19

2. AIM AND OBJECTIVE OF THE STUDY

Nowadays, there is a demand for industrial enzymes, predominantly

microbial origin, are ever increasing due to their applications in a wide variety of

industrial processes. Microbial proteases account for just about 40% of the total

global enzyme sales. Proteases obtained from the microorganisms are more

preferred than proteases of plant and animal sources. Microbial proteases posses

almost all characteristics desired for their biotechnological applications in the

industries. Amongst the various proteases, bacterial protease was the most

significant compared with animal, fungi and plant protease. Therefore, the present

study was undertaken with the following objectives:

• To screen the protease producing microorganisms from bioeffluent.

• To determine the Proteolytic activity of the isolates.

• To identify the isolates by biochemical, cultural and molecular

characterization.

• To identify the isolates by 16s rRNA sequencing

• To optimize the cultural parameters for the enhanced production of

protease.

• To determine alkaline protease enzyme assay.

• To design the substrate from agro industry for production media for the

enhanced production of protease.

• Analysis of protease enzyme and molecular weight determination by SDS -

PAGE.

• To characterize the enzyme by column chromatography, NMR and HPLC.

• To assess the application of purified protease enzyme.

20

3. REVIEW OF LITERATURE

3.1 Characteristics of Protease Enzyme

A protease is any enzyme that conducts proteolysis, that is, begins protein

catabolism by hydrolysis of the peptide bonds that link amino acids together in the

polypeptide chain forming the protein. Microbes have both intra cellular and extra

cellular proteases, the intracellular proteases are responsible for the maintenance of

amino acid pool inside the cell by degrading the unwanted proteins and the extra

cellular proteases hydrolyze proteins outside the cells into peptides and amino acid

required by the cells for their growth. Proteases are classified into two major groups:

the exopeptidases (peptidases) and the endopeptidases (proteinases). The peptidases

hydrolyze the protein from C- or N-terminus releasing single amino acid and the

endopeptidases as the name suggests hydrolyses the peptide bond in the middle of

the amino acid chain. Further the proteases are also classified into alkaline, acid and

neutral proteases based on their pH optima of activity (Banerjee et al., 1993).

It's optimal activity shown in alkaline (basic) pH. Proteases occur naturally in

all organisms. These enzymes are involved in a multitude of physiological reactions

from simple digestion of food proteins to highly-regulated cascades (e.g., the blood-

clotting cascade, the complement system, apoptosis pathways, and the invertebrate

prophenoloxidase-activating cascade). Peptidases can either break specific peptide

bonds (limited proteolysis), depending on the amino acid sequence of a protein, or

break down a complete peptide to amino acids (unlimited proteolysis). The activity

can be a destructive change, abolishing a protein's function or digesting it to its

principal components; it can be an activation of a function, or it can be a signal in a

signaling pathway. Proteases, also known as proteinases or proteolytic enzymes, are

a large group of enzymes. Proteases belong to the class of enzymes known as

hydrolases, which catalyse the reaction of hydrolysis of various bonds with the

participation of a water molecule.

21

Proteases are involved in digesting long protein chains into short fragments,

splitting the peptide bonds that link amino acid residues. Some of them can detach

the terminal amino acids from the protein chain (exopeptidases, such as amino

peptidases, carboxy peptidase A); the others attack internal peptide bonds of a

protein endopeptidases such as trypsin, chymotrypsin, pepsin, papain, elastase

(Mahmoud et al., 1978; El-Enshasy et al., 2008).

Proteases represent the class of enzymes that occupy a pivotal position with

respect to their physiological roles as well as their commercial applications. More

than 75% of industrial enzymes are hydrolases. Protein-degrading enzymes

constitute about 40% of all enzymes sales (Leisola et al., 2001). Further the

proteases are also classified into alkaline, acid and neutral proteases based on their

pH optima of activity. On the basis of the functional group present at the catalytic

site these proteases are classified as serine proteases, cysteine proteases, aspartic

proteases, threonine proteases, glutamic acid proteases and metalloproteases. The

proteases represent one of the three largest groups of industrial enzymes others

being the amylases and lipases. The proteases find their application in detergents,

leather, food, pharmaceutical industries and bioremediation processes (Rao et al.,

1998).

Extra-cellular proteases produced by ferementation processes are

economically comparable to the chemical process. Hence, any substantial reduction

in the cost of 1 ml of fungal spore suspension (10 spores/ml) and production of

enzymes will be a positive stimulus for the incubated at 35 °C for 120 h

commercialization of enzymatic depilation (Brown et al.,1991).

3.2 Microbial proteases

Microbial proteases are among the most important hydrolytic enzymes and

have been studied extensively since the advent of enzymology. They are essential

constituents of all forms of life on earth, including prokoryates, fungi, plants and

22

animals. They can be cultured in large quantities in relatively short time by

established fermentation methods and produce an abundant, regular supply of the

desired product. In recent years there has been a phenomenal increase in the use of

alkaline protease as industrial catalysts. Alkaline proteases (EC.3.4.21–24, 99) are

defined as those proteases which are active in a neutral to alkaline pH range. They

either have a serine center (serine protease) or are of metallo- type

(metalloprotease); and the alkaline serine proteases are the most important group of

enzymes so far exploited (Gupta et al., 2002b). These enzymes offer advantages

over the use of conventional chemical catalysts for numerous reasons; for example

they exhibit high catalytic activity, a high degree of substrate specificity can be

produced in large amounts and are economically viable. Microbial alkaline

proteases dominate the worldwide enzyme market, accounting for two-third of the

share of the detergent industry. Although production is inherent property of all

organisms, only those microbes that produce a substantial amount of extracellular

protease have been exploited commercially. Alkaline proteases of Bacillus sp. origin

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 aminoacid mixtures

(Agrawal et al., 2004).

