Title of Invention	A NOVEL ALKALINE PROTEASE
Abstract	A novel alkaline protease exhibiting a feature of halotolerance and activity as well as stability in the presence of oxidants as well as surfactants is provided. While the protease exhibits elastolytic activity, there is no collagenolytic activity. The alkaline protease is prepared from a strain of Bacillus subtilis, designated as MTCC 5333, using submerged fermentation technique, wherein the production medium is prepared without using any common salt or alkali and the pH of the said medium is the neutral range. The protease finds potential application in leather processing and detergent industries.

Title of Invention

A NOVEL ALKALINE PROTEASE

Abstract

A novel alkaline protease exhibiting a feature of halotolerance and activity as well as stability in the presence of oxidants as well as surfactants is provided. While the protease exhibits elastolytic activity, there is no collagenolytic activity. The alkaline protease is prepared from a strain of Bacillus subtilis, designated as MTCC 5333, using submerged fermentation technique, wherein the production medium is prepared without using any common salt or alkali and the pH of the said medium is the neutral range. The protease finds potential application in leather processing and detergent industries.

Full Text	Field of invention The present invention relates to a novel alkaline protease and a process for the preparation thereof. More particularly, the present invention relates to a novel alkaline protease from bacterial source for industrial applications. In particular, the present invention relates to a novel protease produced from a bacterial strain of Bacillus subtilis MTCC 5333. The protease of the present invention finds potential application in leather processing industry as an eco-benign option for dehairing of hides and skins without using any chemicals. An important feature of this protease is that while it exhibits elastolytic activity, there is no collagenolytic activity. In addition, it exhibits activity and stability in the presence of high concentrations of salt, surfactants and oxidants. This feature enables the protease to be used in the soaking bath itself, thereby contributing to the productivity enhancement. Further, the enzyme is envisaged to have potential application as an additive in detergent industry. Background of invention and Description of Prior art Proteases are conventionally produced from several micro-organisms, plant sources and animal sources. However, commercial production of protease from bacterial and fungal origin have gained much importance because of their extracellular production, submerged cultivation, high yield and short duration of production and easy recovery of the enzyme. As reported by Gupta et al. (Applied Microbiology and Biotechnology, 60: 381-395, 2002) and Tjalsma et al. (Journal of Applied Microbiology, 96: 569, 2004), although wide ranges of microbes are known to produce proteases, major industrial enzymes with increasing market potentials are derived from Bacillus group of bacteria due to their higher capacity and activity. Being a fast growing and non-pathogenic GRAS (Generally Regarded As Safe) microbe, production of protease especially from Bacillus subtilis strains is beneficial in terms of scalability, high yield and commercial exploitation as reported by Gupta et al. (Applied Microbiology and Biotechnology, 60: 381-395, 2002) and De Boer and Diderichsen (Applied Microbiology and Biotechnology, 36: 1-4, 1991). There have been several reports on the different features of the proteases available in the public domain. They are summarized below: Stability against detergents and oxidants Generally, proteases are susceptible to the oxidative inactivation by the oxidants and denaturation by anionic surfactants. Application of proteases in the detergent formulations faces various practical problems because most recent commercial detergent products contain anionic surfactants, bleaching agents, oxidants, enzymes other than proteases and water-softening builders such as zeolites. Therefore, the suitability of the proteases for use as a detergent additive depends on its stability and compatibility with these components at alkaline pH and wide range of temperature, and ideally it should be stable and active in the detergent solution. As reported by Siezen and Leunissen (Protein Science, 6: 501-523, 1997), most of the proteolytic enzymes are classified under serine alkaline proteases (or Subtilisins, E.C. 3.4.21.14) and they mainly function in a narrow range of pH, temperature, and ionic strength. Among them, a few are able to resist chemical oxidants forming a new subfamily of subtilisins named oxidatively stable alkaline proteases, as reported by Okuda et at. (Extremophiles, 8: 229-235, 2004). Saeki et al. (Biochemical Biophysical Research Communications, 279: 313-319, 2000) have reported oxidatively stable subtilisin-like serine proteases, having 43-45 kDa molecular weight from five different strains of alkalophilic Bacillus. These proteases are stable in the presence of 50 mM H202. Saeki et al. (Current Microbiology, 47: 337-340, 2003) have reported a highly oxidant (in 1 M H2O2) and surfactant stable subtilisin of 30 kDa from alkalophilic Bacillus KSM-LD1. Takaiwa et al. (U.S. Pat. No. 6759228) have provided a serine alkaline protease of molecular weight of 43 kDa from Bacillus strains, having stability in various surfactants of 1% concentration and oxidant such as hydrogen peroxide of 50 mM concentration. Nonaka et al. (Journal of Biological Chemistry, 279: 47344-47351, 2004) report an oxidatively stable subtilisin-like alkaline protease of 43 kDa from Bacillus sp. KSM-KP43 that withstands 0.88 M H2O2 concentration. The limitation associated with these wild type proteases is that they are not able to withstand high concentration of oxidants. Also halotolerant property of these enzymes is not reported. In order to circumvent the problem of sensitivity towards oxidants, several attempts have been made through protein engineering methods to improve the functioning of proteases in the presence of oxidants and surfactants. Proteases are engineered by introducing mutation in the form of addition, substitution and deletion of amino acids. There are various commercially available detergent proteases such as Alkalase, Savinase, Esperase and Everlase (Novozymes Biotech, Inc, Denmark), and Maxacal, Purafect OxP ™ and Properase ™ (Genencor Int., USA). These enzymes mainly originate from various members of Bacillus species and some of them like Everlase and Purafect OxP ™ are protein engineered to enhance the enzyme stability and washing performance under these harsh conditions as reported by Maurer (Current Opinion in Biotechnology, 15: 330-334,2004) and Branner et al. (U.S. Pat. No. 5482849). But, protein engineering methods essentially strive to improvise such properties of proteases as stability in the detergent components, surfactants and oxidants to suit only the demands of detergent industry and hence, their potential is confined to limited industrial applications. For example, halotolerant property of the engineered proteases is not reported and this limits their application potential for some industries that involve high salinity. Halotolerance Different industrial applications require proteases, which are able to maintain high activities under moderate to high saline conditions. This feature of a protease is commonly known in the art as 'halotolerance'. Several attempts have been made to produce halotolerant protease from bacterial organisms. Setyorini et al. (Bioscience Biotechnology and Biochemistry, 70: 433-440, 2006) describe two highly halotolerant proteases of 29 and 34 kDa from Bacillus subtilis FP-133 grown in a medium containing high salt concentration and the protease exhibits 80% activity in the presence of 20% NaCI. However, the major limitation associated with most of these proteases is that they do not exhibit stability towards oxidants. This has prompted the researchers to explore possibilities for producing halotolerant protease that exhibits oxidant stability as well. Very few reports on oxidative and surfactant stable halotolerant proteases are available from wild type Bacillus group of bacteria. Joo and Chang (Enzyme Microbial Technology, 36: 766-772, 2005; Journal of Applied Microbiology, 98: 491-497, 2005) have reported an alkaline protease of 28 kDa from alkalophilic Bacillus clausii I-52 exhibiting stability and activity at 20% NaCI concentration besides having oxidant (6% H202) and surfactant stable properties. However, it is quite evident that all these proteases exhibit a lower molecular weight not exceeding 34 kDa. Moreover, producing halotolerant alkaline protease from these bacteria requires salt or alkali and/ or both. The limitation associated with the medium containing high salt concentration or alkali is that it causes damage to the fermentor and its accessories in addition to hampering productivity and economy as it affects the life of the fermentor. This necessitates the requirement of producing halotolerant alkaline proteases from bacterial organism grown in neutral pH as well as in the absence of salt and alkali wherein the fermentation process does not require any control in the form of maintenance of pH in the medium. There are no reports on oxidant and detergent stable, halotolerant alkaline proteases from organisms grown in medium having neutral pH, especially from Bacillus that does not require NaCI or alkali for growth and protease production. Molecular weight Conventionally, the higher the molecular weight of an enzyme, the easier is the purification process, thereby ensuring lesser impurities in the final product. As reported by Kumar and Takagi (Biotechnology Advances, 17: 561-594, 1999), almost all subtilisins, excepting a very few, produced from Bacillus group of bacteria, possess molecular weight, ranging from 15-45 kDa. Ogava et al. (Biochimica et Biophysica Acta, 1624: 109-114, 2003) have produced a subtilisin-like high molecular weight alkaline protease (FT protease) of 72 kDa from Bacillus sp. KSM-P43. Similarly, Okuda et al. (Extremophiles, 8; 229-235, 2004) have produced a 72 kDa subtilisin like alkaline protease from several Bacillus strains. But, halotolerance and oxidant stability are not reported for all these proteases of molecular weight 72 kDa. Elastolytic Activity Elastin is a protein present in the elastic fibers of the arteries, dermis etc. of vertebrates. Elastin is water-insoluble and has a large amount of special bridging structure such as desmosine and lysinonorleucine. The amino acid composition thereof is very special, with 50-60% of alanine and glycine. Elastase is capable of hydrolyzing