into
four groups, (i) papain-like, (ii) trypsin-like with preference for cleavage at the arginine
residue, (iii) specic for glutamic acid, and (iv) others. Cysteine proteases have neutral
pH optima, although a few of them, e.g., lysosomal proteases, are maximally active at
acidic pH. They are susceptible to sulfhydryl agents such as pCMB but are unaffected by
DFP and metal chelating agents. Papain is the best-known cysteine protease.
1.3.4 Metalloproteases
These are the most diverse of the catalytic types of proteases characterized by the requirement of a divalent metal ion for their activity (Page, 1996). Out of 30 families of
metalloproteases, 17 are endopeptidases, 12 are exopeptidases and 1 (M3) belongs to both
endo-and exopeptidases. These proteases exhibit their activity in the neutral to alkaline
pH range. The neutral proteases show specicity for hydrophobic amino acids, while alkaline proteases possess a very broad specicity. All of them are inhibited by chelating
agents such as EDTA but not by sulfhydryl agents or DFP. Matrix metalloproteases play a
prominent role in the degradation of the extracellular matrix during tissue
morphogenesis, differentiation, and wound healing, and may be useful in the treatment of
cancer and arthritis (Browner et al. 1995). Thermolysin, collagenase and elastase are the
well-studied metalloproteases.
1.4 ALKALINE PROTEASES
Alkaline proteases (EC.3.4.21-24, 99) are active in a neutral to alkaline pH range. They
either have a serine centre (serine protease) or of metallo-type (metalloprotease) and
they are the most important group of enzymes exploited commercially (Gupta et al.
2002b). Alkaline proteases are most active at pH values of about pH 10. They are sensitive to DFP and a potato inhibitor but not to TLCK or tosyl-L-phenylalanine
chloromethyl ketone (TPCK). They are all specic against aromatic or hydrophobic
amino acid residues at the carboxyl side of the splitting point (Ward, 1985). These
enzymes also offer advantages over the use of conventional chemical catalysts for numerous reasons. For example they exhibit high catalytic activity, a high degree of substrate
specicity, can be produced in large amounts and are economically viable (Anwar &
Saleemuddin, 1998).
10

Alkaline proteases being a physiologically and commercially important group of

enzymes are used primarily as detergent additives. These enzymes have broad substrate
specicities and will function to some extent under extreme conditions encountered in
domestic washing temperatures of 20 to 70C, a pH up to 11 and at high concentrations of
detergents, polyphosphates, chelating agents such as EDTA and oxidizing agents such as
sodium perborate (Cowan, 1994; Subbarao et al. 2009). In recent years there has also
been a phenomenal increase in the use of alkaline protease as industrial catalysts. In
Japan, 1994 alkaline protease sales were estimated at $116 million. There is expected to
be an upward trend in the use of alkaline proteases so that by the turn of the decade the
total value for industrial enzymes is likely to reach $700 million or more (Kumar &
Takagi, 1999; Turk, 2006). Especially, alkaline proteases of microbial origin, which dominate the worldwide enzyme market, possess considerable industrial potential due to their
biochemical diversity and wide applications in tannery and food industries, medicinal formulations, detergents and processes like waste treatment, silver recovery and resolution
of amino acid mixtures (Agarawal et al. 2004; Gupta et al. 2002a; Rao, 1998).
Alkaline proteases are produced by a wide range of microorganisms including bacteria,
molds, yeasts and also mammalian tissues. Despite this interest in other microbial
sources, survey of the literature conclusively shows that bacteria are by far the most popular source of commercial alkaline proteases to date. Bacterial alkaline proteases are characterized by their high activity at alkaline pH, e.g., pH 10 and their broad substrate specicity. Their optimal temperature is around 60C. These properties of bacterial alkaline
proteases make them suitable for use in the detergent industry (Rao,1998). From all the
alkaliphilic bacteria that have been screened for use in various industrial applications,
members of the genus Bacillus, mainly the strains B. subtilis and B. licheniformis were
found to be predominant and a prolic source of alkaline proteases (Kumar & Takagi,
1999). Some industrially important alkaline proteases produced from various Bacillus sp.
are tabulated in Table 1.1.
Table 1.1: Some industrially important alkaline proteases produced from various
Bacillus sp. (Anwar & Saleemuddin, 1998)
Bacterial Species
PH
Industrial applications
optimum/stability
Bacillus stearothermophilus
Detergents and heavy duty
9.5
laundry powders
10.0-12.5
Detergent formulations
Bacillus sp. Y. (BYA)
Bacillus licheniformis
8.2
Catalyst for N-protected
amino acids
12.0-13.0
Dehairing/leather industry
Bacillus sp. (AH-101)
Detergent formulations
Bacillus sp. (Savinase/Durazym)
9.0-11.0
Bacillus rmus
8.0
Detergent industry
Bacillus sp. (P-001A)
9.5
Production of biomass from
natural waste
8.2
Synthesis of biologically
Bacillus licheniformis
active peptides
(Alcalase)
Bacillus subtilis
8.5
Bating agent in leather
industry
11

1.5 PROPERTIES OF ALKALINE PROTEASES

1.5.1 Optimum Temperature are Thermostability of Alkaline Proteases
The heat stability of enzymes is affected by at least two factors alone or in combination.
The rst one is the primary structure of the enzyme Secondly, specic components such
as polysaccharides and divalent cations. A wide range of microbial proteases from
thermophilic species has been extensively puried and characterized. These include
Thermus sp., Desulfurococcus strain Tok12S1 and Bacillus sp. Among them alkaline proteases derived from alkaliphilic bacilli, are known to be active and stable in highly alkaline conditions (Rahman et al. 1994). Further studies on microbial alkaline proteases have
been done in view of their structural-function relationship and industrial applications, as
they needed stable biocatalysts capable of withstanding various conditions of operation
(Rahmam et al. 1994). Generally alkaline proteases are produced from alkaliphilic
Bacillus are known to be active over a wide range of temperature. The optimum temperatures of alkaline proteases range from 40 to 80C.
1.5.2 Optimum pH of Alkaline Proteases
Enzymes are amphoteric molecules containing a large number of acid and basic groups,
mainly located on their surface. The charge on these groups will vary, according to their
acid dissociation constants, with the pH of their environment. This will affect the total net
charge of the enzymes and the distribution of charges on their exterior surfaces, in addition to the reactivity of the catalytically active groups. These effects are especially important in the neighborhood of the active sites, which will overall affect the activity, structural stability and solubility of the enzyme (Chaplin & Bucke, 1990). The pH optima of
some alkaline proteases produced from Bacillus sp. are given in Table 1. 2..
Table 1.2: pH optimum of various alkaline proteases produced from Bacillus sp.
Bacterial Species
Bacillus stearothermophilus

12

PH
optimum/stability
9.5

Industrial applications
Detergents and heavy duty
laundry powders

Bacillus sp. Y. (BYA)

10.0-12.5

Bacillus licheniformis

8.2

Bacillus sp. (AH-101)

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

Bacillus sp. (P-001A)

9.5

Production of biomass from

natural waste

Bacillus licheniformis
(Alcalase)

8.2

Synthesis of biologically active

peptides

Bacillus subtilis

8.5

Bating agent in leather industry

1.5.3 The Isoelectric Point

The pH referred as isoelectric point (pI) at which the net charge on the molecule is zero, is
a characteristic of each enzyme, where solubility in aqueous solutions is generally minimum. Isoelectric pH values of various alkaline proteases produced from Bacillus sp are
given in Table 1.3.
Table 1.3: Isoelectric pH values of various alkaline proteases produced from
Bacillus sp.microorganism pI Reference
Microorganism
B. pumilus UN-31-C-42
Bacillus sp. PS719

pI
9.0
4.8

Reference
Huang et al. 2003
Hutadilok-Towatana et al.1999

1.5.4 The Molecular Weight

The molecular weights of alkaline proteases generally range from 15 to 30 kDa (Kumar &
Takagi, 1999) with few reports of higher molecular weights of 32.0 kDa (Huang et al.
2003), 33.5 kDa (Rahman et al. 1994). These are tabulated in Table 1.4
Table 1.4: Molecular weights of various alkaline proteases produced from
Bacillus sp.
Source
B. pumilus UN-31-C-42
B. pumilus MK6-5
Bacillus licheniformis MIR 29
Bacillus sp. No. AH-101
B. pumilus MK6-5
B. pumilus UN-31-C-42
B. stearothermophilus F1
Bacillus licheniformis MIR29
Bacillus mojavensis

