INTRODUCTION:

The enzymes are considered as “green chemicals” due to their ecofriendly nature. Also, they possess wide range of applications ranging from industrial sector to house-hold products.⁴. Enzymes are delicate protein molecules necessary for life.³

Proteases, also termed proteinases or peptidases¹ are enzymes occurring everywhere in nature, be it inside or on the surface of living organisms such as plants, animals and microbes. These enzymes carry out proteolysis i.e. breakdown of proteins by hydrolysis of the peptide bond that exists between two amino acids of a polypeptide chain. Proteases are degradative enzymes which catalyze the total hydrolysis of proteins.⁷ Protease are the single class of enzymes which occupy a pivotal position due to their wide applications in detergent, pharmaceutical, photography, leather, food and agricultural industries. An important biotechnological application of protease is in bioremediation processes³.

Microorganisms regarded as an important source of proteases because they can be obtained in large quantities using cultural techniques within a shortest possible and they produce a regular and abundant supply of the desired product.⁵ Among various proteases, bacterial proteases are the most significant, compared to animal and fungal proteases.¹ Proteases also known as peptidyl-peptide hydrolase and constitute 60–65 % of the global enzyme market. Proteases are commercially important enzymes having a wide range of applications in various industrial, biotechnological, medicinal and basic research fields.² Though proteases are produced by a variety of bacteria such as Pseudomonas aeruginosa, Flavobacterium, Clostridium, Achromobacter, Thermo actinomyces and species belonging to Streptomyces, Bacillus sp. is the major source which secretes a variety of soluble extracellular enzymes.²

A myriad of Bacillus species from many different exotic environments have been explored and exploited for alkaline protease production but most potential alkaline protease producing bacilli are strains of B. subtilis.⁵ B.subtitils are involved in the enzyme industries and produce a variety of intracellular and extracellular proteases¹. Bacterial alkaline proteases are characterized by their high activity at alkaline pH, e.g., pH 10, and their broad substrate specificity. Their optimal temperature is around 60°C. These properties of bacterial alkaline proteases make them suitable for use in the detergent industry.⁵

The demand for proteolytic enzymes having appropriate specificity and stability to pH, temperature, metal ions, surfactants and organic solvent is now rising, stimulating the search for more new enzyme sources.⁴ Many researchers are moving towards exploring new sources for producing various enzymes. Conversion of wastes into useful biomass by microorganisms and their enzymes is a new trend, and new protease-producing microorganisms and perfected fermentation technology are needed to meet the ever-growing demand for this enzyme.⁴ Waste products of meat, poultry and fish processing industries can supply a large amount of protein rich material for bioconversion to recoverable products.

Research has been done on many other related sources for protease production; a few to mention are, slaughter house soil, dairy industrial effluent, market waste, sewage waste.⁴ Further physicochemical parameters for maximum protease production from B. subtilis were optimized followed by purification and characterization studies.⁴ It is now known that single amino acid mutations in more than 50 human proteases result in hereditary/genetic diseases. Studies suggest that proteases are responsible for the complex processes involved in the normal physiology of the cell as well as in abnormal pathophysiological conditions. Their involvement in the life cycle of disease- causing organisms has led them to become a potential target for developing therapeutic agents against fatal diseases such as cancer and AIDS.⁷ In this study an attempt was made to study isolation and selection of bacterial strains that is potent producer of proteases and optimization of culture conditions required for enzyme production.³

REVIEW OF LITERATURE:

Microbes are the good sources of proteases. Since, proteases are enzymes of metabolic as well as commercial importance, there is vast literature on their biotechnological and biochemical aspect (ward, 1983; Fox et al, 1991 and Morihora and Oda, 1993). Bacillus species have been successfully used in degradation of proteinaceous wastes into useful biomass of proteases have also been demonstrated by many investigators. Bacillus species are the prolific producers of extracellular hydrolytic enzymes and in particular their production of protease (Godfrey and Reichet, 1983).

Microbial proteases are among the most important hydrolytic enzymes that have been studied extensively since the advent of enzymology. There is renewed interest in the study of proteolytic enzymes, mainly due to the recognition that these enzymes not only play an important role in the cellular metabolic processes but have also gained considerable attention in the industrial community. These enzymes have become widely used in the detergent industry, since their introduction in 1914 as detergent additives (Guptal et.al.,2002).