elastin as it has (a) stronger binding affinity for elastin, and (b) stronger substrate specificity for alanine and glycine as reported by Stone et at. (Methods in Enzymology 82: 588-605, 1982). As reported by Foster (Methods in Enzymology, 82: 559-570, 1982) and Paz et al. (Methods in Enzymology, 82: 571-587, 1982), elastin cannot be easily hydrolyzed by ordinary proteases. This limitation has however been obviated by Tsai et al. (Applied and Environmental Microbiology, 54: 3156-3161, 1988; U.S. Pat. No. 5972683), who have reported an elastase from Bacillus sp. Ya-B. However, the limitation associated with this is that the protease also possesses collagenolytic activity. Proteases having elastolytic activity is an added advantage especially in leather manufacture to produce certain type of leathers. But, collagenolytic activity is undesirable for the purpose. Thus, the above parameters may be summarized as follows. There are proteases available in the art to exhibit the individual features of higher halotolerance upto a level of 20% NaCI, higher oxidant stability to the extent of 2 M, higher molecular weight upto 72 kDa or even elastase activity. But exhibiting of one property seems to be always at the cost of another feature. Hence, the use of these proteases for certain industrial applications gets restricted. For example, leather processing industry has been witnessing a shift towards eco-benign approaches to address the environmental problems effectively. Conventionally, the raw hides and skins are predominantly preserved with the help of salt that results in the arresting of microbial growth due to dehydration. Hence, the processing of these preserved stocks in a tannery starts with water treatment that not only conditions the stock for further aqueous processing by way of rehydration, but also washes the hides/skins. The process is known as soaking in the leather trade. The processing liquor generated after soaking exhibits a very high salinity to the extent of 15-25% due to the presence of abundant salt in the hides/skins to be processed. Soaking is followed by removal of hair and other non-collagenous substances. Usually, depilants pasted on the hides/skins result in loosening of hair, which is removed manually. It requires ample time and labour, thereby affecting the productivity. The use of an enzymatic depilant in the soaking bath itself is a possible eco-benign solution to this problem towards enhancing the productivity. However, it demands that the enzyme is stable as well as active at a high salinity of not less than 25%. The enzyme also has to be stable against detergents, which may be required to be added to soaking bath to enhance rehydration. Moreover, the activity of the enzyme on any other non-collagenous substance will be an added benefit. Further, the economy of use of the enzyme may be enhanced by its purity. Therefore, keeping in view the drawbacks of proteases available in the prior art the inventors of the present invention realized that there exists a dire need for providing a broad spectrum protease which exhibits a combination of all the above features, namely halotolerance, stability to detergents, molecular weight not less than 76 kDa and activity on a non-collagenous substance such as elastin, to ensure its successful industrial application especially with respect to leather. The inventiveness of the present invention lies in providing a surfactant, oxidant stable and halotolerant bacterial alkaline protease having elastolytic activity but not the collagenolytic activity, growing a bacterial strain in a neutral medium containing no salt or alkali. Objects of the Invention The main object of the present invention is thus to provide a novel alkaline protease from bacterial source for industrial applications, which obviates the limitations as stated above. Another object of the present invention is to provide a protease that exhibits halotolerant property. Yet another object of the present invention is to provide a protease which exhibits activity and stability in the presence of oxidants and surfactants. Yet another object of the present invention is to provide a protease that exhibits elastolytic activity coupled to the absence of collagenolytic activity. Still another object of the present invention is to provide a process to prepare a protease from a bacterial strain of Bacillus subtilis, designated as MTCC 5333, isolated from an effluent treatment plant of KAR Tannery, Dindigul, Tamilnadu, India and deposited in MTCC, IMTECH, Chandigarh. Yet another object of the present invention is to provide a process to produce a halotolerant alkaline protease in a medium of neutral pH that does not contain salt or alkali. Summary of the Invention The present invention provides a novel alkaline protease produced from a bacterial strain of Bacillus subtilis, deposited at MTCC, IMTECH, Chandigarh, India and designated as MTCC 5333, the molecular weight of the protease being in the range of 76-84 kDa, wherein the said protease exhibits specificity against substrates such as non-collagenous fibrous protein, globular protein, casein, egg albumin, proteoglycans and elastin; the said protease having stability in pH range of 5.0-12.0, temperature ranging between 10-60 °C; activity in pH range of 6.0-11.0 and temperature ranging between 10-70 °C; wherein the said protease exhibits activity and stability in salt, surfactants and oxidants, and the said protease further exhibits action towards elastin but inactive to collagen. Accordingly, the present invention provides a novel alkaline protease produced from a bacterial strain of Bacillus subtilis, deposited at MTCC, IMTECH, Chandigarh, India and designated as MTCC 5333, wherein the said protease exhibits the following characteristics: [a] molecular weight in the range of 76 to 84 kDa; [b] specificity against substrates such as non-collagenous fibrous proteins, globular proteins, casein, egg albumin, proteoglycans and elastin; [c] stability in pH range of 5.0 to 12.0 and temperature ranging between 10 to 60 degree C; [d] activity in pH range of 6.0 to 11.0 and temperature ranging between 10 to 70 degree C; [e] activity and stability in the presence of salt, surfactants and oxidants; [f] action towards elastin but inert to collagen. The invention further provides a process for the preparation of a novel alkaline protease from bacterial source MTCC 5333, wherein the process steps comprise: [i] culturing Bacillus subtilis, deposited in MTCC, IMTECH, Chandigarh and designated as MTCC 5333 in a culture medium containing essentially a carbon source, nitrogenous source, inorganic salt and preferably an antifoaming agent, maintained at a pH in the range of 6.0 to 7.0, under aerobic submerged culture conditions, at temperature ranging from 25 to 35 degree C, preferably under shaking conditions; [ii] harvesting the culture medium obtained in step [i] after a period of 24 to 36 hrs to obtain the enzyme alkaline protease in the extracellular medium; [iii] separating the enzyme from the culture medium as obtained in step [ii] by known methods followed by purification to obtain the novel alkaline protease in liquid form; [iv] optionally drying the novel alkaline protease in liquid form as obtained in step [iii] to obtain the powdered alkaline protease. The invention also provides a biologically pure bacterial strain of Bacillus subtilis, deposited in MTCC, IMTECH, Chandigarh and designated as MTCC 5333. In an embodiment of the present invention, the bacterial strain used is isolated from an effluent treatment plant of KAR Tannery, Dindigul, Tamilnadu, India. In another embodiment of the present invention, the carbon source used for the production medium may be selected from glucose, sucrose, starch, glycerol, lactose. In still another embodiment of the present invention, the nitrogenous source used for the culture medium may be selected from soya bean meal, casamino acids, casein, corn steep liquor, yeast extract, either individually or in different combination. In yet another embodiment of the present invention, the inorganic salt used may be selected from CaCI2, MgSO4, KH2PO4either individually or in different combination. In still another embodiment of the present invention, the antifoaming agent used may be selected from olive oil, polypropylene glycol 2000, silicone oil, soyabean oil, cotton seed oil. In yet another embodiment of the present invention, the production medium for the protease is prepared without using either alkali or common salt. In still another embodiment of the present invention, the method of separation used may be such as microfiltration, centrifugation. In yet another embodiment of the present invention, the method of purification may be such as ultrafiltration, salting out method, gel-filtration, ion exchange chromatographic methods in any combination and order. In still another embodiment of the present invention, the method of drying the liquid protease may be such as freeze drying, spray drying, air drying, and vacuum drying. In yet another embodiment of the present invention, the molecular weight of the protease may be in the range of 76-84 kDa. In still another embodiment of the present invention, the oxidants against which the protease exhibits stability may be such as hydrogen peroxide, sodium perborate. In yet another embodiment of the present invention, the protease exhibits stability and activity in NaCI solution of strength not more than 6 M. In yet another embodiment of the present invention, the protease exhibits stability and activity in hydrogen peroxide solution of strength not more than 4 M. In still another embodiment of the present invention, the protease exhibits stability and activity in sodium perborate solution of strength not more than 10%. Detailed Description of the Invention The bacterial strain Bacillus subtilis MTCC 5333 is isolated from an effluent sample having pH 11.0, collected from effluent treatment plant of KAR tannery, Dindigul, Tamilnadu, India. Initially, 10 ml aliquot of the effluent sample is mixed along with 100 gms of sterile humic soil as well as 5 gms of casein in a 250 ml Erlenmeyer flask and the mixture is incubated for 3-5 days at 30 °C for enrichment. Following enrichment, the sample is serially diluted and plated onto the medium comprised of 0.5% glucose, 0.25% peptone, 7.5% skim milk and 2% agar. The plates are incubated at 30 °C overnight. The colony displaying the maximal zone of clearance on the skim milk plate is selected, purified by streak plate method. The strain exhibiting maximal zone of clearance is isolated and identified to be Bacillus subtilis. The strain is deposited in MTCC, IMTECH, Chandigarh and is designated as MTCC 5333. The isolate is a Gram positive, obligate aerobe, spore