Molecular weight
Method
Reference
(kDa)
32
SDS-PAGE Huang et al. 2003
28
25/40
30
28
32
33.5
25/40
30

SDS-PAGE Kumar, 2002

SDS-PAGE
SDS-PAGE
SDS-PAGE
SDS-PAGE
SDS
SDS-PAGE
SDS-PAGE

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

1.5.5 Metal Ion Requirement and Inhibitors of Alkaline Proteases

Alkaline proteases require a divalent cation like Ca2+, Mg2+ and Mn2+ or a combination of
these cations, for maximum activity. These cations were also found to enhance the thermal stability of a Bacillus alkaline protease. It is believed that these cations protect the
enzyme against thermal denaturation and play a vital role in maintaining the active conformation of the enzyme at high temperatures (Kumar & Takagi, 1999). Inhibition studies
give insight into the nature of the enzyme, its cofactor requirements, and the nature of the
active site. Alkaline proteases are completely inhibited by phenylmethylsulfonyl uoride
(PMSF) and diisopropyl uorophosphates (DFP). In this regard, PMSF sulfonates the
essential serine residue in the active site, results in the complete loss of activity. This inhibition prole classies these proteases as serine hydrolases. In addition, some of the alkaline proteases were found to be metal ion dependent in view of their sensitivity to metal
chelating agents, such as EDTA. Thiol inhibitors have little effect on alkaline proteases of
13

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

CHAPTER -2: SCREENING OF PROTEASE

PRODUCING MICROORGANISMS

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

2.1.4 Biochemical and phenotypic characterization of Protease Producing Isolate

Morphological studies were conducted under scanning electron microscope (model:
JOEL-JSM 5600). Conventional physiological and biochemical characterization tests
were carried out at Institute of Microbial Technology (IMTECH) Chandigarh, India.
2.1.5 Molecular characterization of the isolate
2.1.5.1Amplication and Sequencing of 16S rRNA Gene
The 16S rRNA gene was PCR amplied according to the (Sathish & Prakasham, 2010)
The DNAs program was used for connecting the sequence of fragments and the BLASTN
program was used for a gene homology search with standard defaults. The nuclear
sequence of the 16S rRNA gene for the strain was deposited in the GenBank database
2.1.5.2 Phylogenetic Analysis of the Strain
The 16S rRNA gene sequence of the isolated strain was used as a query to search for
homologous sequence in the nucleotide sequence databases by running BLASTN
program. The identied sequences were aligned using CLUSTAL-W algorithm in
MEGA 4.0 software. Phylogenetic trees were inferred by using the neighbor-joining
bootstrap analysis with the help of MEGA 4.0 software package based on 1000
resampling.
2.1.6 Analytical Methods
Protease activity was determined using modied Auson-Hagihara method (Hagihara et
al.1958). The protein content of the enzyme preparations was estimated by Lowry
method using Bovine serum albumin as standard (Lowry,1951). The absorbance of the
medium was noted at regular intervals at 600nm. From the absorbance the dry weight was
calculated by using the standard curve of absorbance vs. dry weight.
2.2 RESULTS AND DISCUSSION
2.2.1 Enrichment and isolation of microorganisms
Soil samples from different areas which were collected, are used for isolation of bacterial
strains producing protease enzyme. 1g of soil was suspended in sterile distilled water and
mixed thoroughly for 1h at room temperature before using for isolation studies. 1 %
casein-agar plates were prepared and incubated at 4oC for 15 min to solidify the agar
solution. The plates were brought to room temperature and spread with 0.1 ml of soil
solution under sterile conditions. These plates were incubated at 37oC in an incubator.
After 24h of incubation, the plates were checked for microbial colonies with clear zone of
hydrolysis.
2.2.2 Screening of protease positive cultures by casein-agar plate method
More than 70 microbial strains from different plates were selected and grown in the
casein containing agar slants. Each isolate was further conrmed for its production
pattern using 1 % casein agar plates. Depending on the zone of clearance, 20 strains were
further screened. Further, to evaluate the potential of these isolates for production of
protease enzyme, fermentation studies were performed using YPD medium. The
quantitative estimation of produced protease revealed that among 20 isolates, DKMNR
exhibited the highest proteolytic activity with a clear zone diameter of 25 mm (Fig. 2.1).
Based on the zone diameter and broth studies DKMNR was selected for further studies.
From gure 2.1 it was observed that DKMNR is grown on the casein agar plate and also
observed a clear hydrolysis zone on the plate.
19

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

2.2.4 Molecular characterization of isolate DKMNR (16S Ribotyping)

The gene sequence revealed that it contains 1536 base pairs consisting of Adenine 24.7%, Cytosine - 23.6%, Guanine - 31.3% and Thymine - 20.4%; with AT:CG ratio of
45:55. Blast analysis denoted 99% similarity to the B. subtilis family. Phylogenetic tree
was constructed by taking the sequences obtained in the blast search, using Bacillus
cereus CYPPB-1 (FN430421) as an outer group. Figure 2.3 shows the phylogentic tree
from this it was observed that the isolate DKMNR belongs to the Bacillus subtilis family.
The partial sequence of 16s rRNA gene was submitted to the GenBank database and can
be accessed under Genbank accession number FR717670. The culture was deposited in
MTCC which was designated as Bacillus subtilis DKMNR 10551.

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

CHAPTER-3: ENRICHMENT OF PROTEASE

PRODUCTION BY PROGRESSIVE OPTIMIZATION
METHODS
3.0 INTRODUCTION
The microorganisms from diverse environments were considered as an attractive source
for protease as they can be cultured in large quantities in fewer periods. Furthermore,
microbial protease is extracellular which simplies the downstream processing and have
longer self-time (Gupta et al. 2002a). As a rule, the wild strains usually produce limited
qualities of the desired enzyme to be useful for commercial applications (Ganesh et al.
2008). Some extracellular enzymes are used in the food, dairy, detergent, pharmaceutical,
and textile industries and are produced in large amounts by microbial synthesis.
The amounts of protease produced by the microorganisms vary greatly with strain and
the media used. Thirty to forty percent of the production cost of industrial enzymes is estimated to be the cost of the growth medium (Joo et al. 2002). In order to obtain high and
commercial viable yields of protease it is essential to optimize fermentation media for the
growth of biomass and production of protease (Ghorbel et al. 2003). Optimization of
medium components was done to maintain a balance between the various medium components, thus minimizing the amount of unutilized components at the end of fermentation. Research effort is mainly to evaluate the effect of various carbon and nitrogenous
nutrients as cost-effective substrates on the yield of enzymes. So far no dened medium
has been established for the best production of alkaline proteases from different microbial
sources. Each organism has its own special conditions for maximum enzyme production.
So, it is important to know the suitable nutrients and cultural conditions required to
achieve higher productivity (Subbarao et al. 2008).
It was planned to formulate a suitable production medium for alkaline protease production from isolated Bacillus subtilis DKMNR by optimizing the effect of various cultural
and environmental factors on alkaline protease production with the help of advanced statistical methods.
3.1 MATERIALS AND METHODS
3.1.1 Microorganism
A strain of Bacillus subtilis DKMNR was used in the present study. This culture was
maintained on agar based YPD medium slants at 40C and sub cultured at monthly interval.
3.1.2 Medium for protease production by submerged fermentation
Yeast extract-Peptone-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 8.0 using 0.1N HCl or 0.1N NaOH solution.
3.1.3 Shake ask fermentation
A loop full culture from the (Bacillus subtilis DKMNR) slant was transferred aseptically
into 250 ml Erlenmeyer asks containing 50 ml of sterile production medium the produc22

tion media was composed of (YPD) yeast extract-peptone-dextrose 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 and pH 8 after inoculation with 1% (v/v) culture media, incubated at 300C on
rotary shaker at 120 Rpm. Initially the fermentation was carried out for 24 hrs, the culture
media was separated by centrifugation and the supernatant was used for assaying enzyme
activity
3.1.4 Estimation of protease Activity
The protease was assayed according to the method of modied Auson-Hagihara method
(Hagihara et al.1958). One unit of alkaline protease activity was dened as 1 ug of tyrosine liberated ml -1 under the assay conditions.
3.1.5 Screening of nutrients for the production of alkaline protease from Bacillus
Subtilis DKMNR using Plackett-Burman design
In order to select the carbon and nitrogen sources for enhancement of the protease production was carried out by employing the PB design. Table 3.1 indicates the selected variables and their levels. The experimental plan was shown in the Table 3. 2. Analysis of the
experimental results was performed based on the effect of each variable. The effect of
each selected variable on protease production was determined using the following equation 3.1.