Microorganisms elaborate a large array of proteases, which are intra cellular and/or extra cellular. Intra cellular proteases are important for various cellular and metabolic processes, such as sporulation and differentiation, protein turnover, maturation of enzymes and hormones and maintenance of the cellular protein pool. Extra cellular proteases are important for the hydrolysis of proteins in cell free environments and enable the cell to absorb and utilize hydrolytic products (Kalisz, 1998). At the same time, these extra cellular proteases have also been commercially exploited to assist protein degradation in various industrial processes (Outtrup and Boyce, 1990).

Today proteases account for approximately 40% of the total enzyme sales in various industrial market sectors, such as detergent, food, pharmaceutical, leather, diagnostics, waste management and silver recovery (Gupta etal.,2002). Microbial proteases have been reviewed several times with emphasis on different aspects of proteases. Aunstrup (1980) focused on microbial selection and fermentation, whereas Ward (1985) mainly dealt 21 with the sources of microbial proteases and their possible functional role in nature. Outtrap and Boyce (1990) focused on industrially important proteases, their applications and the role of molecular biology in protease research. The bioindustrial viewpoints of microbial alkaline proteases from sources to cellular role, production, downstream processing, characterization and commercial application have also been reviewed (Anwar and Saleemuddin, 1998).

An alkaline protease isolated from Bacillus species has optimum conditions of temperature and pH for activity (Krishna Suresh Babu et al., 2005).

This review article is based on the work of different researchers who worked on Bacillus subtilis for the optimized production of protease enzyme. The bacterial isolate was isolated from three different sources. The isolate from sea water showed optimum production of the enzyme at 37ºC and pH 9. The isolate from the abattoir waste had optimized production at 40ºC and pH 14. The isolate from the soil showed the optimized production of the enzyme at 40ºC and pH 8. The results clarified that the same species when taken from different areas and placed in a growing medium in lab, needed different parameter conditions to grow and survive. The protease assay proved that the sea water isolate produced the high amount of protease in comparison to other two sources. Hence, according to the result of the present study, sea water is the most suitable medium for production of proteases in high amount and their optimization in industries for future purposes.

In view of the recent trend of developing environmentally friendly technologies, proteases are expected to have extensive applications in leather treatment and in several bioremediation measures. Proteases have gained commercial importance because of their potential applications in a wide range of industries including detergents, food and feed, leather, medical, diagnostic, pharmaceutical, waste management and recovery of silver from X-ray films. The applications of proteases in future may meet the need for a stable biocatalyst capable of withstanding harsh conditions of treatment. Because of the importance of microbial proteases in various industries, it is necessary to search for new enzymes at this time. The present study has taken up with a view to find out new microbial protease which is useful industrially. Objectives of the work included:

1. The isolation of efficient protease producing bacteria.

2. Morphological and biochemical characterization of efficient bacterial isolate.

3. Optimization of physiological conditions for enhanced protease production.

MATERIALS:

Media preparation

Zobell Marine Medium (HIMEDIA laboratories)

Ingredients Gms / Litre	Ingredients Gms / Litre
Yeast extract 1.000 Ferric citrate 0.100 Sodium chloride 19.450 Magnesium chloride 8.800 Sodium sulphate 3.240 Calcium chloride 1.800 Potassium chloride 0.550 Sodium bicarbonate 0.160	Potassium bromide 0.080 Strontium chloride 0.034 Boric acid 0.022 Sodium silicate 0.004 Sodium fluorate 0.0024 Ammonium nitrate 0.0016 Disodium phosphate 0.008 Agar 15.000 Final pH (at 25°C) 7.6±0.2
Nutrient agar medium (HIMEDIA laboratories)	Gelatin agar medium (HIMEDIA laboratories):
Ingredients Gms/Litre	Ingredients Gms / Litre
Sodium chloride 5.000 Beef extract 1.500 Yeast extract 1.500 Agar 15.000 Final pH (at 25°C) 7.4±0.2	Gelatin 30.000 Casein enzymic hydrolysate 10.000 Sodium chloride 10.000 Agar 15.000 Final pH (at 25°C) 7.2±0.2

Materials and Reagents:

Gelatin yeast extract broth (Gelatin, glucose, yeast extract, potassium hydrogen phosphate), sucrose, fructose, cellulose, lactose, starch, xylose, glucose, dextrose, ammonium nitrate, ammonium chloride, ammonium sulphate, yeast extract, potassium nitrate, sodium nitrate, peptone, ferrous sulphate, potassium dihydrogen phosphate, magnesium phosphate, sodium chloride, tris-buffer, aqueous casein solution, trichloroacetic acid, sodium carbonate, folin phenol reagent, beef extract, yeast extract, urea, peptone, potassium chloride, magneese chloride, magnesium chloride, copper sulphate, magnesium sulphate, phosphate buffer, gram’s stain.