forming, motile and rod shaped bacterium. The isolate forms bulging sporangia bearing centrally positioned, oval shaped endospores. Features of colony morphology include round configuration, wavy margin, convex elevations, rough surface and opaque density. It grows well at initial pH values between 5.0-11.0 and over a range of temperatures from 15°- 60° C. Growth of the bacterium does not require NaCI but it also grows well in the presence of 10% NaCI. Following are the biochemical features of the bacterium: it is positive for Voges Proskauer test, citrate utilization, casein hydrolysis, starch hydrolysis, gelatin hydrolysis, nitrate reduction and catalase test but, negative for indole test, methyl red, urea hydrolysis, nitrite reduction and H2S production. The bacterium is fast growing, fermentative and produces acid from the carbohydrates such as dextrose, fructose, cellobiose, galactose, inositol, inulin, maltose, mannitol, mannose, salicin, sorbitol and sucrose. However, acid production from lactose, arabinose and rhamnose is absent. The production medium is prepared using essentially a carbon source, nitrogenous source and inorganic salt. The medium is maintained at a pH in the range of 6.0 to 7.0. The bacterial strain of Bacillus subtilis, deposited at MTCC, IMTECH, Chandigarh and having designation no. MTCC 5333 is grown under aerobic conditions in the said production medium in submerged culture conditions with shaking. The medium is harvested after a period of 24-36 hrs and is subjected to separation by known methods. The separated crude enzyme is partially purified by known methods to obtain the alkaline protease in liquid form, which is optionally dried conventionally to obtain the protease in powder form. The protease is purified by known methods such as ion exchange and gel filtration chromatography and the molecular weight of the purified protease is determined by 10% Sodium Docecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The protease activity is expressed in terms of tyrosine equivalents using Hammersten casein. To 1.9 ml of 1 % casein solution prepared in 0.1 M carbonate buffer of pH 9.5, 0.1 ml of suitably diluted enzyme solution is added and the reaction mixture is kept at 40° C for 10 min. The reaction is terminated by the addition of 3 ml of 5% trichloroacetic acid solution. The absorbance of the trichloroacetic acid soluble filtrate at 280 nm is measured and one unit is expressed as the liberation of one mg tyrosine equivalent of substrate per gram of dried enzyme in 10 min. One unit is also expressed as the liberation of one mg of tyrosine equivalent of substrate per ml in 10 min. The protease activity of the enzyme preparation varies in the range of 20 - 800 units. Elastolytic activity is determined using elastin-Congo Red (Sigma) as the substrate according to Cahan et al. (Journal of Biological Chemistry, 276: 43645-43652, 2001), with suitable modifications. Reaction mixture containing 10 mg of elastin-Congo Red in 2.2 ml of 0.1 M Tris-HCI, 0.5 mM CaCI2, pH 8.0, and 0.1 ml of suitably diluted enzyme solution, are incubated with shaking at 37 °C for 1 hour. The reaction is stopped by adding 0.2 ml of 0.12 M EDTA, followed by centrifugation and measurement of absorbance at 495 nm of the clear supernatant. One unit of activity is the amount of protease that causes an optical density increase of 1 unit/h at 495 nm. Elastolytic activity of the enzyme preparation varies in the range of 10-50 units. Collagenolytic activity is determined using collagen from Bovine Achilles Tendon as reported by Woessner (Archives of Biochemistry and Biophysics, 93: 440-447, 1961). To 2 ml of 0.1% collagen solution prepared in Tris-HCI, pH 8.0, 0.5 ml of suitably diluted enzyme is added and incubated at 37 °C for 1 h. The reaction mixture is filtered and 1.0 ml of Chloramine-T is added to the filtrate and incubated at room temperature for 20 min. To this, 1.0 ml of perchloric acid is added. After 5 min, 1.0 ml of freshly prepared p-Dimethylamino benzaldehyde (DMAS) is added, mixed well and incubated at 60 °C for 20 min. The pink colour developed is read at 557 nm against blank containing distilled water instead of enzyme solution. Protease does not exhibit action towards collagen substrate. The substrate specificity of the protease is evaluated using various substrates such as bovine hemoglobin by a method as reported by Anson (Journal of General Physiology, 22: 79-89, 1938), azocasein by a method as reported by Brock et al. (Applied and Environmental Microbiology, 44:561-569, 1982), azocoll by a method as reported by Chavira et al. (Analytical Biochemistry, 136: 446-450, 1984), hide powder azure by a method as reported by Mozersky and David (Journal of American Leather Chemists Association, 87: 287-294, 1992) and keratin azure by a method as reported by Santo et al. (Current Microbiology, 33: 364-370, 1996), This enzyme is active against casein, hemoglobin, azocasein, azocoll, hide powder azure and keratin azure suggesting that it has a broad specificity towards various substrates. It also has an elastase activity as it hydrolyzes elastin-congo red. However, the protease does not have collagenolytic activity as it does not liberate hydroxy proline from collagen. Table 1 provides a comparative data relating to the protease of the present invention and that produced by Bacillus subtilis FP-133 strain and also by a different strain of Bacillus clausii I-52, considered to be the closest prior art. (Table Removed) Table 1. Comparative account of protease from Bacillus subtilis of the present invention with that of the closest prior art with respect to properties and applications. (Table Removed) The following examples are given by way of illustration only and therefore should not be construed to limit the scope of the present invention. Example 1 A culture medium [pH - 6.4] was prepared by mixing the following ingredients: (Table Removed) pH - 6.4 750 ml medium containing the ingredients was taken in a 2.8 L Fernbach flask and cells of Bacillus subtilis MTCC 5333 were inoculated in the medium. The flask was fitted to a shaker moving at 125 rpm. The temperature was maintained at 30 °C and shaking was continued. After a period of 24 hrs, the shaker was stopped and 600 ml of the medium was transferred to a 20 L fermentor with working volume of 14 L. Polypropylene glycol 2000 was used as antifoam agent. Air was fed to the fermentor at the rate of 0.5 vvm and the temperature was maintained at 30 °C. The liquid was subjected to stirring with an agitator rotating at a speed of 200 rpm. After a period of 30 hrs, the culture medium was harvested and subjected to centrifugation to collect the crude enzyme in liquid form. Using casein substrate, the activity of the crude enzyme was found to be 25 units/ml. This broth containing crude liquid enzyme was subjected to ultrafiltration using 10-kDa membrane to obtain liquid enzyme concentrate. The protease activity of the ultrafiltrate was found to be 150 units/ml. This ultrafiltrate was subjected to salting out employing 60% ammonium sulphate saturation and the resulting precipitate was subjected to freeze drying with 2% maltodextrin to obtain 110 g of dry protease powder. It was stored at 4 °C. The characteristics of the protease were studied and they are mentioned below. Activity with casein : 850 units/g Molecular weight : 78 kDa The concentrate obtained by ultrafiltration and ammonium sulphate saturation was subjected to gel-filtration and ion exchange chromatographic methods using Sephadex G200 and QAE-Dowex an ion exchange matrix respectively to purify the protease and the molecular weight was determined by 10% SDS-PAGE. Halotolerance The effect of NaCI on protease stability was studied by incubating 1 g protease having activity of 850 units each with 50 ml solution of 1, 2.5, 5 and 6 M NaCI solution for 24 hrs at 30 °C and the residual activity was measured. Also, the effect of NaCI concentration on the activity was evaluated by incubating 0.1 ml of enzyme suspension having an activity of 8.5 units/ml with 1.9 ml of assay mixture containing 1% casein and 1-6 M NaCI and the assay was carried out. The stability and activity of protease were about 80 and 70% respectively in the presence of 5 M NaCI. Oxidant stability The stability of protease in the presence of oxidant was examined by incubating 1 g protease each with 50 ml solution of 0.5, 1, 2 and 4 M hydrogen peroxide for 4 hrs at 30 °C and the residual activity was estimated. About 70% of the original activity was retained with 2 M oxidant whereas 45% of the activity was retained with 4 M hydrogen peroxide. Detergent stability The stability of protease in the presence of different commercial detergents was studied by incubating 1 g protease each with 50 ml solution containing commercial detergents at the concentration of 10 mg/ml for 4 hrs at 30 °C and the residual activity was determined. 80-90% of the protease activity was retained in the presence of commercial detergents. Specificity of protease on other substrates: Activity on elastin : 45 units/g Collagenolytic activity using bovine collagen : Nil Activity using hemoglobin substrate: 400 units/g Activity using Azocasein substrate: 230 units/g Activity using Azocoll substrate: 20 units/g Activity using Hide powder azure substrate: 15 units/g Activity using Keratin azure substrate: 12 units/g Example 2 A culture medium was prepared by mixing the following ingredients. (Table Removed) pH-6.5 2.1 L medium containing the ingredients was taken in a 3 L Fermentor and 50 ml of 24 hrs seed inoculum of Bacillus subtilis MTCC 5333 developed in shake flask culture was transferred to the medium. The fermentation was carried out at 30°C and agitation of 250 rpm for 20 hrs after which, 1.5 L of this culture was transferred to 20 L fermentor with a medium volume of 13.5 L. Air was fed at a flow rate of 1 vvm and the temperature was maintained at 30 °C. The medium in the fermentor was subjected to stirring with an agitator rotating at a speed of 250 rpm. After a period of 28 hrs, the culture medium was harvested and subjected to centrifugation to collect the crude enzyme in liquid form. Using casein substrate, the activity of the crude enzyme was found to be 22 units/ml. This broth containing crude liquid enzyme was subjected to ultrafiltration using 10-kDa membrane to obtain liquid enzyme concentrate having activity of 140 units/ml. This ultrafiltrate was subjected to spray drying with inlet and outlet temperatures of 115 °C and 70 °C respectively along with 20% maltodextrin and 5% lactose to obtain about 200 g of dry protease powder. It was stored at 4 °C. The characteristics of the protease were studied and they are mentioned below. Activity : 400 units/g Halotolerance The effect of NaCI on protease stability was studied by incubating 1 g protease having activity of 400 units each with 50 ml solution of 1, 2.5, 5 and 6 M NaCI solution for 24 hrs at 30 °C and the residual activity was measured. Also, the effect of NaCI concentration on the activity was evaluated by incubating 0.1 ml of enzyme suspension having an activity of 4 units/ml with 1.9 ml of assay mixture containing 1% casein and 1-6 M NaCI and the assay was carried out. The stability and activity of protease were about 75 and 65% respectively in the presence of 6 M NaCI. Oxidant stability The stability of protease in the presence of oxidant was examined by incubating 1 g protease each with 50 ml solution of 1, 2.5, 5 and 10% sodium perborate for 4 hrs at 30 °C and the residual activity was estimated. About 80% of the original activity was retained with 5% oxidant whereas 50% of the activity was retained with 10% perborate. Detergent stability The stability of protease in the presence of different cationic, anionic and nonionic surfactants was studied by incubating 1 g protease each with 50 ml solution containing surfactants at the concentration of 10 mg/ml for 4 hrs at 30 °C and the residual activity was determined. 