Where; E (xi) = the concentration effect of the tested variable.

Yi+ and Yi- = the protease production from the trials where the variable (xi) was measured
at high and low concentrations, respectively and N = the number of trials.
Table 3.1: Selected variables for Planckett-Burman design.
S. No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

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

Response surface methodology consists of a group of empirical techniques devoted to the

evaluation of relations existing between a cluster of controlled experimental factors and
the measured responses, according to one or more selected criteria. A prior knowledge
and understanding of the process and the process variables are necessary for achieving a
realistic model. The factors such as pH, incubation temperature, agitation speed, size of
inoculum and concentrations of carbon (glucose) and nitrogen (peptone) supplements
were the major variables effecting the protease production. Thus, these variables were
selected to nd the optimized conditions for higher protease production using Central
Composite Design (CCD).
The range and the levels of the experimental variables investigated in this study are given
in the Table 3.3. The central values (zero level) chosen for experiment design were glucose 15 gl-1, peptone 10 gl-1, pH 9.0, temperature 33C, agitation 200 rpm and inoculum
size 2%, were selected and each of the variables were coded at ve levels 2,
1, 0, 1, and 2 by using Equation 3.2.
xi = (Xi X0)/DXi -------------------------------------------

(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

-126.250 -63.125 63756

X3

237.500 118.750 225625 1 225625.0 70.5254 8.3979 0.001100

X4

300.000 150.000 360000 1 360000.0 112.5281 10.6079 0.000447

X5

-96.250 -48.125 37056

1 37056.3 11.5830 -3.4034 0.027193

X6

-10.000

-5.000

400

400.0

0.1250 -0.3536 0.741490

X7

-43.000 -21.500

7396

7396.0

2.3118 -1.5205 0.203031

10

X8

-350.500 -175.250 491401 1 491401.0 153.6012 -12.3936 0.000244

11

X9

130.500

68121

1 68121.0 21.2931 4.6144 0.009922

12

X10

-60.250 -30.125 14520

1 14520.3 4.5387 -2.1304 0.100160

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

-147.750 -73.875 87320

1 87320.3 27.2944 -5.2244 0.006408

16
17

X14
X15

35.250 17.625 4970

-127.250 -63.625 64770

1 4970.3 1.5536 1.2464 0.280616

1 64770.3 20.2458 -4.4995 0.010826

18

Error

12797

19 Total SS

65.250

4970

4970.3

1.5536 -1.2464 0.280616

1 63756.3 19.9288 -4.4642 0.011125

3199.2

1447329 19
27

Fig. 3.2: The probability plot of effects on protease production.

Based on the above inference for further studies the peptone and glucose concentrations
were selected to optimize the higher production of protease. Nitrogen source plays a
major role on protease secretion. Peptone and yeast extract enhances the protease production by Bacillus sp (Subbarao et al. 2008). Similarly yeast extract and peptone combination enhanced the enzyme production in the fungus (Subbarao et al. 2008). However the
role of urea on production of protease by B. Subtilis DKMNR differs from the usage of
Peptone and yeast extract. The authors observed the inhibition of protease with addition
of urea.
3.2.2 Optimization by Response surface Methodology (RSM):
The RSM is an effective and sequential and stepwise procedure the lead objective of the
RSM was to run rapidly and efciently along the path of improvement towards the general vicinity of the optimum. It is appropriate when the optimal region for running the process was identied before performing RSM experiments. The inuence of different fermentation parameters such as carbon source (glucose), nitrogen source (peptone),
medium pH, incubation temperature, agitation speed and size of inoculum were optimized by central composite design. Table 3.5 depicts the FFCCD experimental design layout and experimental results. A predicted value for each performed experiment was calculated and the correlation between experimental and predicted values is shown in
Fig.3.3.
28

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

Fig. 3.3: Correlation between the observed and predicted values.

The protease experimental data was analyzed by applying multiple regression and the
results of the FFCCD design were tted with a second order full polynomial equation.
The empirical relationship between protease production (Y) and the 6 test variables in
coded units obtained by the application of RSM is given by Equation 3.5.
Y = 1737.32 - 67.1 x1 - 49.25 x2 + 111.7 x3 + 5.15 x4 + 39 x5 + 37.55 x6 - 68.95 x1*x1 +
10.94 x1*x2 - 25.62 x1*x3 + 92.19 x1*x4 - 46.62 x1*x5 + 17.37 x1*x6 - 106.2 x2*x2 - 19.06
x2*x3 - 61.62 x2*x4 + 12.19 x2*x5 + 65.94 x2*x6 - 108.95 x3*x3 - 73.69 x3*x4 - 0.25 x3*x5
+ 31.62 x3*x6 - 107.95 x4*x4 - 30.69 x4*x5 - 23.81 x4*x6 - 125.5 x5*x5+ 43.12 x5*x6 123.57 x6*x6
--------------------------------------- (3.5)
Where Y is the alkaline protease production in U ml-1 was response and x1 - x6 were the
coded values of the test variables as per the Table 3.3.
The ANOVA was conducted for the second order response surface model. The signicance of each coefcient was determined by Student's t-test and p-values, which were
listed in Table 3.6. The larger the magnitude of the t-value and smaller the p-value, the
more signicant is the corresponding coefcient. (Cochran & Cox, 1957).

31

Table 3.6: ANOVA & Regression coefcients

Coefcients

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

1 21012.5 2.52972 0.12599

X1*X4

92.188

X1*X5

-46.625

-93.25

69565

1 69564.5 8.37497 0.00842

X1*X6

17.375

34.75

9660

X2*X3

-19.063

-38.125

11628

1 11628.1 1.39993 0.24937

X2*X4

-61.625

-123.25

121525 1

p-value

X2*X5

12.188

24.375

X2*X6

1061

56400

1 97022.5 11.6807 0.00247

1E-06
0

1 56400.1 6.79009 0.01614

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

1 32004.5 3.85307 0.06242

X4*X5

-30.688

-61.375

30135

1 30135.1 3.62801 0.06997

X4*X6
X5*X6

-23.813
43.125

-47.625
86.25

18145
59513

1 18145.1 2.18452 0.15358

1 59512.5 7.16479 0.01378

Error
Total SS

4753

173755 1
1

182737 22 8306.2
4299893 49

SS = Sum of squires; df = degree of freedom; MS = Mean squire

32

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.

Fig. 3.4 (a-f): Interaction inuence of selected parameters

34

CHAPTER-4: ECONOMICALLY VIABLE PROTEASE

PRODUCTION BY SOLID STATE FERMENTATION
USING AGRO INDUSTRIAL WASTE MATERIALS
AND OPTIMIZATION OF PRODUCTION
CONDITIONS
4.0 INTRODUCTION
Most of the microbial products at industrial scale are generally produced using submerged fermentation due to its apparent advantages in consistent enzyme production characteristics with dened medium and process conditions. Further, it has advantages in
downstream processing in spite of the cost-intensiveness for medium components
(Subbarao et al. 2009; Sathish & Prakasham, 2010; Mahalaxmi et al. 2009). However,
solid-state fermentation has gained renewed interest and fresh attention from researchers
because of its edge in biomass energy conservation, solid waste treatment and its application to produce secondary metabolites over submerged fermentation (Mahalakshmi et al.
2010; Hymavathi et al. 2009). Production of biocatalysts using agro-biotech substrates
under solid-state fermentation conditions provide several advantages in productivity,
cost-effectiveness in labour, time and medium components further the efuent production is less and thus it is eco-friendly (Mahalakshmi et al. 2010; Hymavathi et al. 2009).
However, these production characteristics have to offer a competitive advantage over
existing products.
There are a large number of techniques available to design culture media. They can vary
from the traditional one-variable at- a-time method to more complex statistical and mathematical techniques (Subbarao et al. 2009), involving experimental designs such as full
and partial factorials, Plackett-Burman design, followed by optimization techniques such
as response surface methodology (RSM) (Hymavathi et al. 2009), articial neural networks (ANNs) (Subbarao et al. 2008), fuzzy logic and genetic algorithms (GA)
(Subbarao et al. 2008), among others. Regrettably, no multipurpose technique is known
to be applicable to all situations. Mixture designs were found to be effective tool for
screening and evaluation of the various solid substrates. In a mixture experiment, the
independent factors are proportions of different components of a blend. The interpretation of data in mixture experiments where the components represent proportionate
amounts of the factors differs from classical factorial experiments where the response varies depending on the amounts of each input variable. The key to mixture experiments is
that the mixture components are subject to a constraint requiring that the proportions sum
to one. In mixture experiments, the measured response is assumed to depend only on the
relative proportions of the ingredients or components in the mixture, and not on the
amount of the mixture. However, one can overcome this limitation by adding the amount
of mixture as an additional factor in the experiment, thereby allowing mixture and process variables to being treated together. The advantage of mixture experiments over factorial design is that one can more efciently study the interaction inuence amongst factors on the production, and subsequently eliminate both neutral and negative factors.
Mixture experiments have been the subject of many studies and have enjoyed extensive
application in pharmaceuticals, geology, petroleum, food, and tobacco industries.
35