3.3 Optimization of pH and Temperature for protease production

The proteases constitute a very large and complex group of enzymes, which

differ in properties such as substrate specificity, active site and catalytic mechanism,

pH and temperature optima, and stability profile. Studies relating to such properties

of these proteases are imperative for the successful application of these enzymes in

their respective industry. The enzyme produced by SSF is hence characterised by

various features that influence enzyme activity such as optimum temperature,

optimum pH, effect of metal ions etc (Sandhya et al., 2004).

23

Isolation and characterization of a new strain of Bacillus sp. from alkaline

soil, which was able to producing extra cellular alkaline protease and amylase from

date waste at pH ranging from 8 to 11 and temperatures of 20 to 50 °C. Purification

was conducted by fractionation, concentration, and cation exchange

chromatography. Molecular weight of purified enzyme was measured by SDS-

PAGE (Khosravi-Darani et al., 2008).

Bacillus subtilis KO strain was isolated from molasses that obtained from the

industrial products of Kom Ombo sugar factory. Different experiments were

performed to optimize the cultivation conditions of Bacillus subtilis KO strain and

the medium contents to reduce the cost of protease production. Optimum protease

production was recorded at 48 hr. incubation period, at 40-45 °C, pH ranged from 7-

7.5, 0.1% (w/v) gelatin concentration in the presence of ammonium phosphate as

inorganic source. From the available present data, it could be recognized that this

study may be the unique to use molasses not only to isolate B.subtilis KO strain but

also to grow and maintain it. Moreover, it used as a medium contain only molasses

to produce considerable amount of protease. (Magdi et al., 2009). Molasses is an

interesting raw material it is rich production of protease in low-cost medium by-

product in sugar industries.

3.4 Alkaline Protease

Proteases are by far the most important enzymes in the food industry used in

food proteins modification. Proteases have been used in ancient technology to

improve palatability and storage stability of the available protein sources;

consequently, proteases have a long history of applications in food products and

they are used in baked goods, brewing, cereals, cheese, chocolate/cocoa, egg

products, meat and fish, wine, protein hydrolysis’s, antinutrient factors removal.

Also they are widely used in the detergent, pharmaceutical, clinical diagnostic,

24

leather, cosmetics and fine chemical industries (Fox et al., 1991; Macfarlane et al.,

1992).

Proteases are among the oldest enzymes known to man. These are

degradative enzymes, which catalyze the total hydrolysis of proteins (Raju et al.,

1994). The molecular weight of proteases ranges from 18 – 90 k Da. (Sidney and

Lester, 1972). These enzymes are found in a wide diversity of sources such as

plants, animals and microorganisms but they are mainly produced by bacteria and

fungi. Microbial proteases are predominantly extracellular and can be concentrated

in the fermentation medium. Microbial proteases are the most important industrial

enzymes (Chouyyok et al., 2005) and account for approximately 40% of the total

worldwide enzyme sale (Godfrey and West, 1996). They are generally used in

detergents (Barindra et al., 2006), leather and food industries (Rao et al., 1998;

Haq et al., 2002). They also have medical and pharmaceutical applications.

Enzymes are one of the major groups of products in biotechnology business

sector. The world market for enzymes grows 7.6% per year and will reach $6

billions by 2011 as predicted in the Freedonia Group Inc. business report in 2007.

This is driven by continued robust growth in pharmaceutical enzyme demand, fine

chemical production, bioethanol production and detergent industries (Kumar and

Takagi 1999; Dayanandan et al., 2003). Moreover, they are also used for cleaning

of membranes used in protein ultrafiltration (Kumar and Tiwari, 1999). Of these

different applications, the main use of alkaline protease is mainly focused on

detergent industries. They are used in all types of laundry detergents and in

automatic dish washing detergents. Their function is to degrade proteinaceous stains

(Maurer, 2004).

In general, most of the alkaline proteases applied for industrial purposes face

some limitations, including low activity and stability towards anionic surfactants

like SDS and oxidants; and their high total cost (Joo et al., 2003). Given the wide

25

application of this enzyme, it is reported that in year 2005 the global proteolytic

enzyme demand will increase dramatically to 1.0 – 1.2 billon dollars (Godfrey and

Reichelt, 1985). Therefore, taking this demand into account and knowing the

geographic richness and biodiversity of Iranian local environment, it is assumed that

there is potential for alkalophilic Bacillus sp living in these environments.

Discovering such species, producing proteases with novel characteristics will be of

great value to the enzyme industry for different applications.

