Title of Invention

"A PROCESS FOR THE PREPARATION OF ALKALINE PROTEASE."

Abstract

A process for preparation of alkaline protease by screening of soil and water samples to isolate an alkaline protease producing microorganism, growing alkalophilic strain of Bacillus sphaericus capable of producing alkaline protease deposited at M.T.C.C. and designated as MTCC-B-0014 in a nutrient medium having pH at least 8.5 in a known manner at least for 24 hours, separating residue by known methods and recovering alkaline protease by conventional centrifugation, ultra filtration and salt precipitation methods.

Full Text

The present invention relates to a process for the preparation of alkaline protease. More particularly this invention relates to a process for isolation of a novel alkalophilic strain of Bacillus sphaericus and to produce therefrom an alkaline protease characterized by its activity in alkaline media and stability under high alkaline condition and in presence of chlorine. Alkaline proteases have pronounced applications in detergent and leather industries. Proteases are enzymes which hydrolyze large protein molecules into smaller peptides or their constituent amino acids depending on their site of action and are classified into acidic, neutral or alkaline proteases depending on their pH optima which may lie in acidic, neutral or alkaline pH range respectively. Another system classifies proteases into four classes called cystein, aspartic, metallo- and serine proteases which is based on the amino acid involved in their respective catalytic sites (Morihara, K., Adv. Enzymology, 41: 179-243, 1974). Alkaline proteases find a wide variety of uses in the industry and constitute the single largest group of industrial enzymes in the world market. Their main applications lie in detergent, leather, food and photographic film industries apart from a large variety of other industrial applications. In laundering, proteins are important in two ways : protein based stains such as blood stain and protein as a binder of dirt particles. Hydrolysis of protein by proteases washes away the stain and makes release of these dirt particles by other detergent components easy. Recently two major trends have been observed in detergent market (van Tilburg, R., Progress in Industrial Microbiology, vol. 20 : 31-52, 1984). One is the change in the composition of detergents with a shift away from phosphate based detergents. Due to ever changing market requirements and growing environmental concerns, there has been a shift away from phosphate based detergents. Phosphate based detergents are not only slow in biodegradation but are also required to be used in much greater concentrations. However, lowering of phosphate content eventually leads to lowering of washing efficiency. To compensate for it, enzymes are added to detergents. Second trend is the change towards lower washing temperatures due to the increased use of chemical fiber clothes having low heat stability and recent emphasis on energy savings. Water supplies are chlorinated in many parts of the world including India and many enzymes are known to be inactivated in presence of chlorine. Some detergent compositions may also include chlorine bleaches. In order to prevent chlorine from attacking and inactivating enzymes especially under alkaline conditions, chlorine bleach scavengers are included in detergent formulations as a part of enzyme stabilizing system (Foley, PP and Jones, LA (1999). US Patent No. 5 858 946, January 12). A protease which is stable against chlorine will be advantageous since chlorine bleach scavengers may not be required to be added. The food process industry uses detergent efficiency to compensate for design or operational deficiencies in their cleaning programmes. A major challenge for food process industry is the successful removal of soils that are resistance to conventional treatment and one such soil is protein. Detergents used for such application contain chlorine which degrades protein into smaller peptide chains thereby lowering binding energies and affecting desorption from the surface. Proteases can also be useful for cleaning of such protein soils. However, the protease incompatibility with chlorine is cited as one of the 'reasons for its not so widespread use as a part of cleaning agent in food processing industry (Inhs, DA, Schmidt, W and Richter, FR (1999). US Patent No. 5 861 366, January 19). Hence, stability of