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

Solid substrate used

Protease production
(Ugds1 material)

References

Bacillus sp ecies

Green gram husk

35,000

Prakasham ,2006

Bacillus species JB99

Pigeon pea

12,430

Johnvesly et al. 2002

Bacillus species

Wheat bran

429

Ramesh & Lonsane, 1990

Bacillus species

Lentil husk

168

Ramesh & Lonsane, 1990

Rhizopus oryzae

Wheat bran

358

Tunga et al. 2001

Rhizopus oryzae

Wheat bran

58.7

Aikat & Bhattacharyya, 2002

4.2.2 Mixture designs for substrate optimization

An augmented simplex lattice design was employed for the present investigation. In
order to observe the mixed substrate effect on protease production, three solid substrates
soybean meal (SM), green gram husk (GH) and bengal gram husk (BGH) were used in
these designs, the total proportions of the different substrates made to 100% i.e., 5g material. The design consists of total 10 runs where three with pure mixtures (one for each component), other three with binary blends for each possible two-component blend, another
three with complete blends (all three components are included but not in equal proportions) and last one as centroid (where equal proportions of all three components are
included in this blend). Table 4.2 shows all the experimental runs with the composition of
substrate as per mixture design model and the protease output values. Assuming that, the
measured response of protease production was dependent on the relative proportions of
the components in the mixture, linear through to cubic models were used for analysis of
the mixture design using following equations.
-------Linear model (Eq. 4.3)

----- Quadratic model (Eq. 4.4)

------Special cubic (Eq. 4.5)

---Full cubic (Eq. 4.6)

Where Y is a response, i is a linear, ij is a quadratic and ijk cubic coefcients, ij is a
parameter of the model. The ixi represents linear blending portion and the parameters ij
represents either synergic or antagonistic blending.
Table 4.2 presents the varying concentrations of solid substrate used during fermentation
process and the protease production data for each experiment. The protease production
values varied from 685 to 1045 Ugds1. This variation of protease yield under similar fermentation conditions but with different substrates suggested the importance of substrate
composition on fermentative protease production. Analysis of individual substrate
impact on protease production pattern indicated that green gram husk is the best substrate
with 53% higher yield compared to other two selected materials. Further analysis of the
data Table 4.2 revealed that mixed substrate improved protease yield. This can be evidenced based on higher protease yield from experiment 5 compared to 1 and 2 (Table 4.2).
However, presence of GH in mixed substrate fermentations, supported better production
compared to SM and BGH which can be evidenced from experiments 4,6, 7 and 9 where a
higher protease production was observed than individual or in other combination substrates as sole substratum. These results suggest that green gram husk is playing the vital
39

role in the mixed substrate fermentation.

In view of the variation in protease yield with different substrates and its production by fermentation is associated with availability of nitrogen source, the nutrient release pattern
during sterilization of substrate material was investigated by extracting with known
amount of distilled water and their concentration was analyzed. It was noticed that more
nutrients were released to fermentation medium by the GH was observed compared to
BGH and SM denoting that the higher protease yield by GH may be correlated with availability of nutrients in the composition of substrate.
Table 4.2 Mixed design experimental layout and protease production.
S. No SM (g) GH (g) BGH (g)

Protease activity (U/gds)

Coded

Real

Coded Real Coded Real Observed Predicted Error

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

4138.62 23015.39 22.2445 0.005882 0.9588

0.9074

73184.79 13711.06

Special Cubic 4088.73

Cubic

Table 4.3: ANOVA.

F-Value p-Value
MS

49.89

1609.46 1239.64

1.3114

0.0366 0.860492 0.9593

0.8780

0.7702 0.627401 0.9840

0.8560

MS= Mean square; SS= Sum of square

In spite of the greater R2 values than the quadratic model they have smaller F-value (F special cubic = 0.0366 & F cubic = 0.7702) and higher p-values (P special cubic =0.8605 & P
cubic = 0.6274). Such data suggests that the quadratic model is the most signicant.
Therefore further data analysis was performed using only quadratic model. The noticed
R2 value of the quadratic model is 0.9588 indicating that 3 substrate components altogether would explain about 95.88% of the variability in the response leaving only 4.12%
of the variability remaining unexplained. In the present study, a good co-relation was identied between predicted and experimental protease production with a variation of 5.8 %
.The empirical relationship between protease production (Y) and substrate variables in
coded units is obtained by the application of second order model as per Eq.4.7.
Y = 685.75 SM + 910.38 GH + 808.84 BGH + 684.4 SM GH + 809.32
SM BGH + 506.6 GH BGH
----------------- (Eq. 4.7)
Where Y is the response of protease production in Ugds1 and GH, SM and BGH were the
substrates with the respective coded experimental values testing the experiments as per
the Table 4.2. The signicance of each coefcient in Eq. 4.7 was determined by Student's
t-test and p-values and listed in Table 4.4. The larger magnitude of the t-value and smaller
p-value denote the corresponding coefcient signicance. The observed lower p-value
(<0.05) in the present experiment suggested that all linear and interactive terms were signicant. For the substrates, GH has one the highest t-test value with a magnitude of
910.38 and the p-value (8 10-6) was one of the least which indicates that the largest inuence in the mixture for protease production. Even BGH has highest magnitude of 808.84
and p-value of 1310-5. The interaction of GH with BGH indicated the lowest magnitude
(3.54) and higher p-value (23.910-3). Similarly, the interaction of SM with the BGH has
the highest magnitude (809.32) and lowest p-value (4.810-3) suggesting a large inuence
on protease production which can be seen from the 5th experiment in Table 4.2.
Table 4.4: Quadratic model terms

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.

Fig. 4.2: Triaxial diagrams of protease production as a function of substrate concentration.

The role of each substrate material was optimized for the production of protease in mixture design fermentation using natural agro-wastes such as SM, BGH and GH. Among all
materials, GH presence is essential and SM is the least important component for maximizing protease production among selected substrates. An optimum protease production
of 1045 Ugds1 could be obtained with a 44: 31: 25 ratio of GH: BGH: SM respectively
without any pretreatment of the material. Further work was preceded with respect to optimization of the fermentation parameters to increase the protease production. The parameters such as pH, moisture content, inoculum concentration, incubation time, and incubation temperature, concentration of the optimized carbon and nitrogen sources from the
selected sources were investigated. Keeping the potentiality of this microbial strain in protease production further evaluation was continued using the mixture of soybean meal,
bengal gram husk and green gram husk as solid support/substrate for solid state fermentation.
4.2.3 Role of pH on protease production
Enzyme production by microbial strains strongly depends on the extracellular pH
because culture pH strongly inuences many enzymatic processes and transport of various components across the cell membranes which in turn support the cell growth and product production (Subbarao et al. 2008), pH dependent alkaline protease production studies
by B. subtilis DKMNR in solid state fermentation using the mixture of SM, BGH and GH
42

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.

Fig. 4.3: Effect of pH on the production of protease by isolated B. subtilis

DKMNR.