Alkaline proteases are produced by a wide range of microorganisms

including bacteria, moulds, yeast and also mammalian tissues. Among bacteria,

Bacillus spp are specific producers of extracellular proteases. These are used as

cleansing additives in detergents to facilitate the release of proteinacious materials

in stains due to grime, blood, milk, etc. Bacillus sp can be cultured in a pH range of

7.0 -11.0 and produces extracellular alkaline proteases (Godfrey et al., 1996;

Cochran et al., 1992).

3.5. Protease enzyme production and industry

Microbial proteases can be produced from bacteria, fungi and yeast using

many processes like solid-state fermentation and submerged fermentation (Kumar

and Takagi. 1999; Anwar and Saleemuddin, 2001; Haki and Rakshit, 2003).

B. horikoshii and B. sphaericus producing alkaline proteases are illustrated by the

fact that the market share of this enzyme is about 20% of overall industrial enzymes

business. However, beside some published reports on the production of alkaline

protease by solid state fermentation. The main process for the industrial production

of this enzyme is carried out in large scale by submerged culture (Mehrotra et al.,

1999).

Bacillus subtilis proteases have wider specificity than trypsin and

chymotrypsin. They possess a number of industrially valuable properties including

their ability to excrete several different hydrolytic enzymes into culture medium.

26

The lack of pathogenicity and the ability to grow in simple culture medium can also

be accounted for their application in industry. With increasing industrial demands

for the biocatalysts that can cope with the industrial processes at harsh conditions,

the isolation and the characterization of new promising strains are possible ways to

increase the yield of such enzymes (Gupta et al., 2002b).

Strains of Bacillus, Steptomyces, Aspergillus are the major producers of

alkaline proteases. Proteases from Bacillus (Bacillopeptidases) are mainly used in

detergents. Subtilisin carlsberg (protease from B. licheniformis) and Subtilisin Novo

(protease from B. amyloliquefaciens) are the best known proteases used in

detergents. These proteases have serine at the active site, and are not inhibited by

EDTA (ethylene diamine tetra acetic acid), but are inhibited by DFP (disopropyl

fluro phosphate). These proteases are stable at high temperature, active in alkaline

pH (9-11) and stable in presence of chelating and perforates, which is an important

characteristic of these enzyme for use in detergents. Screening of organism

producing alkaline proteases is done using strongly basic media and the colonies are

tested on protein agar plates pH 10 (Ellaiah et al., 2002).

Bacillus amyloliquifaciens MTCC1488 was used to obtain alkaline protease.

The enzyme was produced by substituting the glucose source with agricultural

wastes like banana waste and zapota fruit peel waste. The maximum production of

enzyme was obtained by using 300gl-1

of banana (Musa sapientum) fruit peel waste

and 250gl-1

of zapota (Achras zapota) waste as the substitute of 10 gl-1

glucose as the

growth media. All the parameters needed for the optimum production of the enzyme

was found out. These included the optimization of process parameters such as the

effect of various substrates, incubation periods, inoculum’s size, temperature,

amount of substrate, NaCl concentration, supplementary carbon and nitrogen

sources, and shaking speed of submerged fermentation (Blesson, 2009).

27

Most commercial bacterial proteases are mainly actives at neutral and

alkaline pH and are produced by organisms of the genus Bacillus and to lesser

extent by Clostridium (Chu et al., 1992). In general, neutral proteases are active in

a narrow pHrange (pH 5-8) and have relative low thermo tolerance. These

properties make bacterial proteases most advantageous for the detergent and leather

industry. Also, alkaline proteases from Brevibacterium linens have been used in the

dairy industry and proteases from Bacillus thermoprotelyticus are used for the

enzymatic synthesis of aspartaine. There is a long list of bacterial proteases

commonly used in the food industry and they are mostly produced by submerged

fermentation, however fungal protease production is an attractive source for

proteases. Solid state fermentation (SSF) and the advantages of fungal enzyme

production in SSF over submerged state fermentation (SmF) system (Viniegra-

González, et al., 2003; Phadatare et al., 1993), Fungal acid proteases have an

optimal pH rang from 4 - 4.5 and they can be stable at pH values from 2.5-6.0. In

general, fungal neutral proteases are metalloproteases inhibited by chelating agents,

however they can supplement the action of plant, animal and bacterial proteases

reducing bitterness in food protein hydrolysates and food protein modifications.

Enzymes produced from microorganisms that can survive under extreme pH

could be particularly useful for commercial applications under highly alkaline

conditions. e.g. in the production of detergents. Screening of water, soil and

sediment samples collected from different alkaline environments around Orissa

state, India for alkaline protease producing bacteria, resulted in isolation of 18

alkaline protease producing alkalophilic strains. Maximum enzyme activity of 66.23

PU\ml was found in a gram –ve bacterial strain at pH 10 and temperature of 37ºC

after an incubation of 48 hours in water bath shaker.Later this strain was identified

by biochemical characterization to be Serratia liquiefaciens. Some fundamental

parameters like effect of pH, temperature, time and inoculum size for maximum

protease production were also studied. Maximum yield of enzyme (74.3 PU\ml) was

28

obtained at a pH of 9 with 1ml inoculum in the media after 48 hours of incubation in

water bath cum shaker maintained at a temperature of 37ºC (Smita et al., 2012).