Glassware’s used:

Test tubes, petri plates, beaker, glass rod, pipette, conical flask, culture tubes, cuvette.

Instruments:

Centrifuge, spectrophotometer, incubator, automated DNA sequencer, shaker, fermenter, autoclave.

Protocol:

Sample collection: (1,2,3) Samples were Collected from Different Locations as Follows:

S No.	Source	Location
1.	Sea water	Cuddalore coast, Tamil Nadu¹
2.	Soil sample	Different places in Banglore.³
3.	Abattoir waste	Local meat shop, Wadala, Mumbai.⁴

Isolation and screening of protease producing bacteria.^1,2,3

The sample were isolated and cultured in lab in the following medium and the plates were incubated:

	Sea water	Soil sample	Abattoir waste
Medium	Zobell marine agar medium	Nutrient agar medium	Gelatin agar medium
Incubation	37ºC, 5 days	38ºC, 2 day	37ºC, 1 day

Identification of protease producing bacteria:^1,2,3

· The bacterial isolate was first identified by using Gram’s staining method.⁴

· The morphological and physiological characteristic of bacterial isolate was studied according to Bergey’s manual of determinative bacteriology.⁴

· Taxonomic identification based on above tests were carried out using 16s rRNA method.⁴

Optimization of selected isolates:^1,2,3

	Sea water	Soil sample	Abattoir waste
temperature	20ºC, 30ºC, 40ºC, 50 ºC, 60ºC, 70ºC, 80ºC	25ºC, 37 ºC, 40 ºC	20ºC, 30ºC, 40ºC, 50ºC, 60ºC, 70ºC, 80ºC
pH	3-12	6-9	3, 5, 7, 9, 10-14
Nitrogen sources	Yeast extract, beef extract, peptone, urea, ammonium chloride, sodium nitrate, ammonium sulphate	Beef extract, tryptone peptone	Ammonium nitrate, ammonium chloride, ammonium sulphate, yeast extract, potassium nitrate, sodium nitrate.
Carbon sources.	Starch, glucose, maltose, lactose, xylose, fructose.	Dextrose, sucrose, lactose, maltose	Sucrose, fructose, glucose, cellulose, lactose, starch
Sodium chloride.	1-10%
Metal ion.	CaCl2, MnSO4, CuSO4, KCl, MgCl2

Note: The Strain Bacillus Subtilis Extracted from Sea Water Existed in A Marine Habitat That is Why Protease Production Increased in Presence of Nacl and Decreased in its Absence.

Enzyme Production:

1. Sea water:¹

· Medium: Glucose (0.5g), peptone (1g), ferrous sulphate (0.1g), magnesium sulphate (0.5g), sodium chloride (3g).

· 10ml of medium was taken in a 100ml flask and sterilized in autoclave at 121ºC for 15 min and after cooling the flask was inoculated with overnight grown bacterial culture.

· The culture was then incubated at 37ºC in shaker for 48 hrs.

· At the end of fermentation period, the culture medium was centrifuged at 5000rpm for 15min to obtain the supernatant.

2. Soil sample:³

· Medium: dextrose (1%), peptone (0.5%), potassium dihydrogen phosphate (0.2%), magnesium sulphate (0.2%), casein (1%).

· It was maintained at 37 ºC for 48hrs in a shaking incubator.

· After inoculation, fermentation was carried out at 37 ºC at 200 rpm for 48 hrs.

· At the end of each fermentation period, the whole culture broth was centrifuged at 10,000 rpm for 15 min to remove cellular debrises and clear supernatant was used as enzyme.

3. Abattoir waste:²

· Medium: gelatin (1%), glucose (1%), yeast extract (0.2%), dipotasium hydrogen phosphate (0.3%).

· It was incubated at 40 ºC upto 32hrs in an orbital shaker at 150 rpm.

· The contents were then centrifuged at 5000 rpm at 4 ºC for 20 min.