43

4.2.4 Role of moisture content on protease production

Among the several factors that are important for microbial growth and enzyme production under solid-state fermentation using a particular substrate, moisture content and
water activity was one of the most critical factors (Sathish et al. 2008). Solid-state fermentation processes are different from submerged fermentation culturing, since microbial growth and product formation occurs at or near the surface of the solid substrate particle having low moisture contents (Pandey et al. 2000). Thus, it is crucial to provide optimized water level that controls the water activity (aw) of the fermenting substrate for
achieving maximum product production. Reports on enzyme production by microbial
species under solid-state fermentation indicated that the availability of water in lower or
higher concentrations affected microbial activity adversely (Ramesh & Lonsane, 1990).
Moreover, water is known to have profound impact on the physico-chemical properties of
the solids and this, in turn, affects the overall process productivity (Pandey et al. 2000).
The data indicated that, characteristic nature of enzyme production along with studied
moisture level and moisture content played a critical role in alkaline protease production
in B. subtilis DKMNR. Maximum enzyme production was observed with 120% moisture
content, which was noticed as 1362 Ugds1 matrix biomass (Fig 4.4). Linearity between
moisture content and enzyme production was observed up to 120 % and thereafter further
increase in moisture level in the fermentation medium resulted in reduction of protease
production. The percent reduction in enzyme production from either side of the optimum
moisture level (120 %) varied (Fig. 4.4). This was evidenced from the fact that, an
increase in 20 % moisture level reduced the production to the tune of only 17 % to that of
optimum production while decrease in same quantity of moisture level caused 30 %
reduction indicating the severity of damage to cell metabolism and subsequent enzyme
production will be more with low water activity in solid state medium than higher water
level to that of critical requirement. The decrease in production with increase in moisture
level in the solid medium might be attributed to decrease in mass transfer of heat and
gases caused by water logging among the inter-particulate area which in turn adversely
affects cellular and biosynthetic activities associated with microbial growth (Mitchell
and Lonsane, 1993). The observed reduction of enzyme production at reduced moisture
level might be associated with reduced availability of water content for microbial growth.
The results further indicated that, moisture content during solid-state fermentation played
a major role in regulating alkaline protease production in the isolated B. subtilis
DKMNR. Though the pattern of protease production with the function of moisture level
in the bacterial strain and comparison of protease production values of strains reported in
the literature were observed to be similar. However, optimum requirement of moisture
content during solid state fermentation process varied with the type of organism and agro
industrial material (Prakasham et al. 2006).

44

Fig. 4.4: Effect of moisture content on the production of protease by B. subtilis

DKMNR.
4.2.5 Role of inoculum concentration on protease production
Initial inoculum level inuenced the cellular metabolic activity, growth of the microorganism and metabolite production (Gupta et al. 2002c). The role of initial inoculum concentration on alkaline protease production under solid-state fermentation environment
with soybean meal, bengal gram husk and green gram husk as medium was investigated
to determine the optimum inoculum requirement. Inoculum level selected for this study
ranged from 0.5 % to 3.5 % from 24 hr grown bacterial cell suspension having an
absorbance of 0.8 at 600 nm.
Alkaline protease production by B. subtilis DKMNR strain under solid-state fermentation conditions varied with initial inoculum level and showed parabolic nature in the studied inoculum range (Fig.5.5). Maximum protease synthesis (1391 Ugds1) was noticed in
2% inoculum supplemented fermentation conditions. The reduction pattern of enzyme
production with increase or decrease in inoculum supplementation from the optimum
level also showed difference. Increase of inoculum level from 2 to 3.5% adversely caused
33 and 48% reduction in the enzyme production. Decrease of the same from 2% to 1.5 and
1% resulted in 36% and 69% reduced protease production, respectively depicting the
importance of inoculum level optimization for efcient protease production under solidstate fermentation conditions.

45

Fig. 4.5: Effect of inoculum concentration on the production of protease by B.

subtilis DKMNR.
4.2.6 Role of incubation temperature on protease production
Temperature is another critical parameter that has to be controlled and varied from organism to organism. The mechanism of temperature control of enzyme production is not well
understood (Chiplonkar et al. 1985). However, studies by (Mahalakshmi et al. 2009)
showed that a link existed between enzyme synthesis and energy metabolism in Bacilli,
which was controlled by temperature and oxygen uptake. The optimum temperature values reported for maximum protease production are given in the Figure 4.6.
Alkaline protease production by B. subtilis DKMNR in solid state fermentation was
increased by optimizing the temperature of the environment. Maximum protease production was 1505 Ugds1 at 32 C. The incubation temperature showed parabolic nature in this
study (Fig.4.6). The increase and decrease of temperature showed a lot of difference in the
protease production. An increment of 4C in the temperature reduced the enzyme production to 39% and decrease of 4C in the incubation temperature also decreased the protease
production to 30%. Therefore, the shifting of temperature from 26 to 40C has enhanced
the protease production in the fermentation media to two-fold when compared to the control experiment.

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

Alkaline protease production data in different inorganic nitrogen sources supplemented

condition indicated that enzyme production was having negative inuence in the presence of ammonium-based and nitrate-based nitrogen sources. However, very little impact
was observed when compared to control (Fig.4.9). Sinha and Satyanarayana, (1991),
noticed ammonium nitrogen associated regulation in protease production in thermophilic
Bacillus licheniformis. These observations clearly suggested that complex nitrogen compounds had an edge over inorganic nitrogen sources in alkaline protease production. This
might be due to the lowering of pH, by ammonium ion caused reduction in the enzyme
yields.
However, genetic nature of microbial species especially with respect to ammonium ion
concentration in the medium and enzyme production could not be ruled out because, protease synthesis was known to be repressed by rapidly metabolizable nitrogen sources
(Nigam and Singh, 1994). These observations were in accordance with the noticed protease production in the presence of complex nitrogen sources in Bacillus licheniformis
(Cheng et al. 1999), and alkalophilic Bacillus species (Fujiwara et al. 1989).
Experiments were conducted to optimize the casein concentration as nitrogen source for
production of alkaline protease production, by varying the concentration of yeast extract
in the medium. The results indicated that the protease production decreased with increase
in casein concentration in the medium. This data suggested that an economic production
of alkaline protease by this bacterium can be obtained with 0.5% casein supplementation
to the mixture of SM, BGH and GH based solid-state fermentation medium though
enzyme yield was 3267 Ugds1. The results are depicted in Figure 4.10.

Fig. 4.9: Effect of different nitrogen sources on the production of protease by isolated B. subtilis DKMNR.
50

Fig. 4.10: Effect of casein concentration on the production of protease by isolated

B. subtilis DKMNR.
4.2.9 Role of incubation time on protease production
Incubation time is one of the essential physiological fermentation parameters to be evaluated for optimal production of any microbial product/metabolite. To determine the optimum incubation time required for alkaline protease production by isolated B. subtilis
DKMNR, the enzyme production pattern was investigated during solid-state fermentation process using soybean meal, bengal gram husk and green gram husk as solid matrix.
It was observed that protease production increased with incubation time from the beginning of the fermentation (Fig. 4.11). Several reports also indicated that, extracellular
enzyme production of those enzymes, that were having biochemical importance to the
producing microbial strains, was related with growth characteristics of the producing
microorganism.
Maximum protease production by this bacterial strain under solid-state fermentation environment with the mixture of media was found to be 3264 Ugds1. Inuence of incubation
time on alkaline protease production under solid-state fermentation with soya bean meal
suggesting that the enzyme production was growing till 48 hrs. The results obtained were
considered with the observations made in thermoalkalophilic Bacillus species JB-99
using Pigeon pea waste as substrate material supplemented with mineral salt solution
(Johnvesly et al. 2002), alkaline protease producing Bacillus species strain GX6638
(Takami et al.1989), Bacillus species AR-009 (Gessesse,1997), and with Streptomyces
thermovulgaris (Yeoman and Edwards, 1994).

51

Fig. 4.11: Effect of incubation time on the production of alkaline protease by isolated B. subtilis DKMNR.