any protease against chlorine will be advantageous. It has been observed that because of widely differing uses and applications detergent market is always looking for enzymes which have properties better than those presently available. Proteolytic enzymes produced by cultivating the members of the genus Bacillus constitute the major source of proteolytic enzymes such as alkaline proteases used in detergent compositions (Kalisz, H.M. Microbial Proteinases. Adv. Biochemical Eng./Biotechnol., Vol 36: 1-65, 1988). As detergent additives, it is also important for the proper functioning of these enzymes that they be active under actual conditions of application such as in presence of commercial laundry detergents and chlorine and under high alkaline conditions. Hence there is an urgent requirement to find out new microorganisms which can produce alkaline proteases which are better suited for the changed requirements of detergent market. Screening of alkalophiles from naturally occurring habitats in different parts of the world is expected to result in isolation of new protease producing organisms useful for many applications. Thus the main objective of this invention is to develop an improved process for the production of alkaline protease using a novel alkalophilic strain of Bacillus sphaericus. Another objective of the present invention is to isolate a novel alkalophilic strain of Bacillus sphaericus used in the production of alkaline protease. Soil and water samples collected from Leh and Ladakh and other alkaline regions of India were plated onto a medium containing nutrient-agar and skim milk, maintained at an alkaline pH. These samples upon incubation produce alkaline protease producing organisms, which were selected on the basis of appearance of the zone of hydrolysis around a colony. These organisms were then tested for their ability to produce alkaline protease in a reproducible manner. Based on the diameter of the zone of hydrolysis formed a few potent organisms were selected and protease production was checked in a liquid medium consisting of glucose, yeast extract, biopeptone, KH2PO4, MgSO4.7H2O and Na2CO3. The organism which produced maximum amount of alkaline protease was selected . The selected isolate was deposited in Microbial Type Culture Collection and Gene Bank (MTCC), a national facility located in Institute of Microbial Technology ( a constituent laboratory of the applicant), Sector 39-A, Chandigarh, India and has been given an accession no. MTCC-B-0014 where B denotes a bacterium. The isolated strain, designated as MTCC-B-0014 was subjected to tests described below. The results of these tests are given in Table 1. 1. Production of catalase, production of urease, production of indole, Voges-Proskauer and Methyl-Red test, gelatin hydrolysis, anaerobic growth, acid from carbohydrates, tolerance of sodium chloride, hydrolysis of starch, decomposition of casein: These tests were conducted according to Bergey's Manual of Systematic Bacteriology, Vol. 2, Williams and Wilkins, Baltimore, 1986. PRODUCTION OF INDOLE: After incubation in indole production medium (1% tryptone medium) added 2 ml. of the following reagent : p-dimethylaminobenzaldehyde 5g ; isoamyl alcohol 75 ml ; cone. HC1 25 ml. The contents of the tube were shaken vigorously and checked for the appearance of pink color in the alcohol layer which separated out on standing. HYDROLYSIS OF GELATIN : Growth on nutrient agar + 0.4% gelatin was flooded with 10 ml of sulfuric acid, saturated with sodium sulfate. Gelatin hydrolysis was indicated by a clear zone under or around the growth. ANAEROBIC GROWTH : The anaerobic growth was checked in an anaerobic jar. VOGES-PROSKAUER AND METHYL RED TEST: The organisms were grown in 5 ml aliquots of VP broth (proteose peptone 7g/l; glucose 5g/l; sodium chloride 5g/l; pH 6.5). VP-Test: Added 3 ml of 40% (w/v) sodium hydroxide and 0.5-1.0 mg of creatine to the culture broth. Kept at room temperature for 30-60 min. and observed for the appearance of red color. MR-Test : Added 5 drops of methyl red solution (Bacto methyl red 0.1 g, 95% ethanol 300 ml, distilled water upto 500 ml) to the culture broth. Positive reaction is indicated by appearance of distinct red color and negative reaction is indicated by yellow color. PRODUCTION OF CATALASE: The growth of nutrient agar was flooded with 10% hydrogen peroxide. Production of gas bubbles indicated the presence of catalase. UREASE TEST : Inoculated the urease test broth consisting of (per litre) urea 20 g, monopotassium phosphate 0.091 g, dipotassium phosphate 0.095g, yeast extract O.lg, phenol red O.Olg. Positive reaction is indicated by change of color from pinkish yellow to deep purple. ACID/GAS FROM CARBOHYDRATES: The organisms were inoculated into slants/tubes of acid production medium. Durham tubes were used for detection of gas. Change in color from blue to yellow indicated the production of acid. The composition of acid production medium was (per litre): diammonium hydrogen phosphate 1 g; potassium chloride 0.2 g; magnesium sulfate 0.2 g; yeast extract 0.2 g; agar 15 g; pH 7.0; added 15 ml of 0.04% (w/v) solution of Bromo-cresol purple and sugars at a final concentration of 0.5%. SODIUM CHLORIDE TOLERANCE: Growth in nutrient broth containing 1%, 2%, 3%, 4% and 5% sodium chloride was checked. HYDROLYSIS OF STARCH : The organisms were grown on nutrient agar containing 1% soluble starch. The plates were flooded with 95% ethanol. Hydrolysis of starch is indicated by the appearance of a clear zone underneath or around the growth. HYDROLYSIS OF CASEIN: The organisms were grown on milk agar plates. Hydrolysis of casein was indicated by appearance of clear zone underneath or around the growth. The milk agar was made by mixing autoclaved skim milk solution (10%) with equal volume of 2X nutrient agar. 2. Oxidase test, nitrate reduction test, nitrite reduction test: These tests were conducted according to Cowan, S.T., (1974), Manual for the identification of Medical Bacteria 2nd ed, Cambridge University Press, Cambridge and described in detail below: OXIDASE TEST: The organisms were grown on nutrient agar, cells were removed with a toothpick and applied on a filter paper. 2-3 drops of 1 % aqueous solution of tetramethyl-p-phenylenediamine dihydrochloride were put on the cells. A positive reaction was shown by the development of a dark purple color within 10 seconds. NITRATE REDUCTION: The organisms were inoculated into nitrite-free broth (1 g potassium nitrate per 1000 ml of Nutrient broth). Observed for gas in the Durham's tubes. Added 1 ml of reagent A (0.8% sulphanilic acid in 5N acetic acid) followed by 1 ml of reagent B (0.6% dimethyl-a-naphthylamine in 5N acetic acid or 0.5% a-naphthylamine in 5N acetic acid). Appearance of red color indicated the presence of nitrite which has been formed by reduction of nitrate. To the tubes not showing red color, added powdered zinc (5 mg/ml) and allowed to stand. Appearance of red color indicated the reduction of nitrate to nitrite, which in turn had been reduced. NITRITE REDUCTION: Inoculated the organisms into broth (1 g potassium nitrite in 1000 ml nutrient broth) and tested for the presence of nitrite as in nitrate reduction test. It has also been observed that the strain designated as MTCC-B-0014 can produce an alkaline protease which is optimally active at pH 10.5 and stable under high alkaline condition and in presence of commercial laundry detergents and chlorine. Table 1: Morphological Characteristics (Table Removed) The process of the present invention involves following steps: a. Collection of soil and water samples, b. Plating the above samples on the alkaline skim milk agar medium plates, c. Selection of strain which produces maximum amount of alkaline protease, d. Characterization of the selected strain on the basis of it's morphological, physiological and biochemical properties, e. Production of alkaline proteases by this strain, designated as MTCC-B-0014, when grown in a medium containing glucose, yeast extract, biopeptone, KH2PO4, MgSO4.7H2O and Na2CO3 ,or, starch, PH-soyatose, KH2PO4, MgSO4.7H2O and Na2CO3. f. Recovery of protease, and g. Use of the protease obtained as above for detergent applications. The details of the process are as follows: Soil and water samples collected from Leh, Ladakh and other alkaline regions of India were used for isolation of alkaline protease producing organisms by plating onto skim milk agar medium. The skim milk agar medium, used for isolation of alkaline protease producing organisms, essentially consists of the following : Skim milk powder, Na2CO3 and nutrient agar dissolved in distilled water and mixed in a fixed proportion or varying proportions. These samples from alkaline regions of the country were serially diluted using normal saline solution (0.8% NaCl in distilled water) and plated on the skim milk agar medium