Enzyme Purification:

The purification of the enzyme was done by ammonium sulphate precipitation method and column chromatography.^3,2

Protease assay:

To study proteoloytic activity, supernatant was used as enzyme source. 1%casein in 0.1 M phosphate buffer and pH 7.0) was used as substrate. 1ml enzyme and substrate was incubated at 50 ºC for 60 min. To stop the reaction 3ml Trichloroacetic acid was used. One unit of protease activity was defined as the increase of 0.1-unit optical density at 1 hr incubation period. Then it was centrifuged at 5000 rpm for 15 min. From this ,0.5ml of supernatant was taken, to this 2.5ml of 0.5 M sodium carbonate was added, mixed well and incubated 20 min. Then it was added with 0.5ml of folin phenol reagent and the absorbance was read at 660 nm using Spectrophotometer (Bharat Pokhrel et al, 2014.). The amount of protease produced was estimated and expressed in microgram of tyrosine released under standard assay conditions. Based on the tyrosine released the protease activity.¹⁰

Techniques Used Above: 16s rRNA gene sequencing method:

The presence of hyper variable regions in the 16S rRNA gene provides a species-specific signature sequence which is useful for bacterial identification process. 16S Ribosomal RNA sequencing is widely used in microbiology studies to identify the diversities in prokaryotic organisms as well as other organisms and thereby studying the phylogenetic relationships between them.

Steps in Ribosomal RNA Sequencing:

· Extraction of DNA

· Polymerase Chain Reaction

· Agarose Gel Electrophoresis

· Elution of DNA

· Radiolabeling Technique

· Restriction Digestion

· Southern Blotting

· Autoradiography

Figure-1

Column Chromatography:

Column chromatography is one of the most useful methods for the separation and purification of both solids and liquids. This is a solid - liquid technique in which the stationary phase is a solid and mobile phase is a liquid. The principle of column chromatography is based on differential adsorption of substance by the adsorbent

Figure-2

Steps for Column Chromatography:

Preparation of the Column:

· Place the column in a ring stand in a vertical position.

· A plug of glass wool is pushed down to the bottom of the column.

· Prepare slurry of silica gel with a suitable solvent and pour gently into the column.

· Open the stop cock and allow some solvent to drain out. The layer of solvent should always cover the adsorbent; otherwise, cracks will develop in the column.

Adding the Sample to the Column:

· Dissolve the sample mixture in a minimum amount of solvent (petroleum ether).

· Remove the solvent by placing the mixture in a rotary evaporator at a low temperature.

· Place the dry powder on a piece of weighing paper and transfer it to the top of the column through the funnel.

Developing the Chromatogram:

· Attach a dropping funnel filled with petroleum ether on to the column. Add petroleum ether continuously from the funnel to the top of the column. Open the stopcock carefully.

· The components of the mixture run down the column forming two separate yellow bands.

Recovering the Constituents:

· Continue running the petroleum ether till both the bands are eluted out separately.

· Collect the constituents in two different R.B flasks. (Ortho nitrophenol is obtained first, followed by para nitro phenol.).

· Evaporate the solvent by placing the mixture in a rotary evaporator

Polymerase Chain Reaction (PCR):

In this technique only the DNA of the organism is examined, not the entire viable microorganism, as a result, the pathogenic microorganism can also be evaluated. Valuable genetic information about the microorganisms can be obtained quickly.

Steps in the PCR process:

A technique for amplification of a specific fragment of DNA of interest by a series of successive cycles. By this process, a single molecule of template DNA can generate over a billion copies of itself after 30 cycles of exponential replication. There are three phases in the cycle, each of which occurs at a different temperature. The phases can be performed in an instrument known as thermocycler, which provides these different temperatures.

Figure-3

1. Denaturation: During this process, the double helical arrangement of the sample DNA (template DNA) is denatured at a temperature of about 94°C- 95°C. The two strands get separated out.

2. Annealing: In this step, the primers, which are the sequences of DNA added to the reaction mixture anneal with the complementary (similar or matching) sequences in the template DNA. This occurs at different temperatures.

3. Extension: This is the final stage of the PCR cycle, occurs at 72°C when the enzyme Polymerase added to the reaction mixture, make the primers extend along the length of the DNA strand.

SDS-PAGE:

The proteins being covered by SDS are negatively charged and when loaded onto a gel and placed in an electric field, it will migrate towards the anode (positively charged electrode) are separated by a molecular sieving effect based on size. After the visualization by a staining (protein-specific) technique, the size of a protein can be calculated by comparing its migration distance with that of a known molecular weight ladder (marker).