52

CHAPTER-5: PURIFICATION AND EVALUATION OF

INDUSTRIAL APPLICATION OF PROTEOLYTIC
ENZYMES

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

5.1.2.3 Polyacrylamide gel electrophoresis

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried
out according to the method of Laemmli using a 10% crosslinked polyacrylamide gel on a
Tarson gel electrophoresis unit (Tarson, India). Silver staining was performed to visualize
protein bands on the gel. Native PAGE was performed according to the method of Davis,
(1964) (with Tris/Glycine buffer, pH 8.3. Coomassie Brilliliant Blue (0.1 %) staining was
used to detect the protein bands on the gel.
5.2 Characterization of puried enzyme
5.2.1 Determination of the pH optimum and pH stability
The pH optimum for puried proteases was assayed by analyzing its activity in the pH
range of 4.5 to 13 using casein as a substrate and buffer systems of 0.05 mol l-1 phosphate
buffer for pH 5.0 to 7.5, Tris-HCl for pH 8.0 to 9.0, glycine-NaOH for pH 9.5 to 11.0,
sodium phosphate for 11.5 to 12.0 and sodium carbonate for 12.5 to 13.0. pH stability
study of the protein was analyzed by pre-incubating 5ml of puried enzyme in 3.5 ml of
selected pH buffer at 37 C for 0-120 min and subsequent analysis of residual activities
under standard assay conditions.
5.2.2 Determination of optimum temperature and thermal stability
To study the temperature optima of enzyme, the enzyme reaction mixture was incubated
at different temperatures ranging from 35C to 90C in glycine-NaOH buffer (pH 11.0)
and measured protease activity using casein (1%) as substrate. For determining thermal
stability, the enzyme was pre-incubated for 1.0 h at different temperatures ranging from
35 to 90 C and the residual activity was measured under standard assay conditions after
incubating with casein.
Thermo-inactivation assays were carried out by preheating 950 l of standard buffer at the
corresponding temperature, then adding 1 g protein in 50 l of the same buffer and preincubating the mixture at the same temperature. Samples were collected every 1.0 h at 75,
80, 85 and 90 C and cooled to 50 C before analyzing the protease activity.
5.2.3 Effect of various metal ions
Effect of different metal ions like Ca2+,Co2+,Cu2+, Ba2+,Mg2+,Mn2+,Fe2+,Hg2+,Na+ and Zn2+,
K2+, Ag2+, Pb2+ and EDTA, impact on the enzyme catalytic behavior was studied by preincubating puried enzyme in a specied ion (10mM nal concentration) containing
buffer solution. After 1 h of incubation, casein was added and residual activity of the
enzyme was measured.
5.2.4 Effect of protease inhibitors
Effect of different protease inhibitors such as phenyl methyl sulphonyl uoride (PMSF),
di isopropyl urophosphate (DFP) (serine protease inhibitor), EDTA (metallo protease
inhibitor), p-chloro mercuric benzoate (pCMB), Ido acetic acid (cysteine protease inhibitor) and -mercaptoethanol on protease activity was investigated by incubating the
enzyme for 30 min at 30 C in the selected protease inhibitor containing reaction mixture
in a nal concentration of 1.0 and 5.0 mM the proteolytic activity was assayed by AusonHagihara method ( Hagihara et al.1958).

54

5.2.5 Effect of surfactants and oxidatives on protease activity

The impact of 1.0% nal concentration of different surfactants and oxidatives (SDS in
w/v, Tween-80, Triton X-100 in v/v and H2O2 in v/v) on proteolytic activity of the puried
alkaline protease was studied by pre-incubating the enzyme in above surfactant solutions
at 37C before being tested for protease activity. After 4 h incubation, casein was added
and residual activity of the enzyme was measured. A parallel control was kept with
enzyme and buffer with substrate and the control activity value was considered as 100%.
5.2.6 Hydrolysis of protein substrates
Protease activity using other than casein as substrate material was assayed by mixing 100
l of enzyme and 1.9 ml of assay buffer containing 1mgml1 BSA, egg albumin and gelatin in a separate reaction mixtures and measured the protease activity. The casein dependent protease activity was considered as control for calculating the percent activity.
5.3 Estimation of kinetic parameters
5.3.1 Determination of activation energy
The activation energy of the isolated protease was determined by measuring the enzyme
activity at different temperatures. Arrhenius plot constructed between the reciprocal of
the temperature and enzyme activity was used to determine the activation energy
(Subbarao et al. 2009)
5.3.2 Determination of Vmax and Km values
The kinetic parameters, Vmax and Km, of the puried alkaline protease were determined
by measuring of enzyme activity at different concentrations of the substrate (casein, 0.41.6 %). The kinetic parameters (Km and Vmax) values were determined using Michaelismenton equation through non-linear regression analysis using the program enzyme kinetics module 1.3 in Sigmaplot -10.0.
5.4 Evaluation of industrial applications
5.4.1 Detergent stability
Different commercially available detergents like Henko (Henkel spice, India), Ariel
(Procter& Gamble, India) Surf, surf excel, Rin (Hindustan Lever Ltd., India) were used
to study the compatibility of the puried alkaline protease. The enzyme was incubated in
one percent of above detergent (wv1 solution) in tap water at pH 9.0 and at room temperature for 1-2 h before measuring the enzyme activity. Enzyme activity without any detergent was taken as 100%
5.4.2. Wash Performance Test
The application of protease as a detergent additive was studied on white cotton cloth
pieces (3cm x 3cm) stained with human blood, egg yolk and chocolate. The stained cloth
pieces were taken in separate plates. One petriplate was added of distil water (15 ml), the
second plate was added of distil water 15 ml containing 1ml of surf excel detergent (5
mg ml1) protease free; whereas the third was added of distil water (15 ml) containing
1ml surf excel detergent (5 mg ml1) protease free added with 5ml of enzyme solution.
After incubation at 500 c for 15 minutes, the cloth piece were taken out, rinsed with distil
water, and dried. Examination of various pieces visually exhibited the effect of enzyme in
the removal of stains. Untreated cloth pieces stained with blood, egg yolk and chocolate
were taken as control.

55

5.4.3. Dehairing of goat skin

The precipitate of enzyme by ammonium sulphate (1% w w1 in 10% water) was applied
on esh side of water soaked goat hide (6cm x 6cm) incubate at a temperature 37 0C in a
dry place. Loosening of hair and epidermis were observed by scraping using a blunt scalpel.
5.5RESULTS AND DISCUSSION:
5.5.1 Enzyme purication and molecular weight
Table 5.1 summarizes the results of the alkaline protease purication. This enzyme was
puried 2.45-fold starting from the culture ltrate and achieved near homogeneity by
ammonium sulfate precipitation (60%), ion exchange chromatography using Sephadex
G-100 and gel ltration. The specic activity of the nally puried enzyme was 486.18
Umg1 of protein indicating 30% recovery. Puried alkaline protease was homogeneous
as evidenced from by a single protein band in SDS-PAGE. The apparent molecular mass
of the puried enzyme was estimated to be 43.0 kDa (Figure 5.1). The above results suggest that this is a monomeric enzyme. These results are in accordance with the literature
reports where molecular masses of Bacillus genera proteases were rarely more than 50
kDa (Subbarao et al. 2009) reported that protease from the Bacillus circulans has 39.5
kDa.
Table 5.1: Purication of alkaline protease produced by Bacillus subtilis DKMNR
Total
activity
171548
Crude
(NH4)2SO4
85051
Sephadex-G100 18961

Total
protein
(mg)
864
234
39

Specic activity Recovery Purication

(%)
fold (%)
(U mg-1)
198.55
100
1
363.47
60.24
1.83
486.18
30
2.45

Fig. 5.1: SDS-PAGE analysis of the puried protease. Lane A: Marker; B:

Crude; C:Ammonium sulphate precipitated and dialyzed sample; D: Puried protease by Sephadex G-100
56

5.5.2 Characterization of puried enzyme

5.5.2.1 Effect of substrate concentration on protease activity
The substrate concentration also critically affected the enzyme activity. The constant
amount of puried protease enzyme from the Bacillus subtilis DKMNR was added to the
different concentrations of casein solution. The protease has shown the highest activity of
1697 U mg-1 min-1 at the highest concentration of substrate (1.6%). The nature of the curve
indicates that the increase in substrate concentration increases the protease activity at certain point after that it reaches the saturation. From Figure.5.2 it was observed that the isolated protease follows the pseudo rst order mechanism.

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

Fig. 5.3: Effect of pH on protease activity obtained from Bacillus subtilis

DKMNR
5.5.2.3 pH stability studies
pH dependent enzyme stability studies were performed by incubating it at a range of pH
solutions from 10.5 to 12.0 for 0 120 min at room temperature and measuring the
proteolytic activity at pH 11.0. The catalytic activity prole denoted that the enzyme
activity varied with incubation time and storage pH (Fig.5.4).
Incubation of protease for rst min in 10.5 to 11.5 pH solution did not show any reduction
in activity prole whereas approximately 30 percent reduction in activity was noticed
with the enzyme incubated in the pH range of 10.5 to 11.5 indicating its stable nature at
this pH range (Fig.5.4). The increase in incubation time to 90 min revealed that enzyme
activity was adversely affected and showed only 40 % and 50% of its activity at pH 10.5
and 11.5 respectively, with a maximum stability in the pH range of 10.5 to 11.0.

Fig. 5.4: Inuence of pH and incubation time on protease activity.