3.6 Agro-Residue as substrate in Enzyme Production

Bioconversion of the agro-residue offers the possibility of creating

marketable value-added products. In this regard, Sago residue which contains solid

and liquid materials produced abundantly as a by-product from the sago starch

processing industry. Utilization of sago residue not only reduce the polluting effects

from the sago processing industries, but will also provide an economic solution for

waste management system at sago processing mills. This review focuses on the

developments in processes and products for the value addition of sago residues

through biotechnological means (Awg-Adeni, 2010).

The effect of different concentrations of Sugar Mill effluent on growth, yield,

biochemical contents and enzymatic activities of green gram have been studied. The

increasing pace of industrialization in public and private sectors along with

urbanization, population explosion and green revolution are reflected in varying

degree of pollution of water, soil and air. The sugar mill effluent is having a higher

amount of organic and inorganic elements. The pigment analyses viz. chlorophyll

‘a’, chlorophyll ‘b’, total chlorophyll, protein, amino acid, sugar contents and

enzymes activities were analysed at 15 and 60days. The yield parameters of green

gram plants were recorded at the time of harvest. All morphological growth

parameters, biochemical contents, enzyme activities and yield parameters were

found to increase at 10% effluent concentration and it decreased from 25% effluent

concentration onwards. So, these results reflect that the sugar mill effluent is toxic

to crop and it can be used for irrigation purpose after a proper treatment with

appropriate dilutions (Baskaran et al., 2009).

Proteases are advantageous due to the ease downstream processing, moreover

an alkaline protease from Conidiobolus coronatus was found to be similar to the

29

ones contained on Indian detergents retaining 43% of its activity at 50 °C for 50 min

in the presence of calcium and glycine (Ottsen and Rickert, 1970). In the cheese

making industry, chymosin is preferred due to its high specificity for casein, which

is responsible for its excellent performance in cheese making. However, extensive

research has been done and it has resulted in the fungal enzymes production by

Mucor michei (GRAS) ( Ikasari and Mitchell, 1994). Also, in the milk industry

acid, alkaline and neutral fungal proteases produced by Aspergillus oryzae have

been also used (Malathi and Chakraborty, 1991). In the baking. industry, endo- and

exoproteinases from Aspergillus oryzae have been used to modify wheat gluten by

limited proteolysis such enzymatic treatment reduces the mixing time and increases

the volume of the loaf.

3.7 Importance of Protease Enzyme in Biotechnology

Biotechnological importance of these enzymes has been realized by the

leather industries for the purpose of dehairing and bating hides as a substitute toxic

chemical (Bhosale et al., 1995). In food industry proteases are used as crude

preparation. In pharmaceutical industry they are used as ingredients of ointments for

debridement of wards and in medicine preparation (Jany et al., 1986).

Bacillus sp, as one of the best alkaline protease producers, have shown

various physiological capabilities (Holt et al., 1994; Ram et al., 1994; Sneath et

al., 1986). The detection and isolation methods of Bacillus sp are based on

resistance of their endospores to high temperatures ranging 70 – 80 °C. This

condition destroys all vegetative forms of the other known bacteria and other

endospores (Emtiazi et al., 2005; Holt et al., 1994; Sneath et al., 1986). Proteases

are the most useful industrial enzymes that are produced 500 tones annually. In

industry, proteases are produced from bacteria and fungi. The alkaline proteases,

with an optimum activity in pH 9 - 11, have numerous applications in daily life of

peoples such as food complementary of beast and poultry, confectionary, bakery,

30

fermentation industries, leathering, detergent industry, biotransformation and so on.

In this research a wild strain of Bacillus cereus and Bacillus polymixa, isolated from

the soils of Isfahan (Iran), showed the best alkaline protease activity in industrial

mediums such as sweet sorghum extract and molasses (Keivan Beheshti Maal et

al., 2009). Alkaline proteases have also been used in the recovery of silver from

photographic plates and peptide synthesis.

However, the recent cleaning technologies include enzyme containing

formulations and zeolite based detergents. Of these, the enzyme detergents often

referred to as “Green Chemicals”, are proving extremely useful in keeping a check

on the environmental pollution. Addition of alkaline protease to detergents

considerably increases (35-40%) the cleaning effect (particularly in removing stains

containing proteins, e.g., blood, cocoa, milk, eggs and sauces) and increases the

consumption of surface active substances, thereby improving the ecological

situation (Kumar and Parack, 2003). Biocatalysts hydrolyze peptide bonds in

proteins and hence are classified as hydrolases and categorized in the subclass

peptide hydrolases or peptidases (Ellaiah et al., 2002). Because of this functional

property, they are widely used in laundry detergents, leather processing, protein

recovery or solubilization, meat tenderization, and the biscuit and cracker industries

(Johnvesly et al., 2001). However, other application potentials of these enzymes

depend on the nature of catalytic activity with respect to reactant medium, which led

to the classification of proteases as acidic, neutral and alkaline. Among these

different biocatalysts, alkaline proteases have wide application spectra and novel

properties due to their exotic catalytic nature. Hence, these proteases and their

producing organisms attracted attention of scientific community to understand the

protein chemistry and protein engineering to enhance their utilization niche

(Germano et al., 2003).