Figure-4

Steps of SDS-PAGE:

Assembling the Glass Plates:

1. Assemble the glass plate on a clean surface. Lay the longer glass plate (the one with spacer) down first, then place the shorter glass plate on top of it.

2. Embed them into the casting frame and clamp them properly Make sure that the that the bottom ends of the glass plates are properly aligned.

3. Then place it on the casting stand.

Casting the gels:

Prepare 10% of resolving gel and 4.5% of stacking gel.

1. Prepare the separating gel solution by combining all reagents. Do not add Ammonium per sulfate and TEMED.

2. Add APS and TEMED to the monomer solution (just before pouring) and mix well by swirling gently. Pour the solution till the mark. (It is ok if you introduce air bubbles, add a layer of isopropanol or distilled water on top of the gel so as to level the poured gel.)

3. Allow the gel to polymerize for 20-30 minutes.

4. Prepare stacking gel. Mix all reagents except APS and TEMED. Drain the isopropanol with strips of filter paper.

5. Add APS and TEMED to the monomer solution (just before pouring) and mix well by swirling gently. (Make sure you keep the comb ready by the side.)

6. Place a comb in the stacking gel sandwich. Allow it to polymerize for 10 minutes.

Preparation of Samples:

Mix your protein in the ratio 4:1 with the sample buffer. Heat your sample by either:

a) Boiling for 5-10 minutes. (Works for most proteins.)

b) 65°C for 10 minutes.

c) 37°C for 30 minutes.

Running the Gel:

1. To assemble, take out the gels from the casting frame and clamp them in the gel apparatus. (Make sure that the short plate always faces inside and if you have got only one gel to run use the dummy plate that is available to balance).

2. When the plates are secured, place them in the cassette and then lock it.

3. Place them in the gel running tank.

4. Fill the inner chamber of the tank with buffer. (Now it is easy to remove the comb, since it is lubricated).

5. Remove the comb carefully (without breaking the well). [Now the gel is ready to load the samples]

6. Rinse the loading tip a few times with distilled water. (Make sure that all the water is poured out before loading the samples.)

7. Insert the loading tip to a few mm from the well bottom and deliver the samples into the well. Rinse the syringe with distilled water after loading for a few times.

8. Attach the power supply by putting the lid (Make sure that the connection is in correct way i.e., black black and red - red). Set the voltage upto 180 V and run for 1 hour. (Don't allow the dye front to go out of the gel).

Staining the Gel: After running, switch off the power supply and take out the gel plates, remove the gel. Place the gel in the staining solution for 30 minutes. Destain the gel until the bands are properly seen. Determine the approximate molecular weight of the visualised protein bands by comparing them with the molecular weight ladders(markers).

Folin- lowry Assay: Lowry’s assay for total protein is one of the most commonly performed colorimetric assays. This procedure is sensitive because it employs two colour forming reactions. It uses the Biuret reactions in which Cu2+ in presence of a base reacts with a peptide bond of protein under alkaline conditions resulting in reduction of cupric ions (Cu2+) to cuprous ions (Cu+), and Lowry’s reaction in which the Folin Ciocaltaeu reagent which contains phosphomolybdic complex which is a mixture of sodium tungstate, sodium molybdate and phosphate, along with copper sulphate solution and the protein, a blue purple colour is produced which can be assessed by measuring the absorbance at 650-700nm.

Figure-5

Working Steps:

1. Arrange the reagent solutions prepared, on the table.

2. Label the test tubes with the volume taken and arrange them in a test tube rack.

3. Pipette out the standard protein solution from the standard flask into the test tubes labelled [0.2ml-1ml].

4. Pipette a known volume of the unknown solution to the tube labelled “unknown” arranged in the test tube rack.

5. To the test tube labelled ‘Blank’, add 1ml of distilled water using a micropipette.

6. Volume in each test tube is made up to 1 ml by adding distilled water.

7. Add 5ml of alkaline copper reagent to all the test tubes. Vortex and incubate for 10 minutes at room temperature.

8. The solution in all the test tubes has turned blue in colour.

9. After incubation, add 600ul of Folin’s Ciocalteau reagent to all test tubes using micropipette. Vortex and incubate for 20 minutes at room temperature.

10. After incubation, the color intensity varies accordingly with the concentration of protein present in the tubes.

11. Now record the absorbance of each solution at 650 nm using a colorimeter.

12. Plot the absorbance against amount of protein in milligrams to get a standard calibration curve. Check the absorbance of unknown sample and determine the concentration of the unknown sample from the standard curve plotted.