58

5.5.2.4 Effect of temperature on protease activity and stability

The inuence of incubation temperature on protease activity was studied by incubating
the enzyme for 20 min in specied temperature prior to determination of proteolytic
activity. Protease activity was not affected by pre incubation of the enzyme at 35 to70C
but decreased markedly at higher temperatures more than 70oC (Fig.5.5). Analysis of the
data revealed a phase activity prole with initial constant activity phase in the temperature range of 35 to 450C followed by rapid accelerated phase in the range of 45 to 60C and
slow accelerated phase in the range of 60 to 70C (Fig. 5.5). This type of three phase activity has not been reported earlier.
5.5.2.5 Thermostability studies
The thermo stability of protease was studied by incubating it at various temperatures from
75 to 90oC for 0 to 210 min at optimum pH. The catalytic activity prole denoted that the
enzyme activity varied with incubation time and temperature (Fig. 5.6). Incubation of protease at 75oC for more than 60 min did not show any effect on the enzyme but at temperatures from 80 to 90oC caused a severe decline of protease activity when the incubation
time was below 60 min. While, with increase in incubation time to 120 min and higher
revealed that enzyme activity was affected much and showed only 53 % and 41% of its
activity at incubation time of 150 and 210 min, respectively. The enzyme activity
decreased abruptly as the incubation time was increased.

Fig. 5.5: Effect of temperature on protease enzyme from 35oC to 90oC.

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

Residual activity (%)

5 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

Tween-20, enhanced its residual activity to 21 & 6 % respectively. In presence of strong

anionic surfactant SDS 1 % the enzyme retained 84 % of its residual activity. In general
the protease species of Bacillus are unstable against the oxidants and bleaching agents
(Anwar & Saleemuddin, 1998). However, the enzyme under investigation did not show
any inhibition for presence of 1% hydrogen peroxide (Table 5.3). Suggesting the puried
enzyme is stable for tested cationic, anionic, nonionic and for different commercially
available detergents Hence, protease of Bacillus subtilis DKMNR is most suitable in the
formulations of the commercial detergents as most of the commercial detergents contain
cationic and anionic components in its formulations.
Table 5.3: Residual activity of puried alkaline protease at different
concentrations of surfactants
Surfactants

Residual activity (%)

Control

100

Triton X-100

121

Tween-20
SDS
H2O2

106
84
97

5.5.2.9 Hydrolysis of proteinaceous substrates

The substrate specicity of Bacillus subtilis DKMNR protease varied with tested natural
proteins like casein, BSA, egg albumin, and gelatin. This protease exhibited the highest
activity towards casein. Considering the casein dependent activity as control, BSA and
Egg albumin hydrolysis activity was assayed. In Figure 5.8 the data revealed that enzyme
showed only 75, 56 and 26 % of activity with BSA, egg albumin and gelatin as substrates
as shown in Figure 5.8. The present observations are in agreement with the report on
Bacillus subtilis PE-11(Adinarayana, et al. 2003).

Fig. 5.8: Inuence of isolated protease on hydrolysis of various proteins.

62

5.5.3 Kinetic parameters of pure protease

5.5.3.1 Activation energy
The activation energy of puried protease of Bacillus subtilis DKMNR was investigated
using the Arrhenius plot by plotting the residual activity in natural logarithm (lnv) versus
inverse of the temperature (T-1K) after tting the data in linear regression (Figure 5.9).
The observed correlation coefcient was higher than 0.95 which indicate the deactivation
follows the rst order kinetics. From Figure 5.9 activation energy was calculated as
41.082 kJ/mol/k such variation of enzyme activation energy indicates the conformational
changes especially at the catalytic site which improves afnity towards substrate binding.
This could be further evidenced based on the observation that the noticed change of rate
of enzyme activity (62 and 31 U per increase of one degree centigrade in the temperature
range of 45 to 600C and 60 to 700C, respectively).
5.5.3.2 Km and Vmax values
The protease enzyme was characterized further for its Km and Vmax towards casein as a
substrate at 70 0C. The Km and Vmax values were determined by using Michaelis-menton
equation through nonlinear regression analysis. The results were recorded as Km of
0.7614 mg mL-1 and Vmax of 2582 mol min-1. In the literature the similar kind of studies
for protease obtained from the B. circulans (Subbarao et al. 2009).

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

Table 5.4: Relative activity of puried alkaline protease at different incubation

time intervals
Effect of Detergents

Relative activity at different incubation times

1h

2h

Henko

90

83

Surf

75

72

Surf excel

84

76

Ariel

80

69

Rin

84

46

5.5.4.2 Wash performance test

To check the contribution of the enzyme in improving the washing performance of the
detergent, the use of protease DKMNR as a detergent additive was studied as shown in
Fig 5.10. Puried enzyme removed blood stain within 15 min by incubating at 50 0Cwith
the supplementation of laundry detergents solution (surf excel). Where as in Fig 5.10 b,
laundry detergents when we use without the addition of puried enzyme the stain removal
is not proper as compared to Fig 5.10 c. These results show the efciency of B. subtilis
DKMNR protease in stain removal efciency. (Subbarao et al. 2009) also showed the signicant improvement effect of the supplementation of proteolytic preparation of Botrytis
cinerea, in a laundry detergent.

64

Fig. 5.10: Washing performances of alkaline proteases from B. subtilis DKMNR in

the presence of detergent surf excel a) Cloth stained with blood b) Blood stain cloth
washed with detergent only c) Blood-stained cloth washed with detergent added
with puried enzyme solution d) Cloth stained with egg yolk e) egg yolk- stained
cloth washed with detergent only f) Egg yolk-stained cloth washed with detergent
added with puried enzyme solution g) Cloth stained with chocolate h) Chocolate
stained cloth washed with detergent i) Chocolate stained cloth washed with detergent added with puried enzyme solution
On the other hand, incubation of protease with egg yolk stained cotton cloth piece showed
removal of the stains with the usage of detergent (surf excel) with in 15 min when incubated at 50 0C. Rapid stain removal was noticed with the supplementation of commercial
available detergent as shown in Figure 5.10f. Similar results were noticed with protease
from B. circulans, B. subtilis PE-11 and Pseudomonas aeruginosa (Subbarao et al. 2009)
indicating the role of B. subtilis DKMNR in industrial application especially in detergent
preparation.
While the effect of protease solution was also put evidence on chocolate by the test of spot
on cotton cloth piece as a substrate, it was seen in the Figure 5.10g that the protease enable
to removal chocolate stain easily Figure 5.10i when compared to Figure 5.10h without the
addition of protease to the detergent.
This protease solution showed high capability for removing proteins and stain from
cloths therefore it could be used as an alkaline protease in detergent powder or solution.
Its ability to act in the presence of detergents might be exploited. Ferid Abidi, (2008)
reported usefulness of protease from Botrytis cinerea for the removal of chocolate stains
from cotton cloth in the presence and absence of detergents. Therefore, preparation containing DKMNR protease activity could be consider as a potential candidate for use as
cleansing additive in detergents to facilitate the release of proteinaceous stains.
5.5.4.3 Dehairing of goat skin
Enzymatic dehairing process has been gaining importance as an alternative chemical
methodology in present day scenario as this process is signicant in reduction of toxicity
in addition to improvement of leather quality. Several microbial proteases are evaluated
for their dehairing character and it was noticed that only those enzymes with pH stability
under alkaline conditions especially in the range of 9.0 and with non keratinase and non
collagenolytic properties were having edge over others (Nilegaonkar et al. 2007). The
enzyme produced by B. circulans revealed robustness towards alkaline pH, detergent and
65