31

Proteases have an important position in the enzyme industry as they have a

determinant role in the microbial and human physiological needs as well as the great

commercial market applications. Since they are indispensable for living organisms,

proteases occur in a wide diversity of plants, animals and microorganisms (Rao et

al., 1998). Papain, bromelain, keratinases and ficsin represent some of the plant

proteases (Neurath, 1994). However, the use of plants as a source of proteases is

governed by factors no easily controlled such as land availability and climatic

conditions as well as its excretation is a time-consuming process. Pancreatic,

trypsin, chymotrypsin, pepsin and rennin are the most important proteases of animal

origin. However, their production depends on the availability of livestock for

slaughter. Therefore, microbial proteases are preferred above enzymes from plant

and animal sources since they present most of the desired characteristics for

biotechnological applications (Ganesh and Takagi, 1999).

Proteases are being an industrial candidate, which are widely used in food,

bakery, and beverage and detergent industry. In leather industry, alkaline proteases

are exhibiting a prominent role in unhairing and bating processes. An extensive use

of filamentous fungi, especially Aspergillus species has been studied elaborately.

Although, the significant application of alkaline protease produced from these

strains in leather industry is being limited. A. flavus and A. terreus found as the

potential strains for production of tannery protease in submerged fermentation. To

improve the productivity of this enzyme in liquid broth, various media ingredients

have been optimized. The crude and partially purified proteases preliminarily

characterized and used for unhairing processes at lab scale in tannery. The protease

obtained from these strains showed the good activity in wide alkaline condition

suggesting the possibility of using in leather and detergent industry (Chellapandi,

2010).

32

For industrial production of alkaline protease, technical media are usually

employed that contained very high concentration of carbohydrates, proteins, and

other media components. To develop an economically feasible technology, research

efforts are mainly focused on: (1) improvement in the yields of alkaline proteases;

and (2) optimization of the fermentation medium and production conditions in

industrial scale. For this purpose, soybean meal (Glycine max) was recognized as a

potentially useful and cost-effective medium ingredient, because is is produced as

by-product of oil industry. Soybean meal contains 40% protein and rich in other

organic and inorganic components. Moreover, the production process was scaled up

in different level from shake flask up to 500-L stirred tank bioreactor (Kaur et al.,

2001; Aijun et al., 2005).

Proteases represent one of the 3 largest groups of Industrial enzymes and find

application in detergents, leather industry, food industry; pharmaceutical industry

and bioremediation processes (Anwar and Saleemuddin, 1998; Gupta et al., 2002a).

Their importance in conducting the essential metabolic and regulatory functions is

evident from their occurrence in all forms of living organisms (Wandersman, 1989).

Extracellular proteases are important for the hydrolysis of external proteins and

enable the cell to absorb and utilize the hydrolytic products (Kalisz, 1988). They are

widely distributed in nature. Microorganisms are the most preferred source of these

enzymes in fermentation bioprocesses not only because of their fast growth rate but

also for their ability to engineer genetically to generate new enzymes with desirable

abilities or simply for enzyme overproduction (Rao et al., 1998). Microbial

proteases play an important role in biotechnological processes and they account for

approximately 59% of the total enzymes used (Spinosaa, 2000).

In recent years the potential of using microorganisms as biotechnological

sources of industrially relevant enzymes has stimulated interest in the exploration of

extracellular enzymatic activity in several microorganisms (Akpan et al., 1999;

33

Pandey et al., 2000). Amylases are important enzymes employed in the starch

processing industries for the hydrolysis of polysaccharides such as starch into

simple sugar constituents (Akpan et al., 1999; Mitchell and Lonsane, 1990).

Although amylases can be obtained from several sources, such as plants and

animals, the enzymes from microbial sources generally meet industrial demand

(Pandey et al., 2000).

Green mussel (Perna viridis) was screened for protease production by

culturing on skim milk agar containing 5.0 and 10.0 mM of PMSF (phenyl methyl

sulphonyl fluoride). the applications of alkaline serine protease in detergent and

solvent industry were tested and it was revealed that the purified enzyme can be

used as an additive in detergent Industry (Padmapriya et al., 2012). Actinomycete

being a protease producing bacteria has the potential for use in industrial purpose,

pharmaceuticals, cytotoxic agent and its proteolytic activity (Balachandran et al .,

2012).

Forty six strains of actinomycetes were isolated from the soil collected from

Northern Himalayas, India. Isolation of actinomycetes was performed by serial

dilution plate technique. Forty six isolated actinomycetes cultures were grown in

ISP 2 medium to study the morphology and biochemical characteristics. Isolated

strains were studied for protease enzyme production in skim milk agar medium with

solubilising capacity. Seven isolates were studied for melanin pigmentation and

different NaCl concentration. Effects of environmental conditions on protease

enzyme production of seven isolated strains were also studied at different pH,

temperature and metal ions (mercaptoethanol, dithiothreitol, iodoacetamide,

MgSO4, CaCl2 and EDTA). Seven isolates were also studied for lytic enzyme

activity using different bacteria and yeast such as Pseudomonas aeruginosa,

Enterococcus feacalis, Escherishia coli, Candida albicans, Bacillus subtilis,

Klebsiella pneumonia and Staphylococcus aureus. Isolates ERIA-31 and ERIA-33

34

produced more protease enzyme activity in modified nutrient agar media compared

to other actinomycetes cultures. ERIA-31 and ERIA-33 were tested for cytotoxic

effect in human adenocarcinoma cancer cell line (A549). IC50 for ERIA-31 was

57.04 µg /mL and IC50 for ERIA-33 was 55.07 µg/mL. Therefore ,Actinomycete

being a protease producing bacteria has the potential for use in industrial purpose,

pharmaceuticals, cytotoxic agent and its proteol (Balachandran et al., 2012) .