RESULT AND DISCUSSION:

In the present study, the protease producing strain Bacillus subtilis was isolated from three different sources viz., sea water, soil and abattoir waste.the morphological and biochemical tests revealed that it is a gram-positive bacterium. The strains were provided with different growing mediums for optimized production of the protease.

Isolation of Bacterial Culture: In the present study, the bacterial strains were isolated from different sources and plated on separate medium

Sea water	Soil sample	Abattoir waste.
Zobell marine agar medium	Nutrient agar medium	Gelatin agar plates

Screening of Protease Producing Organisms: In the present study, the isolated colonies were incubated at different parameters and then used for further studies.

Sea water	Soil sample	Abattoir waste.
37ºC, 5 days	35ºC, 2 day	37ºC, 1 day

Effect of pH on Protease Production: In the present study the effect of pH by Bacillus subtilis revealed that optimum pH all the strains were different and they showed optimum protease production at different levels.

Sea water	Soil sample	Abattoir waste.
9	8	14

Figure 6: Effect of pH on protease production from Bacillus subbilis

Figure 7: Optimization of production media for various pH

Figure 8: Optimization of production media for various temperatures

Effect of temperature on enzyme production:

In the present study, the production medium of all strains were supplied with various temperature levels according to which maximum yield by each strain was seen at that the following temperatures.

Sea water	Soil sample	Abattoir waste
37 ºC	40 ºC	40 ºC

Figure 9: Effect of temperature on protease production from Bacillus subtitis

Figure 10: Optimization of production media for various temperatures

Figure 11

Effect of carbon source on protease activity: In the present study, many different carbon sources were used according for all the strains. Among which the following sources proved to be the most significant source for the given below strains

Sea water	Soil sample	Abattoir waste
glucose	dextrose	glucose

Figure 12: Effect of Carbon sources on the activity of protease enzyme from Bacillus subtilis

Figure 13: Optimization of production media for various carbon sources

Figure 14

Effect of nitrogen source on protease activity:

In the present study, the supplementaed nitrogen source enhanced the production of enzyme. The highest production of protease were seen on the following sources.

Sea water	Soil sample	Abattoir waste
Beef extract	peptone	Yeast extract

Figure 15 : Effect of nitrogen sources on protease production from Bacillus subtilis

Figure 16 : Optimization of production media for various nitrogen sources

Figure 16.

Effect of NaCl on protease activity: One of the isolate used in the present study was isolated from sea water and due to this reason the medium of this strain showed reduced protease production in the absence of sodium chloride whereas increased production in the presence of it. The maximum yied was seen at 7% NaCl.

Enzyme production and purification: The sample were inoculated in enzyme production medium and were undergone with fermentation period at certain parameters. Centrifugation was followed by the fermentation period. The supernatants were used as enzyme sources and were purified by column chromatography.

Protease assay: Amount of protease produced according with the parameters optimally used. The results revealed that thebest and highest amount of protease was produced by strains isolated from sea water.

	Sea water
pH	123.5 U/ml
temperature	117.4 U/ml
Carbon	199.01 U/ml
Nitrogen	118.42 U/ml

Figure 17.

APPLICATIONS:

Proteases have a large variety of applications, mainly in the detergent and food industries. In view of the recent trend of developing environmentally friendly technologies, proteases are envisaged to have extensive applications in leather treatment and in several bioremediation processes. The worldwide requirement for enzymes for individual applications varies considerably. Proteases are used extensively in the pharmaceutical industry for preparation of medicines such as ointments for debridement of wounds, etc. Proteases that are used in the food and detergent industries are prepared in bulk quantities and used as crude preparations, whereas those that are used in medicine are produced in small amounts but require extensive purification before they can be used.⁹

Proteases execute a large variety of functions, extending from the cellular level to the organ and organism level, to produce cascade systems such as haemostasis and inflammation, which are responsible for the complex processes involved in the normal physiology of the cell as well as in abnormal pathophysiological conditions. Their involvement in the life cycle of disease- causing organisms has led them to become a potential target for developing therapeutic agents against fatal diseases such as cancer and AIDS. Microbial proteases are increasingly used in treatment of various disorders namely cancer, inflammation, cardiovascular disorders, necrotic wounds etc. Proteases are used an immune–stimulatory agents. Increased antibiotic concentration at a target site when protease was concomitantly used with an antibiotic. Proteases are used extensively in the pharmaceutical industry for preparation of medicines such as ointments for debridement of wounds. It is also used in denture cleaners and as contact-lens enzyme cleaners. Proteases have a large variety of applications, mainly in the detergent and food industries. Proteases are envisaged to have extensive applications in leather treatment and in several bioremediation processes. Proteases that are used in the food and detergent industries are prepared in bulk quantities and used as crude preparations; whereas those that are used in medicine are produced in small amounts but require extensive purification before they can be used.⁶