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

CHAPTER 6: FERMENTATION STUDIES FOR

IMPROVED PRODUCT FORMATION

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

6.2 Solid state Fermentation (SSF)

Solid-state fermentation (SSF) may be dened as the fermentation involving solids in
absence or near absence of free water. But the substrate, which is used for growth and
metabolism of the microorganisms, must have enough moisture (Pandey et al. 2000). SSF
holds potential for the production of enzymes and credited as the beginning of the fermentation technique in ancient time. Therefore it is not surprising that all the fermentation processes used in ancient time were based on the principles of SSF.
SSF can be the best being employed for the processing of the agroindustrial residues
(Gessesse, 1997). Because solid-state processes have lower energy requirements, produce lesser wastewater, and are environment friendly to resolve the problem of solid
waste disposal. Further utilization of agro industrial residues in the SSF offer a unique process development for value addition of these low cost residues (Johnvesly et al. 2002). At
present SSF processes are used at commercial scale for the production of microbial products such as feed, fuel, food, industrial chemicals and pharma products. Its application in
bioprocess such as bioleaching, bioremediation, bio pulping etc, has offered several
advantages (Pandey et al. 2000).
The key aspect of the SSF is the selection of proper substrate, which should be in-soluble
and should acts both as a physical support and source of nutrients. The substrate should be
a solid material, which can be naturally occurring such as agro crops materials, agro
industrial residues or inert supports (Pandey et al. 2000; Johnvesly et al. 2002; Gessesse,
1997). There are two major considerations for the selection of substrate; one is a specic
substrate, which requires suitable value addition or disposal. The second could be related
with the goal of producing a specic product from suitable substrate.
Agro industrial residues are generally considered the best substrates for SSF processes.
Some of the substrates that have been used in SSF process includes Cane bagasse, Wheat
bran, Maize bran, Gram bran, Wheat bran, Rice straws, Rice husk, Soy hull, grape vine,
Trimmings, Saw dust, Banana waste, Tea waste, Palm oil waste, Sugar beet pulp, Sweet
sorghum pulp, Apple pomace, peanut meat, coconut & Mustard oil cake, wheat & Corn
ours, Steamed rice and Starch etc (Gessesse, 1997; Aikat & Bhattacharyya, 2000;
Pandey et al. 2000; Johnvesly et al. 2002; Germano et al. 2003).
However wheat bran holds the key and the most commonly used in various processes.
The selection of substrates for enzymes depends upon several factors, mainly the cost of
substrate. The substrate may provide the needed nutrients to the microorganisms growing
in it, but some of the nutrients may be present in sub optimal concentrations or even
absent in the substrate. In such cases this can be overcome by supplying nutrients externally.
The other important factor is the moisture content; the water activity (aw) of the medium
has been attributed as a fundamental parameter for mass transfer of the water and solute
transfer across the microbial cells. This parameter could be used to modify the metabolic
production or excretion of microbial product so that water has profound impact on the
physicochemical properties of the solids and this in turn effect the overall process productivity. Other parameters that inuence the product production under SSF include incubation temperature, medium pH and available surface area (Tunga et al. 2001).

68

6.3 Strategies to improve the enzyme productivity

Production of protease in bulk amounts is one of the important parameters to be considered for industrial purposes. The primary stage in the development of an industrial fermentation process is to isolate strain(s) capable of producing the target product in higher
quantities. Next is the development of suitable fermentation medium. In general, no
dened medium was established for the best production of any metabolite because the
genetic diversity present in different microbial sources causes each organism or strain to
have its own special conditions for maximum product production. Therefore, it is essential to have a detailed investigation on growth and metabolite production pattern of newly
isolated microbial strain under different environmental conditions to achieve maximum
production benet. Literature reports indicated that extra-cellular protease production in
microorganisms was also strongly inuenced by media components, e.g. variation in C/N
ratio, presence of some easily metabolizable sugars, such as glucose (Beg et al. 2002;
Gupta et al. 2002c) and metal ions Gupta et al. 2002b; Prakasham et al. 2005a). Protease
synthesis was also affected by rapidly metabolizable nitrogen sources such as amino
acids in the medium (Gupta et al. 2002a). Besides these, several other physical factors,
such as aeration, inoculum density, pH, temperature and incubation, also affect the
amount of protease produced (Hameed et al. 1999; Puri et al. 2002). While designing a
medium for screening proteases, it is essential that the medium should contain likely
inducers of the product and be devoid of constituents that may repress enzyme synthesis
because approximately 30-40% of the production cost of the industrial enzymes accounts
for growth medium (Gupta et al. 2002b). Hence, each organism should be studied in detail
for its growth requirements and enzyme production titers.
It was reported that approximately ninety percent of the industrial enzymes are produced
using submerged fermentation (SMF) techniques with the enzyme titers in the range of
grams per liter, even though fed-batch and solid state fermentation techniques are
reported to be more efcient than SMF (Gupta et al. 2002c; Holker and Lenz, 2005;
Vasudeo et al. 2011). A comprehensive account of culture conditions for protease production reported in the literature from various microorganisms is listed in Table 6.1
Table 6.1: Alkaline protease producing microorganisms and their optimized
conditions
Microorganism

pH

Tem- Agita- Incuba- Preferred Preferred

peratur tion
tion
e (C) (rpm) period Nitrogen carbon
source
source

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

Tem- Agita- Incuba- Preferred Preferred

peratur tion
tion
e (C) (rpm) period Nitrogen carbon
source
source

B.pseudorm
us

37

100

66

Yeast
extract

Casein Gupta et
acid
al. 2002a
hydrolysat
e

Bacillus sp.

10

37

180

96

Yeast
extract

Glucose Tari et al.

2006

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

Sweet sor- Molasses Keivan et

ghum
al. 2009
extract

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

6.4 Optimization of fermentation medium

Optimization of nutritional and environmental conditions by the classical method of
changing one independent variable (nutrient, antifoam, pH, temperature, RPM, aeration,
agitation, etc) while xing all others at a certain level can be extremely time consuming
and expensive for a large number of variables. To make a full factorial search, which
would examine each possible combination of independent variable at appropriate levels,
could require a large number of experiments xn, where x is the number of levels and n is
the number of variables. Other alternative strategies of conventional medium optimization must, therefore, be considered which allow more than one variable to be changed at a
time. Several investigators have discussed about these methods (Praksham et al. 2006;
Sreenivasrao et al. 2004).
When more than ve independent variables are to be investigated, the Plackett & Burman
(1946) design may be used to nd out the most important variables in a system, which are
then optimized in further studies. Romsomsa et al. (2010), Guangrong et al. (2008) conducted modeling studies and expressed the results in sets of mathematical equations.
Designing of an appropriate fermentation medium was of critical importance as medium
composition inuences product concentration yield and volumetric productivity
(Akhnazarova and Kafarov, 1982). The commonly used optimization method is 'one at a
time' method (Prakasham et al. 2006). But in one at a time method interactions among the
different parameters is not possible. However, alkaline protease producing microorganisms and their optimized conditions are mentioned in Table 2.2.
It was impractical to optimize all parameters and to establish the best possible conditions
by inter-relating all parameters, as this involves numerous experiments to be carried out
with all possible combinations (Sreenivasrao et al. 2004). Experimental designs based on
statistical tools are known to provide economic and practical solutions in such cases
(Prakasham et al. 2006).
Optimization procedures such as response surface methodology (RSM), orthogonal
array, articial neural network, genetic algorithms, etc., were developed to optimize the
biotechnological processes consisting of an empirical modeling system developed based
on full factorial central composite parameters that inuence the production process
(Sreenivasrao et al. 2004). Statistical methods show better performance than one at a time
method even though it has some limitations. For the application of RSM, a model must be
assumed to determine the relative inuence of the various medium components for the
objective function and the common model used was full second order polynomial. The
number of experiments is given by LN (N factor at L levels) therefore in practice only two
or three levels could be applied and plotting was limited to two or three levels. The level
of orthogonal array design or uniform design was also limited by the factor.
In recent years, there was a great amount of research and development effort focusing on
the use of statistical software packages (Puri et al. 2002). Some of the studies performed
using statistical optimizations for improving the metabolite production are listed in Table
6.2.

71

Table 6.2: Statistical methods used to improve protease production by microorganisms

Microorganism

Design

Software

Rhizopus oryzae LevenbergMarqua Not specied

rdt technique
B. subtilis
IIQDB32
Bacillus sp.
RGR- 14

Yield
improvement

Reference

2.5-fold

Banerjee et
al.1999
Vermelho et al.
1996
Puri et al. 2002

PlakettBurman

Not specied

3.0-fold

Face-centered central composite

design

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

Pillai et al. 2011

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

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APPENDIX -I

ABBREVIATIONS
ANNs

Articial Neural Networks

ANOVA

Analysis of Variance

BSA

Bovine Serum Albumin

CCD

Central Composite Design

DFP

Di- Isopropyl Fluorophosphate

EDTA

Ethylene Diamine Tetra Acetic acid

FFCCD

Full Factorial Central Composite Design

GA

Genetic Algorithms

GRAS

Genetically Regarded As Safe

HCN

Hydrogen Cyanide

IMTECH

Institute of Microbial Technology, India

kDa

Kilo Daltons

pCMB

p-Chloro Mercuric Benzoate

PMSF

Phenyl Methyl Sulfonyl Fluoride

RPM

Rotation per Minute

RSM

Response Surface Methodology

SDS-PAGE

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis

SmF

Submerged Fermentation

SSF

Solid State Fermentation

TLCK

Tosyl- Llysine Chloromethyl Ketone

TPCK

Tosyl-L-Phenylalanine Chloromethyl Ketone

YPD

Yeast Peptone Dextrose

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