3.8. Industrial Applications of Alkaline Proteases from Bacillus Strains

3.8.1. Food Industry

Food industry represents one of the economic sectors where microbial

metabolites have found a wide variety of applications. This is the case of some

enzymes, such as amylases, cellulases, pectinases and proteases which have played

a very important role as food additives. Most of these enzymes have been produced

by submerged cultures at industrial level. However, the production and application

studies of those enzymes produced by solid state fermentations are scarce in

comparison with submerged fermentation (Kumar et al., 2005).

Alkaline proteases can hydrolyse proteins from plants and animals to produce

hydrolysates of well-defined peptide profiles. These protein hydrolysates play an

important role in blood pressure regulation and are used in infant food formulations

specific therapeutic dietary products and the fortification of fruit juices and soft

drinks.In recent years there has been substantial interest in developing enzymatic

methods forthe hydrolysis of soya protein, gelatin, casein, whey and other proteins

in order to prepare protein hydrolysates of high nutritional value. In developing

commercial products from these proteins, emphasis is placed on achieving a

consistent product in high yields, having desirable flavour, nutritional and/or

functional properties (Ward,1991).

Alkaline protease from B. licheniformis is used for the production of highly

functional protein hydrolysates (Ward, 1991). This commercial alkaline protease,

35

Alcalase, has a broad specificity with some preference for terminal hydrophobic

amino acids. Using this enzyme, a less bitter hydrolysate and a debittered enzymatic

whey protein hydrolysate were produced (Kumar and Takagi, 1999). Soluble meat

hydrolysate can also be derived from lean meat wastes and from bone residues after

mechanical deboning by solubilization with proteolytic enzymes. Alcalase has been

found to be the most appropriate enzyme in terms of cost, solubilization, and other

relevant factors. In an optimized process with Alcalase at a pH of 8.5 and

temperature of 55-60ºC, a solubilization of 94 % was achieved. The resulting meat

slurry was further pasteurised to inactivate the enzyme and found wide application

in canned meat production, soups and seasoning (Kumar and Takagi, 1999).

Another alkaline protease from B. amyloliquefaciens resulted in the

production of a methionine-rich protein hydrolysate from chickenpea and soy

protein, which found major application in hypoallergenic infant food formulations

(Kumar and Takagi, 1999). In another study, Rebecca et al., (1991) reported the

production of fish hydrolysate of high nutritional value, using B. subtilis proteases.

Perea et al., (1993) on the other hand used alkaline protease for the production of

whey protein hydrolysate, using cheese whey in an industrial whey bioconversion

process.

3.8.2. Detergent Industry

Proteases to be used as detergent additive should be stable and active in the

presence of surfactants, bleaching agents, bleach activators, fillers, fabric softeners

and various other formulation of a typical detergent. In textile industry, proteases

may also be used to remove the stiff and dull gum layer of sericine from the raw silk

fiber to achieve improved luster and softness. Protease treatment also modifies the

surface of wool and silk fibers to provide unique finishes. The alkaline proteases

also have potential application in removal of gelatin from the used photographic

films vis-à-vis recovery of silver from them (Anwar and Saleemuddin, 1998).

36

Enzymes have long been of interest to the detergent industry for their ability

to aid the removal of proteinaceous stains and to deliver unique benefits that cannot

otherwise be obtained with conventional detergent technologies (Gupta et al.,

2002b). The use of enzymes in detergent formulations is now common in developed

countries, where more than half of the presently available ones contain enzymes

(Chaplin and Bucke, 1990). Detergent enzymes account for approximately 30% of

total worldwide enzyme production (Horikoshi, 1996) and 89% of the total protease

sales in the world; a significant share of the market is captured by subtilisins and/or

alkaline proteases from many Bacillus species (Gupta et al., 2002b).Ideally alkaline

proteases used in detergent formulations should have high activity and stability over

a broad range of pH and temperature, should be effective at low levels (0.4-0.8%)

and should also be compatible with various detergent components along with

oxidizing and sequestering agents. They must also have a long shelf life (Kumar and

Takagi, 1999).

Alkaliphilic Bacillus strains are good sources of alkaline proteases with the

properties that fulfil the essential requirements to be used in detergents; therefore

themain industrial application of alkaliphilic proteases has been in the detergent

industry since their introduction in 1914 as detergent additives. (Ito et al., 1998;

Horikoshi, 1996). The major use of detergent–compatible alkaline proteases is in

laundry detergent formulations. Detergents available in the international market

such as Dynamo®, Eraplus® (Procter & Gamble), Tide® (Colgate Palmolive),

contain proteolytic enzymes,the majority of which are produced by members of the

genus Bacillus (Anwar and Saleemuddin ,1998).