plates. These plates were incubated and checked for appearance of zone of hydrolysis around each colony after every 24 hours. The diameter of the zone of hydrolysis was measured and potent protease producers were selected. Microbial cultures so obtained were purified by restreaking individual colonies. Loopful of each microorganism was inoculated in a known medium, Medium II (Horikoshi, K. and Akiba, T., in 'Alkalophilic microorganisms: a new microbial world'. Japan Scientific Societies Press, Tokyo, 1982) being a composition of glucose, yeast extract, biopeptone, KH2PO4, MgSO4.7H2O and pH adjusted to about 10.0 with Na2CO3 and incubated in an orbital shaker. At 24, 48 and 72 hours, samples of fermented broth were withdrawn and centrifuged. The supernatant was used for analysis of alkaline protease. The culture producing maximum amount of protease activity was selected. This process was repeated a number of times to select the most potent protease producer. The alkaline protease producing isolate, deposited in MTCC under accession no. MTCC-B-0014 was characterized as Bacillus sphaericus on the basis of its physiological and biochemical characteristics. A variety of media can be used for production of protease by Bacillus sphaericus. Nitrogen sources such as yeast extract, peptone, Biopeptone, soyaflour, PH-soyatose, vegetable protein concentrates and assimilable carbon and energy source such as glucose, sucrose, starch, CMC, fructose, glycerol, lactose, mannitol could be used. The medium may contain: (a) glucose (10-20 g/1, yeast extrat (5-10 g/1), biopeptone (5-10 g/1) and inorganic phosphate or, (b) starch (10-20 g/1), PH-soyatose (5-10 g/1) and inorganic phosphate. Further, certain amount of metal salts such as magnesium is preferably added. Na2CO3 was added to adjust the pH to 10.0±0.2. A vigorous aeration is generally maintained during fermentation. The fermentation temperature is suitably maintained in the range of 25° - 37°C and preferably at 30°C. A productive fermentation typically is about 24-72 hours in length and preferably within the range 24 to 36 hours in a fermenter. To recover the enzyme, the fermentation broth is centrifuged to remove cellular materials. Then, the enzyme is concentrated by ultrafiltration and the retentate from this step is precipitated by adding inorganic salts such as ammonium sulphate. The enzyme was found to be very stable in high alkaline conditions and in presence of chlorine and laundry detergents. Accordingly the present invention provides a process for preparation of alkaline protease, which comprises; screening of soil and water samples to isolate an alkaline protease producing microorganism, growing alkalophilic strain of Bacillus sphaericus capable of producing alkaline protease having characteristics as here in described, deposited at M.T.C.C. and designated as MTCC-B-0014 in a nutrient medium having pH at least 8.5 in a known manner at least for 24 hours, separating residue by known methods and recovering alkaline protease by conventional centrifugation, ultra filtration and salt precipitation methods. In a preferred embodiment, the water and soil samples taken as above should be plated on the alkaline skim milk agar medium, consisting of nutrient agar, skim milk and Na2CO3, and incubated at about 30°C . In an another preferred embodiment the strain MTCC-B-0014 produced alkaline protease extracellularly after 24-72 hours of growth in a medium, which essentially consisted of glucose, biopeptone, yeast extract, KH2PO4, MgSO4.7H2O and Na2CO3, or, starch, PH-soyatose, KH2PO4, MgSO4.7H2O and Na2CO3. In yet another preferred embodiment the strain MTCC-B-0014 was grown at pH range between 8.5-10.5 , preferably between 9.5-10.0. In yet another preferred embodiment the growth of the strain designated as MTCC-B-0014 was carried out in a temperature range between 25-37°C, preferably at 30°C. The nutrient medium used has mainly either of the two composition (g/1) as given below: glucose, 10.0; yeast extract, 5.0; biopeptone, 5.0; KH2PO4, 1.0; MgSO4.7H2O, 0.2; Na.CO2, 10.0, or , starch, 10.0; PH-soyatose, 10.0; KH2PO4, 1.0; MgSO4.7H2O, 0.2;Na2CO3,10.0. The protease is recovered by centrifugation, ultrafiltration and ammonium sulfate salt precipitation. The process of the present invention is further illustrated by the following examples, which should not however be construed to limit the scope of the present invention. Unless otherwise stated, percentage