60-70% protease activity was retained with cationic surfactants and 80-90% protease activity was retained in the presence of anionic surfactants. However, the activity was enhanced by 10-40% in the case of nonionic surfactants. Specificity of protease on other substrates: Activity on elastin : 18units/g Collagenolytic activity using bovine collagen : Nil Activity using hemoglobin substrate: 200 units/g Activity using Azocasein substrate: 125 units/g Activity using Azocoll substrate: 12 units/g Activity using Hide powder azure substrate: 7 units/g Activity using Keratin azure substrate: 4 units/g The protease was used in dehairing of salted goat skins. 5 Nos. of wet salted goatskins, weighing 10 kg were suspended in a 100% dehairing water float containing 2.5% protease having 1000 units (based on casein assay) and 0.4% Tween 80 on the salted weight of skins for 8 hrs at room temperature. The pH of the float was adjusted to 9.0 using 0.4 % sodium carbonate. Dehairing was carried out in a usual manner. Example 3 A culture medium was prepared by mixing the following ingredients. (Table Removed) pH-6.0 14 L medium containing the ingredients was taken in a 20 L fermentor and 700 ml of 24 hrs culture of Bacillus subtilis MTCC 5333 developed in Fernbach flask culture was transferred to the medium. The fermentation was carried out at 30 °C and agitation of 200 rpm for 20 hrs after which, 10 L of this culture was transferred to 300 L fermentor with a medium volume of 200 L. Air was fed at a flow rate of 1 vvm and the temperature was maintained at 30 °C. The medium in the fermentor was subjected to stirring with an agitator rotating at a speed of 150 rpm. After a period of 28 hrs, the culture medium was harvested and subjected to continuous centrifugation to collect the crude enzyme in liquid form. Using casein substrate, the activity of the crude enzyme was found to be 20 units/ml. This broth containing crude liquid enzyme was subjected to ultrafiltration using 10-kDa membrane to obtain liquid enzyme concentrate having activity of 130 units/ml. This ultrafiltrate was stored at 4 °C. The characteristics of the protease were studied and they are mentioned below. Activity with casein : 130 units/ml Halotolerance The effect of NaCI on protease stability was studied by incubating 20 ml protease concentrate having activity of 2500 units each with 30 ml solution of 2, 4 and 8 M NaCI for 24 hrs at 30 °C and the residual activity was measured. Also, the effect of NaCI concentration on the activity was evaluated by incubating 0.1 ml of diluted enzyme suspension having an activity of 6.5 units/ml with 1.9 ml of assay mixture containing 1% casein and 1-5 M NaCI and the assay was carried out. The stability and activity of protease were about 80 and 70% respectively in the presence of 5 M NaCI. Oxidant stability The stability of protease in the presence of oxidant was examined by incubating 25 ml protease each with 25 ml solution of 1, 2, 4 and 8 M hydrogen peroxide for 4 hrs at 30 °C and the residual activity was estimated. About 65% of the original activity was retained with 2 M oxidant whereas 40% of the activity was retained with 4 M hydrogen peroxide. Detergent stability The stability of protease in the presence of commercial detergents was studied by incubating 25 ml protease concentrate with 25 ml solution containing detergents at the concentration of 40 mg/ml for 4 hrs at 30 °C and the residual activity was determined. 60-80% of the protease activity was retained in the presence of commercial detergents. Specificity of protease on other substrates: Activity on elastin : 15 units/ml Collagenolytic activity using bovine collagen : Nil Activity using hemoglobin substrate: 55 units/g Activity using Azocasein substrate: 60 units/g Activity using Azocoll substrate: 2 units/g Activity using Hide powder azure substrate: 3 units/g Activity using Keratin azure substrate: Nil Example 4 A culture medium was prepared by mixing the following ingredients. (Table Removed) pH-6.4 1.5 L medium containing the ingredients except casein was taken in a 3 L fermentor and 100 ml of 24 hrs culture of Bacillus subtilis MTCC 5333 developed in Erlenmeyer conical flask was transferred to the medium. The fermentation was carried out at 30 °C and agitation of 250 rpm for 10 hrs after which, 500 ml of 4% casein solution was fed at a flow rate of 0.7 ml/min continuously for 12 hrs using peristaltic pump. Air was fed at a flow rate of 0.5 vvm and the temperature was maintained at 30 °C. The medium in the fermentor was subjected to stirring with an agitator rotating at a speed of 300 rpm. After a period of 12 hrs, the culture medium was harvested and subjected to continuous centrifugation to collect the crude enzyme in liquid form. The activity of the crude enzyme was found to be 32 units/ml using casein substrate. This broth containing crude liquid enzyme was subjected to ultrafiltration using 10-kDa membrane to obtain liquid enzyme concentrate having activity of 180 units/ml. This ultrafiltrate was spray dried employing inlet and outlet temperatures of 115 °C and 75 °C respectively with 20 % maltodextrin and 5% lactose to get 50 g protease powder having activity of 450 units/g and it was stored at 4 °C. The characteristics of the protease were studied and they are mentioned below. Activity with casein : 450 units/g Halotolerance : Stability with 6 M NaCI for 24 hrs at 30°C: 70% Activity in the presence of 6 M NaCI: 55% Oxidant stability: The following stability studies with oxidants were carried out for 4 hrs at 30 °C Stability with 2 M H2O2: 85% Stability with 4 M H2O2: 40% Stability with 5% perborate : 70% Stability with 10% perborate : 30% Specificity of protease on other substrates: Activity on elastin : 30 units/g Collagenolytic activity using bovine collagen : Nil Activity using hemoglobin substrate: 180 units/g Activity using Azocasein substrate: 125 units/g Activity using Azocoll substrate: 13 units/g Activity using Hide powder azure substrate: 10 units/g Activity using Keratin azure substrate: 8 units/g The protease was used in dehairing of salted hides. 4 Nos. of dry salted cow hides, weighing approximately 48 kg were immersed in dehairing suspension comprising 150% water, 3.0% protease having 1350 units (based on casein assay) and 1% Tween 80 on the salted weight of hides for 10 hrs at room temperature. The pH of the float was adjusted to 8.0 using 0.5 % sodium carbonate. Dehairing is carried out in a usual manner. Example 5 A culture medium was prepared by mixing the following ingredients. (Table Removed) pH-6.7 600 ml medium containing the ingredients was taken in a 2.8 L Fernbach flask and cells of Bacillus subtilis MTCC 5333 were inoculated in the medium. The flask was kept for incubation in a shaker at 150 rpm. The temperature was maintained at 30 °C and shaking was continued. Olive oil was used as antifoam agent. After a period of 24 hrs, the shaker was stopped and 500 ml of the medium was transferred to a 20 L fermentor with working volume of 12 L. Air was fed to the fermentor at the rate of 1 vvm and the temperature was maintained at 30 °C. The liquid in the fermentor was subjected to stirring with an agitator rotating at a speed of 200 rpm. After a period of 30 hrs, the culture medium was harvested and subjected to centrifugation to collect the crude enzyme in liquid form. The activity of the crude enzyme was found to be 20 units/ml using casein substrate. This broth containing crude liquid enzyme was subjected to ultrafiltration using 10-kDa membrane to obtain liquid enzyme concentrate. The protease activity of the ultrafiltrate was found to be 120 units/ml. This ultrafiltrate was subjected to salting out employing 60% ammonium sulphate saturation and the resulting precipitate was subjected to freeze drying with 2% lactose to obtain 90 g of dry protease powder. It was stored at 4 °C. The characteristics of the protease were studied and they are mentioned below. Activity with casein : 700 units/g Halotolerance Stability with 4 M NaCI for 24 hrs at 30 °C: 85% Activity in the presence of 4 M NaCI: 75% Oxidant stability The following stability studies with oxidants were carried out for 4 hrs at 30 °C Stability with 2 M H2O2: 70% Stability with 4 M H2O2: 50% Stability with 5% perborate : 75% Stability with 10% perborate : 35% Specificity of protease on other substrates: Activity on elastin : 45 units/g Collagenolytic activity with bovine collagen : Nil Activity with hemoglobin substrate: 280 units/g Activity with Azocasein substrate: 170 units/g Activity with Azocoll substrate: 12 units/g Activity with Hide powder azure substrate: 18 units/g Activity with Keratin azure substrate: 10 units/g The protease was used in dehairing of salted and soaked hides. (a) 4 Nos. of wet salted buff hides, weighing approximately 60 kg were immersed in dehairing suspension comprising 200% water, 3.0% enzyme having 2000 units (based on casein assay), and 1.5% Tween 80 on the salted weight of hides and the hides were dehaired after 12 hrs by the usual procedure. (b) 5 Nos. of wet salted buff halves, weighing approximately 30 kg were initially soaked using 300% water for 3 hrs and the wet weight was noted. The presoaked halves were subjected to main soaking comprising 300% water, 0.01% wetting agent, 0.01% preservative on the wet weight and the pH was adjusted to 8.0 using sodium carbonate and left overnight (18 hrs) at ambient temperature. The soaked hides were green-fleshed using conventional method and the soaked defleshed weight was noted. The defleshed hides were