The main producers of alkaline proteases using species of Bacillus are the

companies such as Novo Industry A.I.S. and Gist Brocades. Novo produces three

proteases, Alcalase from B. licheniformis, Esperase from an alkalophilic strain of a

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B. licheniformis and Savinase from an alkalophilic strain of a B. amyloliquefaciens.

Gist Brocades on the other hand produces and supplies Maxatase from B.

licheniformis. Alcalase and Maxatase (both mainly subtilisin) are recommended to

be used at 10-65ºC and pH 7-10.5. Savinase and Esperase can be used at up to pH

11 and 12 respectively (Chaplin and Bucke, 1990).

Conventionally, detergents have been used at elevated washing temperatures,

but at present there is considerable interest in the identification of alkaline proteases,

which are effective over wider temperature ranges (Oberoi et al., 2001). For

example there is considerable current interest on the exploration of proteases that

can catalyse reactions in cold water. This allows their use in detergents, which can

be used in normal tap water without the requirement for increasing the temperature

of the water. The search for such enzymes is very much a challenge at this time

(Haki and Rakshit, 2003).

Banerjee and his colleagues (1999) have studied on an alkaline protease from

a facultative thermophilic and alkalophilic strain of Bacillus brevis. The alkaline

protease from B. brevis having maximum activity at pH 10.5, showed a high level of

thermostability at 60ºC. The enzyme showed compatibility at 60ºC with all of the

commercial detergents tested. It could also remove blood strains completely when

used with detergents. All the tests were studied in the presence of Ca2+ and glycine

and the data obtained in this study implies that the protease of B. brevis has most of

the properties to be used as a detergent enzyme. In another study, Gupta and his

friends (1999) reported a bleach-stable & thermotolerant alkaline protease from a

new variant of Bacillus sp., having potential application in detergent formulations.

The alkaline protease from newly isolated Bacillus SB5 displayed stability in

the presence of 10% (v/v) H2O2 (oxidizing agent) and 1% SDS (sodium dodecyl

sulphate, surfactant). The enzyme had an optimum activity at pH 10 and 60ºC to

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70ºC, where this was further increased in the presence of all ionic and non-ionic

detergents, surfactants and commercial detergents tested (Gupta et al., 2002b).

Oberoi and his colleagues (2001) also produced an alkaline, SDS-stable

protease from Bacillus sp. RGR-14 that was also suggested to be an ideal detergent

additive for detergent formulations. The enzyme was active over a wide temperature

range in alkaline conditions. In addition being SDS-stable it was also stable towards

oxidizing agents such as H2O2 and sodium perborate. The alkaline protease from B.

clausii I-52 is significant for industrial perspective because of its ability to function

in broad pH and temperature ranges in addition to its tolerance and stability in

presence of an anionic surfactant like SDS and oxidants like peroxides and

perborates. The enzymatic properties of this protease therefore suggest its suitable

application as additive in detergent formulations (Joo et al., 2003).

The in place cleaning of ultrafiltration (UF) and reverse osmosis (RO)

membranes forms one of the most important aspects of modern dairy and food

industries. However, the major limitation in the application of UF in the dairy

industry is the decline in flux with time due to fouling of the membranes. Thus,

protein deposition and precipitation of minerals were inferred as major contributors

to this phenomenon of fouling. An effective protease-based cleaning that can

hydrolyze milk protein into fragments small enough to be rinsed off from the UF

system, is required to overcome fouling problem (Kumar and Tiwari, 1999).

An alkaline protease was produced from an alkalophilic Bacillus strain MK5-

6. The ability of this alkaline protease to clean UF membranes fouled during milk

processing was determined in combination with two alkaline UF membranes

cleaner, Alconax and Ultrasil. The alkaline enzyme that is extremely stable in

Alcanox was optimal at 5 g/L in cleaning the fouled membranes when added to the

same membrane cleaner. For these reasons, they believe the alkaline protease

preparation from alkalophilic Bacillus strain MK5-6 as an attractive candidate to be

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used as membrane cleaner additive (Kumar and Tiwari, 1999). Durham produced

Subtilisin GX from alkaliphilic Bacillus sp. GX6644 (ATCC 53441) that was found

to possess properties suitable as a detergent additive (Durham, 1987).

3.8.3. Leather Industry

Alkaline proteases with elastolytic and keratinolytic activity can be used in

leather processing industries. During bating, the hide is softened by partial

degradation of the interfibrillar matrix proteins (elastin & keratin). Therefore

enzyme preparations with low levels of elastase and keratinase activity but no

collagenase activity are particularly applicable for thisprocess. (Cowan, 1994).

Bating is traditionally an enzymatic process involving pancreatic proteases.

However, recently, the use of microbial alkaline proteases has become popular. The

substitution of chemical depilatory agents in the leather industry by proteolytic

enzymes produced by Bacillus sp. could have important economical and

environmental impacts (Anwar and Saleemuddin, 1998) where the dehairing process

is accelerated by the use of alkaline proteases.