data relates, as they do in the previous description, to weight. Example -1 One of the samples- a soil sample, was taken and tested for the presence of the alkaline protease producers by plating on the alkaline skim milk agar plates. It was serially diluted using sterile normal saline and then plated on the alkaline skim milk agar medium prepared by adding separately sterilized skim milk (0.5 %) to nutrient agar, pH of the medium was adjusted to 10.0 by adding appropriate amount of separately autoclaved 10 % solution of Na2CO3. Plates were incubated at 30°C for 72 hours. The growth of protease producing colonies were marked by appearance of a zone of hydrolysis around them. Diameter of this zone was measured every 24 hours. Based on this about 25 colonies were selected as potent protease producers and they were purified by repeated streaking on the plates of same medium. The selected isolates were grown in a medium consisting of glucose 10 g/1, yeast extract 5 g/1, biopeptone 5 g/1, KH2PO4 1 g/1, MgSO4.7H2O 0.2 g/1 and separately autoclaved Na2CO3 10 g/1, at 30°C and 200 rpm in an orbital shaker. pH of the medium was about 10.0 . Production of alkaline protease was checked at 24, 48 and 72 hours by azocasein hydrolysis assay . In the azocasein hydrolysis assay, reaction mixture containing 20µ1 of 5% azocasein solution, 470 µl of 0.05M glycine-NaOH buffer of pH 10.5 and lOµl of suitably diluted enzyme solution was incubated at 37°C for 30 minutes. The reaction was terminated by addition of 5OOµl 10% TCA (Trichloroacetic acid) solution, followed by incubation in ice for 15 minutes. After centrifugation at 10000 rpm for 5 minutes, 800 µl of supernatant was mixed with 200µl of 1.8N NaOH solution and the absorbance of this mixture was measured at 420 run. Proteolytic activity in Units/ml was calculated as increase in absorbance caused by one ml of the enzyme per hour of reaction under the given conditions. The isolate which produced maximum amount of alkaline protease activity was selected and deposited as mentioned earlier and has been given an accession no. MTCC-B-0014 . Example 2: Alkaline protease producing isolate obtained as in Example 1 and identified as Bacillus sphaericus was maintained on milk agar petri plates of the following medium: Nutrient Agar Skim milk 0.2 % pH : 10.0 (with addition of 10% (w/v) solution of Na2CO3). The plates were incubated at 30°C for 24-30 hours and a loopful of this culture was inoculated into a 500 ml conical flask containing 100 ml of sterile medium of following composition: ' Glucose 1% Biopeptone 0.5% Yeast extract 0.5% KH2PO4 0.1% MgSO4.7H2O 0.02% Na2CO3 1.0% (Sterilized separately and added before inoculation) This composition was not limited to these quantities alone, they could include other ingredients with similar properties and quantities. This culture flask was incubated at 30° C in an orbital Shaker at 200 rpm for 24 hours and 10.0 ml of this culture was used to inoculate 90 ml of sterile medium so as to make 100 ml final volume of above said composition in each of 500 ml flasks. These flasks were incubated at 30° C and 200 rpm in an orbital Shaker for 12 hours and 500 ml of this culture was used to inoculate 4.5 liter of sterile media so as to make total volume of 5 liter with above said composition in a 7 liter fermenter. Fermentation is carried out at 30°C and 300 rpm. Aeration was kept constant at 1 VVM (volume per volume per minute). Protease production was monitored at 4 to 6 hrs interval using azocasein hydrolysis assay. Maximum enzyme activity was observed at 30-36 hours after which it declined and the fermentation was terminated at this point. Example 3 Alkaline protease producing isolate obtained as in Example 1 and identified as Bacillus sphaericus was maintained on milk agar petri plates of the following medium: Nutrient Agar Skim milk 0.2 % pH : 10.0 (with addition of 10% (w/v) solution of Na2CO3 ). The medium for pre-inoculum, inoculum as well as production medium consisted of (g/1) starch, 10.0; PH-Soyatose (Warkem, Bombay), 10.0; KH2PO4, 0.2; MgSO4.7H2O, 0.2 and Na2CO3, 10.0. The solution of Na2CO3 (100 g/1) was sterilized separately and then added into the medium. The initial pH of the medium was 10.0±0.2. For protease(caseinase) assay, samples are centrifuged at 10,000 rpm for 10 minutes and alkaline protease activity is assayed in supernatant (extracellular fraction) using casein (6.25 g/L) as substrate dissolved in 0.05M Glycine-NaOH buffer (pH=10.0). 