immersed in dehairing suspension after green fleshing comprising 100% water, 2.0% enzyme and 1% Triton X 100. The pH of dehairing float was adjusted to 9.0 to 10.0 using approximately 0.4% sodium carbonate and handled for three or four times and left overnight. The hides were dehaired after 12 hrs by the usual procedure. Example 6 A culture medium was prepared by mixing the following ingredients. (Table Removed) pH-6.5 The 14 L medium containing the above ingredients was taken in 20 L fermentor and it was fed with 5% of 20 hrs inoculum developed in Fernbach flasks. Air was flown to the fermentor at the rate of 1 vvm and the temperature was maintained at 30 °C. The liquid in the fermentor was subjected to stirring with an agitator rotating at a speed of 200 rpm. After a period of 30 hrs, the culture medium was harvested and subjected to centrifugation to collect the crude enzyme in liquid form. The activity of the crude enzyme was found to be 20 units/ml. This broth containing crude liquid enzyme was subjected to ultrafiltration using 10-kDa membrane to obtain liquid enzyme concentrate having activity of 100 units/ml. This ultrafiltrate was subjected to spray drying along with 20% maltodextrin and 5% lactose to obtain about 200 g of dry protease powder. The characteristics of the protease were studied and they are mentioned below. Activity with casein : 350 units/g Halotolerance Stability with 5 M NaCI for 24 hrs at 30°C: 70% Activity in the presence of 5 M NaCI: 65% Oxidant stability The following stability studies with oxidants were carried out for 4 hrs at 30°C Stability with 2 M H2O2: 70% Stability with 4 M H2O2: 40% Stability with 5% perborate : 70% Stability with 10% perborate : 45% Specificity of protease on other substrates: Activity on elastin : 15 units/g Collagenolytic activity with bovine collagen : Nil Activity with hemoglobin substrate: 125 units/g Activity with Azocasein substrate: 80 units/g Activity with Azocoll substrate: 5 units/g Activity with Hide powder azure substrate: 8 units/g Activity with Keratin azure substrate: 5 units/g The protease was used as an additive in laundry detergent formulation. For use as a detergent additive, the washing efficiency of the protease was determined by washing tests on 100 Nos. of 10 x 5 cm cotton fabric pieces that were heavily soiled with blood/milk/egg yolk. The stained fabric pieces were washed with 1 liter of water containing 0.2 g (75 units based on casein assay) enzyme and 6 g of commercially available detergent base formulation for 20 min. pH of the washing solution was 10.0. Washing was carried out at 25 °C. After the washing process, the test fabric was rinsed twice with tap water. The washing efficiency was determined by measuring the protein content in the wash solution. The protease was found to be efficient in the removal of protein stains. There was a rise of 20% protein removal when the protease was added along with detergent compared to detergent alone wash. The main advantages of the present invention are the following: • Production of protease from a fast growing industrial Bacillus subtilis MTCC 5333 strain is beneficial in terms of scalability and high yield and the process of production is safer as the bacterium is "Generally Regarded as Safe" (GRAS) microbe. • The production medium in which the strain Bacillus subtilis MTCC 5333 is grown, requires commercially available nutrients and does not require alkali or salt to produce high yields of halotolerant alkaline protease. • The protease is having halotolerance and it is exhibiting its activity and stability in the presence of 6 M salt concentration. Hence the protease can find its potential applications in industry where high saline conditions are employed. • The protease is having oxidant stability and it is stable in the presence of oxidants such as hydrogen peroxide and perborate. It is also stable in the presence of variety of surfactants and detergent components. • • Properties such as halotolerance, stability in oxidants, surfactants and alkaline conditions in addition to broad substrate specificity enable the protease to be used as an effective additive in laundry and heavy duty detergent formulations. • The halotolerant property and stability towards surfactants enable the protease to be used in the soaking bath itself, thereby contributing to the productivity enhancement in dehairing operations of leather manufacture. • The protease exhibits elastase activity but it has no collagenolytic activity. This feature enables the protease to be used in ecobenign lime-sulfide free enzymatic dehairing of skins and hides in leather processing. • The protease possesses halotolerance, stability in surfactants, detergent components, oxidants and alkaline conditions and broad substrate specificity including activity towards elastin and absence of collagenolytic activity and combination of all the properties enables the protease to be used in leather, detergent and other industries. We Claim: 1. A novel alkaline protease produced from a bacterial strain of Bacillus subtilis, deposited at MTCC, IMTECH, Chandigarh, India and designated as MTCC 5333, wherein the said protease exhibits the following characteristics: [a] molecular weight in the range of 76 to 84 kDa; [b] specificity against substrates such as non-collagenous fibrous proteins, globular proteins, casein, egg albumin, proteoglycans and elastin; [c] stability in pH range of 5.0 to 12.0 and temperature ranging between 10 to 60 degree C; [d] activity in pH range of 6.0 to 11.0 and temperature ranging between 10 to 70 degree C; [e] activity and stability in the presence of salt, surfactants and oxidants; [f] action towards elastin but inert to collagen. 2. A novel alkaline protease as claimed in claim 1, wherein the oxidants against which the protease exhibits stability are such as hydrogen peroxide, sodium perborate. 3. A novel alkaline protease as claimed in claim 1, wherein the protease exhibits stability and activity in NaCI solution of strength not more than 6 M. 4. A novel alkaline protease as claimed in claim 1, wherein the protease exhibits stability and activity in hydrogen peroxide solution of strength not more than 4 M. 5. A novel alkaline protease as claimed in claim 1, wherein the protease exhibits stability and activity in sodium perborate solution of strength not more than 10%. 6. A novel alkaline protease as claimed in claim 1, wherein it is useful for dehairing of skins, hides and surfaces as well as in removing proteinaceous stains. 7. A biologically pure bacterial strain of Bacillus subtilis, deposited in MTCC, IMTECH, Chandigarh and designated as MTCC 5333, wherein the said isolate exhibits the following characteristics: [a] gram positive, obligate aerobe, spore forming, motile, and rod shaped bacterium; [b] colony morphology having round configuration, wavy margin, convex elevations, rough surface and opaque density; [c] biochemical characteristics: positive for Voges Proskauer test, citrate utilization, casein hydrolysis, starch hydrolysis, gelatin hydrolysis, nitrate reduction and catalase test but negative for indole test, methyl red, urea hydrolysis, nitrite reduction and H2S production; produces acid from the carbohydrates such as dextrose, fructose, cellobiose, galactose, inositol, inulin, maltose, mannitol, mannose, salicin, sorbitol and sucrose but not from lactose, arabinose and rhamnose; [d] grows well at initial pH values between 5.0-11.0 and over a range of temperatures from 15 to 60 degree C. 8. A biologically pure bacterial strain as claimed in claim 7, wherein it is isolated from an effluent sample having pH 11.0, collected from effluent treatment plant of KAR tannery, Dindigul, Tamilnadu, India. 9. A biologically pure bacterial strain as claimed in claim 7, wherein the said strain is stable at a pH ranging from 5.0 to 11.0. 10. A biologically pure bacterial strain as claimed in claim 7, wherein the said strain is stable at a temperature ranging from 15 to 60 degree C. 11. A biologically pure bacterial strain as claimed in claim 7, wherein the said strain is tolerant to salt concentrations upto 6M. 12. A process for the preparation of a novel alkaline protease as claimed in claim 1, from bacterial source, wherein the process steps comprise: [i] culturing Bacillus subtilis, deposited in MTCC, IMTECH, Chandigarh and designated as MTCC 5333 in a culture medium containing essentially a carbon source, nitrogenous source, inorganic salt and preferably an antifoaming agent, maintained at a pH in the range of 6.0 to 7.0, under aerobic submerged culture conditions, at temperature ranging from 25 to 35 degree C, preferably under shaking conditions; [ii] harvesting the culture medium obtained in step [i] after a period of 24 to 36 hrs to obtain the enzyme alkaline protease in the extracellular medium; [iii] separating the enzyme from the culture medium as obtained in step [ii] by known methods followed by purification to obtain the novel alkaline protease in liquid form; [iv] optionally drying the novel alkaline protease in liquid form as obtained in step [iii] to obtain the powdered alkaline protease. 13. A process as claimed in claim 12, wherein the bacterial strain used is isolated from an effluent treatment plant of KAR Tannery, Dindigul, Tamilnadu, India. 14. A process as claimed in claim 12, wherein the carbon source used for the culture medium is selected from glucose, sucrose, starch, glycerol, lactose. 15. A process as claimed in claim 12, wherein the nitrogenous source used for the culture medium is selected from soya bean meal, casein, corn steep liquor, casamino acids, yeast extract, either individually or in different combination. 16. A process as claimed in claim 12, wherein the inorganic salt used is selected from CaCI2, MgSO4, KH2PO4, either individually or in different combination. 17. A process as claimed in claim 12, wherein the antifoaming agent used is selected from olive oil, polypropylene glycol 2000, silicone oil, soyabean oil, cotton seed oil. 18. A process as claimed in claim 12, wherein the production medium for the protease is prepared without using either alkali or common salt. 19. A process as claimed in claim 12, wherein the method of separation used is such as microfiltration, centrifugation. 20. A process as claimed in claim 12, wherein the method of purification is such as ultrafiltration, salting out method, gel-filtration, ion exchange chromatographic methods in any combination and order. 21. A process as claimed in claim 12, wherein the method of drying the liquid protease is such as freeze drying, spray drying, air drying, vacuum drying. 22. A novel alkaline protease and a process for the preparation thereof substantially as herein described with reference to the foregoing examples.