3.8.4. Medical usage

Alkaline proteases are also used for developing products of medical

importance. It was stated in Gupta et al., (2002b) that Kudrya and Simonenko

(1994) exploited the elastolytic activity of B. subtilis 316M for the preparation of

elastoterase, which was applied for the treatment of burns, purulent wounds,

carbuncles, furuncles and deep abscesses. Kim et al. (2001) reported the use of

alkaline protease from Bacillus sp. strain CK 11-4 as a thrombolytic agent having

fibrinolytic activity (Gupta et al., 2002b).Furthermore, Bacillus sp. has been

recognized as being safe to human. (Kumar and Takagi, 1999).

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3.8.5. Management of Industrial and Household Waste

Alkaline proteases provide potential application for the management of

wastes from various food processing industries and household activities. Feather is

composed of over 90% protein, the main component being keratin, a fibrous and

insoluble protein. Worldwide several million tons of feather is generated annually as

waste by poultry-processing industries. Feathers constitute approximately 5% of the

body weight of poultry and can be considered a high protein source for food and

feed, provided their rigid keratin structure is completely destroyed. Pretreatment

with NaOH, mechanical disintegration, and enzymatic hydrolysis results in total

solubilization of the feathers. The end product is a heavy, greyish powder with a

very high protein content, which could be used as a feed additive (Kumar and

Takagi, 1999).

Considering its high protein content, this waste could have a great potential

as a source of protein and amino acids for animal feed as well as for many other

applications. In some countries, feather is used as animal feed supplement in the

form of feather meal. Development of enzymatic and/or microbial methods for the

hydrolysis of feather to soluble proteins and amino acids is extremely attractive, as

it offers a cheap and mild reaction condition for the production of valuable products

(Gessesse et al., 2003). Gessesse et al. (2003) isolated an organism from an

alkaline soda lake in the Ethiopian Rift Valley Area and identified as Bacillus

pseudofirmus. B. pseudofirmus AL-89, and the protease it produces offers an

interesting potential for the enzymatic and/or microbiological hydrolysis of feather

to be used as animal feed supplement (Gessesse et al., 2003). Dalev (1994) reported

also an enzymatic process using a B.subtilis alkaline protease in the processing of

waste feathers from poultry slaughterhouses.

A formulation containing proteolytic enzymes from B. substilis, B.

amyloliquefaciens and Streptomyces sp. and a disulfide reducing agent

41

(thioglycolate) that enhances hair degradation, helps in clearing pipes clogged with

hair-containing deposits and is currently available in the market. It was prepared and

patented by Genex (Gupta et al., 2002b).

3.8.6. Photographic Industry

Alkaline proteases play a crucial role in the bioprocessing of used X-ray or

photographic films for silver recovery. These waste films contain 1.5-2.0% silver by

weight in their gelatin layer, which can be used as a good source of silver for a

variety of purposes. Conventionally, this silver is recovered by burning the films,

which causes undesirable environmental pollution. Furthermore, base film made of

polyester cannot be recovered using this method. Since the silver is bound to

gelatin, it is possible to extract silver from the protein layer by proteolytic

treatments. Enzymatic hydrolysis of gelatin not only helps in extracting silver, but

also in obtaining polyester film base that can be recycled (Gupta et al., 2002b).

Fujiwara and co-workers studied on this interesting application of alkaline

proteases. They reported the use of an alkaline protease to decompose the gelatinous

coating of X-ray films, from which silver was recovered (Horikoshi, 1999). Protease

B18’ had a higher optimum pH and temperature, around 13.0 and 85ºC. The enzyme

was most active toward gelatin on film at pH 10 (Fujiwara et al., 1991). Singh et

al., (1999) isolated an obligate alkaliphilic Bacillus sphaericus strain from alkaline

soils in the Himalayas, which produced an extracellular alkaline protease. The

alkaline protease of this strain efficiently hydrolysed the gelatin layer of used X-ray

films within 12 min at 50 ºC and at pH 11.0 (Singh et al., 1999).

3.8.7. Peptide Synthesis

Amino acids are of increasing importance as dietary supplements for both

humans and domestic animals. Only the L-amino acids can be assimilated by living

organisms, since the chemical synthesis of amino acids produces a racemic mixture,

it is necessary to separate the isomers before commercial use. Alcalase is a

42

proteolytic enzyme isolated from a selected strain of B. licheniformis, its major

component being subtilisin Carslberg. It was determined that Alcalase was stable in

organic solvents and could be of use as a catalyst in the solution of N-protected

amino acids having unusual side chains (Anwar et al., 1998).

3.8.8. Silk Degumming

One of the least explored areas for the use of proteases is in the silk industry

where only a few patents have been filed describing the use of proteases for the

degumming process of silk. The conventional silk degumming process is generally

expensive and therefore an alternative method suggested, is the use of protease

preparations for degumming the silk prior to dyeing. In a recent study, the silk

degumming efficiency of an alkaline protease from Bacillus sp. RGR-14 was

studied. After 5h of incubation of silk fiber with protease from Bacillus sp., the

weight loss of silk fiber was 7.5%. Scanning electron microscopy of the fibers

revealed that clusters of silk fibers had fallen apart as compared with the smooth and

compacted structure of untreated fiber (Gupta et al., 2002b).