The food industries are the major protease using industries. However, they have also found widespread application in laundry detergents. The thermo stability and their activity at high pH and the alleviation of pollution characteristic have made proteolytic enzymes an ideal candidate for laundry applications. Alkaline proteases are supplemented in different brands of detergents for use in home and commercial establishments. Enzymes have been added to laundry detergents since last 50 years to facilitate the release of proteinaceous material in stains such as those of milk and blood. The proteinaceous dirt coagulates on the fabric in the absence of proteinases as a result of washing condition. The enzyme removes not only the stain, such as blood, but also other materials including proteins from body secretion and food such as milk, egg, fish and meat. An ideal detergent enzyme should be stable and active in the detergent solution and should have adequate temperature stability to be effective in a wide range of washing temperature.

Usually the surgical instruments are washed or cleaned by sterilization or by using chemical steriliants. However, chemical steriliants cannot remove microbes that usually get trapped behind the bioburden that is encrusted on or within surgical instruments. However, the recent technologies include enzyme-containing formulations and zeolite based detergents. Of these, the enzyme detergents often referred to as “Green Chemicals” are proving useful in keeping a check on the environmental pollution and thus improving ecological situation. In leather industry, removal of hair and unwanted adhering subcutaneous layer by chemicals causes a problem. Hence the need for alternatives to sulphide dehairing is being sought.

Tanners are hesitant to use the enzyme because of certain disadvantages in using them at commercial level for reasons of the stability of the enzyme at different environmental conditions such as pH, temperature and duration consistent performance and the cost of production and application. The important factor in choosing an enzyme as a dehairing agent depends on the specificity of the enzyme used, which should not attack the collagenous matter. Numerous studies carried out from time to time to recover silver from photographic films as well as from x-ray films are patented. The silver recovery methods from these wastes includes: burning the films directly oxidation of metallic silver followed by electrolysis stripping the silver-gelatin layer using microbial enzymes specifically alkaline proteases and stripping the gelatin silver layer using different chemicals. Recovery of silver by burning the films creates environmental pollution and health hazards. On the other hand, enzyme from microbial source breaks the gelatin layer embedded with silver in films creating pollution free stripping. The amount of silver varies from 515g/kg of film. Enzymatic method although slow is free from pollution and cost-effective too.

The use of proteases in the food industry dates back to antiquity. They have been routinely used for various purposes such as cheese making, baking, preparation of soya hydrolysates, and meat tenderization. The major application of proteases in the dairy industry is in the manufacture of cheese.

Besides their industrial and medicinal applications, proteases play an important role in basic research. Their selective peptide bond cleavage is used in the elucidation of structure function relationship, in the synthesis of peptides, and in the sequencing of proteins. In essence, the wide specificity of the hydrolytic action of proteases finds an extensive application in the food, detergent, leather, and pharmaceutical industries, as well as in the structural elucidation of proteins, whereas their synthetic capacities are used for the synthesis of proteins.

FUTURE SCOPE:

Proteases are a unique class of enzymes, since they are of immense physiological as well as commercial importance. They possess both degradative and synthetic properties. Since proteases are physiologically necessary, they occur ubiquitously in animals, plants, and microbes. However, microbes are a goldmine of proteases and represent the preferred source of enzymes in view of their rapid growth, limited space required for cultivation, and ready accessibility to genetic manipulation. Microbial proteases have been extensively used in the food, dairy and detergent industries since ancient times. There is a renewed interest in proteases as targets for developing therapeutic agents against relentlessly spreading fatal diseases such as cancer, malaria, and AIDS. Advances in genetic manipulation of microorganisms by SDM of the cloned gene opens new possibilities for the introduction of predesigned changes, resulting in the production of tailor-made proteases with novel and desirable properties. The development of recombinant rennin and its commercialization by Pfizer and Genencor is an excellent example of the successful application of modern biology to biotechnology.⁹