Full Text

Field of invention
The present invention relates to a novel alkaline protease and a process for the preparation thereof. More particularly, the present invention relates to a novel alkaline protease from bacterial source for industrial applications. In particular, the present invention relates to a novel protease produced from a bacterial strain of Bacillus subtilis MTCC 5333. The protease of the present invention finds potential application in leather processing industry as an eco-benign option for dehairing of hides and skins without using any chemicals. An important feature of this protease is that while it exhibits elastolytic activity, there is no collagenolytic activity. In addition, it exhibits activity and stability in the presence of high concentrations of salt, surfactants and oxidants. This feature enables the protease to be used in the soaking bath itself, thereby contributing to the productivity enhancement. Further, the enzyme is envisaged to have potential application as an additive in detergent industry.
Background of invention and Description of Prior art
Proteases are conventionally produced from several micro-organisms, plant sources and animal sources. However, commercial production of protease from bacterial and fungal origin have gained much importance because of their extracellular production, submerged cultivation, high yield and short duration of production and easy recovery of the enzyme.
As reported by Gupta et al. (Applied Microbiology and Biotechnology, 60: 381-395, 2002) and Tjalsma et al. (Journal of Applied Microbiology, 96: 569, 2004), although wide ranges of microbes are known to produce proteases, major industrial enzymes with increasing market potentials are derived from Bacillus group of bacteria due to their higher capacity and activity. Being a fast growing and non-pathogenic GRAS (Generally Regarded As Safe) microbe, production of protease especially from Bacillus subtilis strains is beneficial in terms of scalability, high yield and commercial exploitation as reported by Gupta et al. (Applied Microbiology and Biotechnology, 60: 381-395, 2002) and De Boer and Diderichsen (Applied Microbiology and Biotechnology, 36: 1-4, 1991).

There have been several reports on the different features of the proteases available in the public domain. They are summarized below:
Stability against detergents and oxidants
Generally, proteases are susceptible to the oxidative inactivation by the oxidants and denaturation by anionic surfactants. Application of proteases in the detergent formulations faces various practical problems because most recent commercial detergent products contain anionic surfactants, bleaching agents, oxidants, enzymes other than proteases and water-softening builders such as zeolites. Therefore, the suitability of the proteases for use as a detergent additive depends on its stability and compatibility with these components at alkaline pH and wide range of temperature, and ideally it should be stable and active in the detergent solution.
As reported by Siezen and Leunissen (Protein Science, 6: 501-523, 1997), most of the proteolytic enzymes are classified under serine alkaline proteases (or Subtilisins, E.C. 3.4.21.14) and they mainly function in a narrow range of pH, temperature, and ionic strength. Among them, a few are able to resist chemical oxidants forming a new subfamily of subtilisins named oxidatively stable alkaline proteases, as reported by Okuda et at. (Extremophiles, 8: 229-235, 2004). Saeki et al. (Biochemical Biophysical Research Communications, 279: 313-319, 2000) have reported oxidatively stable subtilisin-like serine proteases, having 43-45 kDa molecular weight from five different strains of alkalophilic Bacillus. These proteases are stable in the presence of 50 mM H202. Saeki et al. (Current Microbiology, 47: 337-340, 2003) have reported a highly oxidant (in 1 M H2O2) and surfactant stable subtilisin of 30 kDa from alkalophilic Bacillus KSM-LD1. Takaiwa et al. (U.S. Pat. No. 6759228) have provided a serine alkaline protease of molecular weight of 43 kDa from Bacillus strains, having stability in various surfactants of 1% concentration and oxidant such as hydrogen peroxide of 50 mM concentration. Nonaka et al. (Journal of Biological Chemistry, 279: 47344-47351, 2004) report an oxidatively stable subtilisin-like alkaline protease of 43 kDa from Bacillus sp. KSM-KP43 that withstands 0.88 M H2O2 concentration. The limitation associated with