0.4 ml of casein solution is taken in an eppendorf tube and pre-incubated at 50°C for 5-10 minutes. 0.1 ml of suitably diluted enzyme solution was added into this and the proteolytic reaction was carried out in Eppendorf tubes at 50°C for 10 minutes and stopped with the addition of 5% (W/V) trichloroacetic acid. The mixture was kept in ice for 15 minutes and then centrifuged at 10,000 rpm for 10 minutes. 0.2 ml of supernatant is taken and quantified as tyrosine equivalent by the method of Lowry (Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., J. Biol. Chem., 193, 265 (1951)). One AU of enzyme activity is defined as the amount of enzyme that liberates peptides equivalent to one µmole of tyrosine per minute. A loopful of freshly prepared culture from skim milk agar plates were used to inoculate a 250 ml flask containing 50 ml medium for preinoculum production. After 24-27 hours of incubation at 30° and 200 rpm on a rotary shaker, 10 ml of this pre-culture was used to inoculate 500 ml flasks containing 100 ml medium. The flasks were incubated at 200 rpm and 30°C for 18 h to serve as inoculum for the fermenter. A fermenter with a working volume of 14 liter was employed for protease production. The initial pH of the medium was adjusted to 10.0 by addition of sodium carbonate solution (100 g/liter). 500 ml of 12 h grown inoculum was used for inoculating the fermenter. The fermenter was aerated at 1.0 VVM and agitated at 300 rpm for 24 hours. During this period, Bacillus sphaericus produced 78000 AU of protease(caseinase) per liter. After 24 hour fermentation, the bacterial cells were harvested by centrifugation (at 6000g for 30 minutes). The culture supernatant was concentrated 5-fold by tangential-flow ultrafiltration. Retentate from this step was further concentrated by ammonium sulphate precipitation at 70 % saturation level. Using centrifugation, ultrafiltration and ammonium sulphate precipitation, >85% recovery of enzyme activity was obtained. Example 4 (a) Effect on pH on B. sphaericus protease activity and stability : Alkaline protease assays were performed at different pH in the range 7.5-11.5. pH was adjusted by using 0.05 M Tris-HCl buffer for pH 7.5-9.0 and 0.05 M Glycine-NaOH buffer for pH 9.5-11.5. . Relative activity (%) at the given pH was calculated as % of the protease activity obtained at optimum pH. The effects of pH on protease activity towards azocasein were examined at 37°C. The enzyme was active over a broad pH range with an optimum at about 10.5 (Table.2). Table 2 Effect of pH on protease activity. (Table Removed) 1.0 ml of enzyme obtained as in example 3 was mixed with 4.0 ml of buffer of given pH and incubated for one hour at 37°C, and assayed for residual activity at 37°C and 10.5 pH. Relative residual activity (%) at the given pH was calculated as % of protease activity obtained at optimum pH of 10.5. This enzyme was found to be stable over a wide range of pH values (pH 7.5-11.0). Table 3 Effect of pH on protease stability. (Table Removed) (b) B. sphaericus protease stability in presence of chlorine 200 ul of enzyme solution obtained as in example 3 was mixed with 200 ul of sodium hypochlorite solution (hypo solution was made in terms of parts per million of available chlorine) and incubated at 37°C for one hour. The residual azocaseinase enzyme activity was measured using azocasein hydrolysis assay. In the azocasein hydrolysis assay, reaction mixture containing 20µl of 5 % azocasein solution, 470µl of 0.05M glycine-NaOH buffer of pH 10.5 and 10 µl of suitably diluted enzyme solution was incubated at 37°C for 30 minutes. The reaction was terminated by addition of 500 µl 10% TCA (Trichloroacetic acid) solution, followed by incubation in ice for 15 minutes. After centrifugation at 10000 rpm for 5 minutes, 800 µl of supernatant was mixed with 200 ul of 1.8N NaOH solution and the absorbance of this mixture was measured at 420 nm. Proteolytic activity in Units/ml was calculated as increase in absorbance caused by one ml of the enzyme per hour of reaction under the given conditions. It was observed that the addition of sodium hypochlorite at level of 500 ppm in terms of available chlorine didnot exert significant effect on stability of this protease. Example 5 Efficacy of B. sphaericus protease in commercial laundry detergents was measured by using alkaline protease obtained from Bacillus sphaericus, designated as