The advent of techniques for rapid sequencing of cloned DNA has yielded an explosive increase in protease sequence information. Analysis of sequences for acidic, alkaline, and neutral proteases has provided new insights into the evolutionary relationships of proteases. Despite the systematic application of recombinant technology and protein engineering to alter the properties of proteases, it has not been possible to obtain microbial proteases hat are ideal for their biotechnological applications. Industrial applications of proteases have posed several problems and challenges for their further improvements. The biodiversity represents an invaluable resource for biotechnological innovations and plays an important role in the search for improved strains of microorganisms used in the industry. A recent trend has involved conducting industrial reactions with enzymes reaped from exotic microorganisms that inhabit hot waters, freezing Arctic waters, saline waters, or extremely acidic or alkaline habitats. The proteases isolated from extremophilic organisms are likely to mimic some of the unnatural properties of the enzymes that are desirable for their commercial applications.⁹

Exploitation of biodiversity to provide microorganisms that produce proteases well suited for their diverse applications is considered to be one of the most promising future alternatives. Introduction of extremophilic proteases into industrial processes is hampered by the difficulties encountered in growing the extremophiles as laboratory cultures. Revolutionary robotic approaches such as DNA shuffling are being developed to rationalize the use of enzymes from extremophiles. The existing knowledge about the structure-function relationship of proteases, coupled with gene-shuffling techniques, promises a fair chance of success, in the near future, in evolving proteases that were never made in nature and that would meet the requirements of the multitude of protease applications.

A Century after the pioneering work of Louis Pasteur, the science of microbiology has reached its pinnacle. In a relatively short time, modern biotechnology has grown dramatically from a laboratory curiosity to a commercial activity. Advances in microbiology and biotechnology have created a favorable niche for the development of proteases and will continue to facilitate their applications to provide a sustainable environment for mankind and to improve the quality of human life.⁹

Proteases are a unique class of enzymes, since they are of immense physiological as well as commercial importance. They possess both degradative and synthetic properties. Since proteases are physiologically necessary, they occur ubiquitously in animals, plants, and microbes. However, microbes are a goldmine of proteases and represent the preferred source of enzymes in view of their rapid growth, limited space required for cultivation, and ready accessibility to genetic manipulation. Microbial proteases have been extensively used in the food, dairy and detergent industries since ancient times. There is a renewed interest in proteases as targets for developing therapeutic agents against relentlessly spreading fatal diseases such as cancer, malaria, and AIDS. The development of recombinant rennin and its commercialization by Pfizer and Genencor is an excellent example of the successful application of modern biology to biotechnology. Analysis of sequences for acidic, alkaline, and neutral proteases has provided new insights into the evolutionary relationships of proteases.⁶

Despite the systematic application of recombinant technology and protein engineering to alter the properties of proteases, it has not been possible to obtain microbial proteases that are ideal for their biotechnological applications. Industrial applications of proteases have posed several problems and challenges for their further improvements. The biodiversity represents an invaluable resource for biotechnological innovations and plays an important role in the search for improved strains of microorganisms used in the industry. A recent trend has involved conducting industrial reactions with enzymes reaped from exotic microorganisms that inhabit hot waters, freezing Arctic waters, saline waters, or extremely acidic or alkaline habitats. The proteases isolated from extremophilic organisms are likely to mimic some of the unnatural properties of the enzymes that are desirable for their commercial applications. The existing knowledge about the structure function relationship of proteases, coupled with gene-shuffling techniques, promises a fair chance of success, in the near future, in evolving proteases that were never made in nature and that would meet the requirements of the multitude of protease applications.⁶

CONCLUSION:

The enzymes may serve as the model system and may pave the way for novel ways for eco-friendly industrial applications.² Proteases are industrially important enzymes with many applications especially in detergent industry.¹ The present study shows the isolation of bacterial strain of Bacillus subtilis from sea water, soil and degraded abattoir waste. Successfully, optimized environmental factors and nutrient conditions yielded maximum protease production. But, among all the strains which gave optimized production of protease, strain isolated from sea water produced the enzyme in high amounts as compared to the others. Hence, it can be said that this enzyme can be produced in large scale from the microorganism Bacillus subtilis isolated from sea water.³ Based on the present study, it is concluded that Bacillus subtilis has wide scope for the industrial production of protease.

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Received on 26.06.2025 Revised on 14.07.2025

Accepted on 29.07.2025 Published on 08.08.2025

Available online from August 14, 2025

Research J. Science and Tech. 2025; 17(3):193-206.

DOI: 10.52711/2349-2988.2025.00027

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