these wild type proteases is that they are not able to withstand high concentration of oxidants. Also halotolerant property of these enzymes is not reported.
In order to circumvent the problem of sensitivity towards oxidants, several attempts have been made through protein engineering methods to improve the functioning of proteases in the presence of oxidants and surfactants. Proteases are engineered by introducing mutation in the form of addition, substitution and deletion of amino acids.
There are various commercially available detergent proteases such as Alkalase, Savinase, Esperase and Everlase (Novozymes Biotech, Inc, Denmark), and Maxacal, Purafect OxP ™ and Properase ™ (Genencor Int., USA). These enzymes mainly originate from various members of Bacillus species and some of them like Everlase and Purafect OxP ™ are protein engineered to enhance the enzyme stability and washing performance under these harsh conditions as reported by Maurer (Current Opinion in Biotechnology, 15: 330-334,2004) and Branner et al. (U.S. Pat. No. 5482849). But, protein engineering methods essentially strive to improvise such properties of proteases as stability in the detergent components, surfactants and oxidants to suit only the demands of detergent industry and hence, their potential is confined to limited industrial applications. For example, halotolerant property of the engineered proteases is not reported and this limits their application potential for some industries that involve high salinity.
Halotolerance
Different industrial applications require proteases, which are able to maintain high activities under moderate to high saline conditions. This feature of a protease is commonly known in the art as 'halotolerance'. Several attempts have been made to produce halotolerant protease from bacterial organisms. Setyorini et al. (Bioscience Biotechnology and Biochemistry, 70: 433-440, 2006) describe two highly halotolerant proteases of 29 and 34 kDa from Bacillus subtilis FP-133 grown in a medium containing high salt concentration and the protease exhibits 80% activity in the presence of 20% NaCI. However, the major limitation associated with most of these proteases is that they

do not exhibit stability towards oxidants. This has prompted the researchers to explore possibilities for producing halotolerant protease that exhibits oxidant stability as well.
Very few reports on oxidative and surfactant stable halotolerant proteases are available from wild type Bacillus group of bacteria. Joo and Chang (Enzyme Microbial Technology, 36: 766-772, 2005; Journal of Applied Microbiology, 98: 491-497, 2005) have reported an alkaline protease of 28 kDa from alkalophilic Bacillus clausii I-52 exhibiting stability and activity at 20% NaCI concentration besides having oxidant (6% H202) and surfactant stable properties. However, it is quite evident that all these proteases exhibit a lower molecular weight not exceeding 34 kDa. Moreover, producing halotolerant alkaline protease from these bacteria requires salt or alkali and/ or both.
The limitation associated with the medium containing high salt concentration or alkali is that it causes damage to the fermentor and its accessories in addition to hampering productivity and economy as it affects the life of the fermentor. This necessitates the requirement of producing halotolerant alkaline proteases from bacterial organism grown in neutral pH as well as in the absence of salt and alkali wherein the fermentation process does not require any control in the form of maintenance of pH in the medium.
There are no reports on oxidant and detergent stable, halotolerant alkaline proteases from organisms grown in medium having neutral pH, especially from Bacillus that does not require NaCI or alkali for growth and protease production.
Molecular weight
Conventionally, the higher the molecular weight of an enzyme, the easier is the purification process, thereby ensuring lesser impurities in the final product. As reported by Kumar and Takagi (Biotechnology Advances, 17: 561-594, 1999), almost all subtilisins, excepting a very few, produced from Bacillus group of bacteria, possess molecular weight, ranging from 15-45 kDa. Ogava et al. (Biochimica et Biophysica Acta, 1624: 109-114, 2003) have produced a subtilisin-like high molecular weight alkaline protease (FT protease) of 72 kDa from Bacillus sp. KSM-P43. Similarly, Okuda et al.

(Extremophiles, 8; 229-235, 2004) have produced a 72 kDa subtilisin like alkaline protease from several Bacillus strains. But, halotolerance and oxidant stability are not reported for all these proteases of molecular weight 72 kDa.
Elastolytic Activity
Elastin is a protein present in the elastic fibers of the arteries, dermis etc. of vertebrates. Elastin is water-insoluble and has a large amount of special bridging structure such as desmosine and lysinonorleucine. The amino acid composition thereof is very special, with 50-60% of alanine and glycine. Elastase is capable of hydrolyzing elastin as it has (a) stronger binding affinity for elastin, and (b) stronger substrate specificity for alanine and glycine as reported by Stone et at. (Methods in Enzymology 82: 588-605, 1982).
As reported by Foster (Methods in Enzymology, 82: 559-570, 1982) and Paz et al. (Methods in Enzymology, 82: 571-587, 1982), elastin cannot be easily hydrolyzed by ordinary proteases. This limitation has however been obviated by Tsai et al. (Applied and Environmental Microbiology, 54: 3156-3161, 1988; U.S. Pat. No. 5972683), who have reported an elastase from Bacillus sp. Ya-B. However, the limitation associated with this is that the protease also possesses collagenolytic activity. Proteases having elastolytic activity is an added advantage especially in leather manufacture to produce certain type of leathers. But, collagenolytic activity is undesirable for the purpose. Thus, the above parameters may be summarized as follows.
There are proteases available in the art to exhibit the individual features of higher halotolerance upto a level of 20% NaCI, higher oxidant stability to the extent of 2 M, higher molecular weight upto 72 kDa or even elastase activity. But exhibiting of one property seems to be always at the cost of another feature. Hence, the use of these proteases for certain industrial applications gets restricted. For example, leather processing industry has been witnessing a shift towards eco-benign approaches to address the environmental problems effectively.

Conventionally, the raw hides and skins are predominantly preserved with the help of salt that results in the arresting of microbial growth due to dehydration. Hence, the processing of these preserved stocks in a tannery starts with water treatment that not only conditions the stock for further aqueous processing by way of rehydration, but also washes the hides/skins. The process is known as soaking in the leather trade. The processing liquor generated after soaking exhibits a very high salinity to the extent of 15-25% due to the presence of abundant salt in the hides/skins to be processed. Soaking is followed by removal of hair and other non-collagenous substances. Usually, depilants pasted on the hides/skins result in loosening of hair, which is removed manually. It requires ample time and labour, thereby affecting the productivity. The use of an enzymatic depilant in the soaking bath itself is a possible eco-benign solution to this problem towards enhancing the productivity. However, it demands that the enzyme is stable as well as active at a high salinity of not less than 25%. The enzyme also has to be stable against detergents, which may be required to be added to soaking bath to enhance rehydration. Moreover, the activity of the enzyme on any other non-collagenous substance will be an added benefit. Further, the economy of use of the enzyme may be enhanced by its purity.
Therefore, keeping in view the drawbacks of proteases available in the prior art the inventors of the present invention realized that there exists a dire need for providing a broad spectrum protease which exhibits a combination of all the above features, namely halotolerance, stability to detergents, molecular weight not less than 76 kDa and activity on a non-collagenous substance such as elastin, to ensure its successful industrial application especially with respect to leather.
The inventiveness of the present invention lies in providing a surfactant, oxidant stable and halotolerant bacterial alkaline protease having elastolytic activity but not the collagenolytic activity, growing a bacterial strain in a neutral medium containing no salt or alkali.

Objects of the Invention
The main object of the present invention is thus to provide a novel alkaline protease
from bacterial source for industrial applications, which obviates the limitations as stated
above.
Another object of the present invention is to provide a protease that exhibits halotolerant
property.
Yet another object of the present invention is to provide a protease which exhibits
activity and stability in the presence of oxidants and surfactants.
Yet another object of the present invention is to provide a protease that exhibits
elastolytic activity coupled to the absence of collagenolytic activity.
Still another object of the present invention is to provide a process to prepare a
protease from a bacterial strain of Bacillus subtilis, designated as MTCC 5333, isolated
from an effluent treatment plant of KAR Tannery, Dindigul, Tamilnadu, India and
deposited in MTCC, IMTECH, Chandigarh.
Yet another object of the present invention is to provide a process to produce a
halotolerant alkaline protease in a medium of neutral pH that does not contain salt or
alkali.
Summary of the Invention
The present invention provides a novel alkaline protease produced from a bacterial strain of Bacillus subtilis, deposited at MTCC, IMTECH, Chandigarh, India and designated as MTCC 5333, the molecular weight of the protease being in the range of 76-84 kDa, wherein the said protease exhibits specificity against substrates such as non-collagenous fibrous protein, globular protein, casein, egg albumin, proteoglycans and elastin; the said protease having stability in pH range of 5.0-12.0, temperature ranging between 10-60 °C; activity in pH range of 6.0-11.0 and temperature ranging between 10-70 °C; wherein the said protease exhibits activity and stability in salt, surfactants and oxidants, and the said protease further exhibits action towards elastin but inactive to collagen.