MTCC-B-0014, as given in example 3 was used for detergent applications. To 1.0 ml of this enzyme of known enzyme activity, 4.0 ml of 5.0 g/1 solution (prepared in distilled water) of a commercially available detergent powders, such as Nirma, Key and Super Wheel, were added. This mixture was incubated at 37°C for one hour. Residual enzyme activity was assayed by routine enzyme assay as described in example 1. Distilled water in place of detergent solution was added in the control set which was incubated similarly and residual enzyme activity was checked. Residual activity in the presence of detergent solution was calculated as percentage of the residual activity in the control set. In the azocasein hydrolysis assay, reaction mixture containing 20µ1 of 5% azocasein solution, 470 µl of 0.05M glycine-NaOH buffer of pH 10.5 and lOµl of suitably diluted enzyme solution was incubated at 37°C for 30 minutes. The reaction was terminated by addition of 500µl 10% TCA (Trichloroacetic acid) solution, followed by incubation in ice for 15 minutes. After centrifugation at 10000 rpm for 5 minutes, 800 µ1 of supernatant was mixed with 200|al of 1.8N NaOH solution and the absorbance of this mixture was measured at 420 nm. Proteolytic activity in Units/ml was calculated as increase in absorbance caused by one ml of the enzyme per hour of reaction under the given conditions. Table 4 Efficacy of B. sphaericus protease in commercial laundry detergents. (Table Removed) The alkaline protease produced as in example 3, and used as above, was found to retain more than 90% activity compared to control set. Example 6 For purification of B. sphaericus protease, ammonium sulphate precipitated enzyme obtained as in example 3 was subjected to extensive dialysis against Tris-Hcl buffer (0.025 M, pH 9.0). This dialyzed enzyme preparation was applied to a Phenyl Agarose column (1.0X10 cm) connected to a Gradifrac protein purification system of Pharmacia, Sweden. Tris-HCl buffer (0.05 M, pH 9.0) containing 1.0 M NaCl was used for loading the sample at a flow rate of 0.5 ml/minute. Fractions of 8.0 ml were collected. The column was washed with the same buffer to remove unbound proteins, Elution of bound proteins was carried out by applying a linear gradient of 0-50% V/V ethylene glycol at a flow rate of 1.0 ml/minute and followed at 280 nm. Active fraction(s) of protease from phenyl agarose column were pooled and loaded on to a Q Sepharose (Pharmacia) ion-exchange column(2.5 xl.O cm) equilibrated with Tris-HCl (0.05 M, pH 9.0) buffer and connected to a Gradifrac Protein purification system. The buffer used was Tris-HCl (0.05 M, pH 9.0) with a flow rate of 0.5 ml/minute. After washing the column with the same buffer, elution of molecular mass=68 kDa) apparently consisted of smaller subunits. ,. Isoelectric point of the major protease i.e., protease B was found to be 8.6. Advantages: 1. Alkaline protease produced by Bacillus sphaericus was active over a wide alkaline pH range. 2. Alkaline protease produced by Bacillus sphaericus was stable under high alkaline condition and in solutions of laundry detergents and chlorine (at the level of 500 ppm) We claim; 1. . A process for preparation of alkaline protease, which comprises; screening of soil and water samples to isolate an alkaline protease producing microorganism, growing alkalophilic strain of Bacillus sphaericus capable of producing alkaline protease having characteristics as here in described, deposited at M.T.C.C. and designated as MTCC-B-0014 in a nutrient medium having pH at least 8.5 in a known manner at least for 24 hours, separating residue by known methods and recovering alkaline protease by conventional centrifugation, ultra filtration and salt precipitation methods. 2. A process as claimed in claim 1 , wherein nutrient medium used has the composition (g/1) as given below: glucose, 10.0; yeast extract, 5.0; biopeptone, 5.0; KH2PO4, 1.0; MgSO4.7H2O, 0.2; Na2CO3, 10.0, or, starch, 10.0; PH-soyatose, 10.0; KH2PO4, 1.0; MgSO4.7H2O, 0.2; Na2CO3,10.0. 3. A process as claimed in claiml to 2 ,wherein pH of the medium is 9.0 - 10.5. 4 A process as claimed in claims 1 to 3 where in growing is effected under aerobic submerged condition with stirring at a temperature of 25 to 37°C for 24 to 72 hours. 5. A process for preparation of alkaline protease substantially as described herein with reference to the examples 2 to 4.

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