Accordingly, the present invention provides a novel alkaline protease produced from a bacterial strain of Bacillus subtilis, deposited at MTCC, IMTECH, Chandigarh, India and designated as MTCC 5333, wherein the said protease exhibits the following characteristics:
[a] molecular weight in the range of 76 to 84 kDa;
[b] specificity against substrates such as non-collagenous fibrous proteins, globular
proteins, casein, egg albumin, proteoglycans and elastin;
[c] stability in pH range of 5.0 to 12.0 and temperature ranging between 10 to 60
degree C;
[d] activity in pH range of 6.0 to 11.0 and temperature ranging between 10 to 70
degree C;
[e] activity and stability in the presence of salt, surfactants and oxidants;
[f] action towards elastin but inert to collagen.
The invention further provides a process for the preparation of a novel alkaline protease from bacterial source MTCC 5333, wherein the process steps comprise:
[i] culturing Bacillus subtilis, deposited in MTCC, IMTECH, Chandigarh and designated as MTCC 5333 in a culture medium containing essentially a carbon source, nitrogenous source, inorganic salt and preferably an antifoaming agent, maintained at a pH in the range of 6.0 to 7.0, under aerobic submerged culture conditions, at temperature ranging from 25 to 35 degree C, preferably under shaking conditions;
[ii] harvesting the culture medium obtained in step [i] after a period of 24 to 36 hrs to obtain the enzyme alkaline protease in the extracellular medium;
[iii] separating the enzyme from the culture medium as obtained in step [ii] by known methods followed by purification to obtain the novel alkaline protease in liquid form;
[iv] optionally drying the novel alkaline protease in liquid form as obtained in step [iii] to obtain the powdered alkaline protease.

The invention also provides a biologically pure bacterial strain of Bacillus subtilis, deposited in MTCC, IMTECH, Chandigarh and designated as MTCC 5333.
In an embodiment of the present invention, the bacterial strain used is isolated from an effluent treatment plant of KAR Tannery, Dindigul, Tamilnadu, India.
In another embodiment of the present invention, the carbon source used for the production medium may be selected from glucose, sucrose, starch, glycerol, lactose.
In still another embodiment of the present invention, the nitrogenous source used for the culture medium may be selected from soya bean meal, casamino acids, casein, corn steep liquor, yeast extract, either individually or in different combination.
In yet another embodiment of the present invention, the inorganic salt used may be selected from CaCI2, MgSO4, KH2PO4either individually or in different combination.
In still another embodiment of the present invention, the antifoaming agent used may be selected from olive oil, polypropylene glycol 2000, silicone oil, soyabean oil, cotton seed oil.
In yet another embodiment of the present invention, the production medium for the protease is prepared without using either alkali or common salt.
In still another embodiment of the present invention, the method of separation used may be such as microfiltration, centrifugation.
In yet another embodiment of the present invention, the method of purification may be such as ultrafiltration, salting out method, gel-filtration, ion exchange chromatographic methods in any combination and order.

In still another embodiment of the present invention, the method of drying the liquid protease may be such as freeze drying, spray drying, air drying, and vacuum drying.
In yet another embodiment of the present invention, the molecular weight of the protease may be in the range of 76-84 kDa.
In still another embodiment of the present invention, the oxidants against which the protease exhibits stability may be such as hydrogen peroxide, sodium perborate.
In yet another embodiment of the present invention, the protease exhibits stability and activity in NaCI solution of strength not more than 6 M.
In yet another embodiment of the present invention, the protease exhibits stability and activity in hydrogen peroxide solution of strength not more than 4 M.
In still another embodiment of the present invention, the protease exhibits stability and activity in sodium perborate solution of strength not more than 10%.
Detailed Description of the Invention
The bacterial strain Bacillus subtilis MTCC 5333 is isolated from an effluent sample having pH 11.0, collected from effluent treatment plant of KAR tannery, Dindigul, Tamilnadu, India. Initially, 10 ml aliquot of the effluent sample is mixed along with 100 gms of sterile humic soil as well as 5 gms of casein in a 250 ml Erlenmeyer flask and the mixture is incubated for 3-5 days at 30 °C for enrichment. Following enrichment, the sample is serially diluted and plated onto the medium comprised of 0.5% glucose, 0.25% peptone, 7.5% skim milk and 2% agar. The plates are incubated at 30 °C overnight. The colony displaying the maximal zone of clearance on the skim milk plate is selected, purified by streak plate method. The strain exhibiting maximal zone of clearance is isolated and identified to be Bacillus subtilis. The strain is deposited in MTCC, IMTECH, Chandigarh and is designated as MTCC 5333.

The isolate is a Gram positive, obligate aerobe, spore forming, motile and rod shaped bacterium. The isolate forms bulging sporangia bearing centrally positioned, oval shaped endospores. Features of colony morphology include round configuration, wavy margin, convex elevations, rough surface and opaque density. It grows well at initial pH values between 5.0-11.0 and over a range of temperatures from 15°- 60° C. Growth of the bacterium does not require NaCI but it also grows well in the presence of 10% NaCI. Following are the biochemical features of the bacterium: it is positive for Voges Proskauer test, citrate utilization, casein hydrolysis, starch hydrolysis, gelatin hydrolysis, nitrate reduction and catalase test but, negative for indole test, methyl red, urea hydrolysis, nitrite reduction and H2S production. The bacterium is fast growing, fermentative and produces acid from the carbohydrates such as dextrose, fructose, cellobiose, galactose, inositol, inulin, maltose, mannitol, mannose, salicin, sorbitol and sucrose. However, acid production from lactose, arabinose and rhamnose is absent.
The production medium is prepared using essentially a carbon source, nitrogenous source and inorganic salt. The medium is maintained at a pH in the range of 6.0 to 7.0. The bacterial strain of Bacillus subtilis, deposited at MTCC, IMTECH, Chandigarh and having designation no. MTCC 5333 is grown under aerobic conditions in the said production medium in submerged culture conditions with shaking. The medium is harvested after a period of 24-36 hrs and is subjected to separation by known methods. The separated crude enzyme is partially purified by known methods to obtain the alkaline protease in liquid form, which is optionally dried conventionally to obtain the protease in powder form.
The protease is purified by known methods such as ion exchange and gel filtration chromatography and the molecular weight of the purified protease is determined by 10% Sodium Docecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE).
The protease activity is expressed in terms of tyrosine equivalents using Hammersten casein. To 1.9 ml of 1 % casein solution prepared in 0.1 M carbonate buffer of pH 9.5, 0.1 ml of suitably diluted enzyme solution is added and the reaction mixture is kept at

40° C for 10 min. The reaction is terminated by the addition of 3 ml of 5% trichloroacetic acid solution. The absorbance of the trichloroacetic acid soluble filtrate at 280 nm is measured and one unit is expressed as the liberation of one mg tyrosine equivalent of substrate per gram of dried enzyme in 10 min. One unit is also expressed as the liberation of one mg of tyrosine equivalent of substrate per ml in 10 min. The protease activity of the enzyme preparation varies in the range of 20 - 800 units.
Elastolytic activity is determined using elastin-Congo Red (Sigma) as the substrate according to Cahan et al. (Journal of Biological Chemistry, 276: 43645-43652, 2001), with suitable modifications. Reaction mixture containing 10 mg of elastin-Congo Red in 2.2 ml of 0.1 M Tris-HCI, 0.5 mM CaCI2, pH 8.0, and 0.1 ml of suitably diluted enzyme solution, are incubated with shaking at 37 °C for 1 hour. The reaction is stopped by adding 0.2 ml of 0.12 M EDTA, followed by centrifugation and measurement of absorbance at 495 nm of the clear supernatant. One unit of activity is the amount of protease that causes an optical density increase of 1 unit/h at 495 nm. Elastolytic activity of the enzyme preparation varies in the range of 10-50 units.
Collagenolytic activity is determined using collagen from Bovine Achilles Tendon as reported by Woessner (Archives of Biochemistry and Biophysics, 93: 440-447, 1961). To 2 ml of 0.1% collagen solution prepared in Tris-HCI, pH 8.0, 0.5 ml of suitably diluted enzyme is added and incubated at 37 °C for 1 h. The reaction mixture is filtered and 1.0 ml of Chloramine-T is added to the filtrate and incubated at room temperature for 20 min. To this, 1.0 ml of perchloric acid is added. After 5 min, 1.0 ml of freshly prepared p-Dimethylamino benzaldehyde (DMAS) is added, mixed well and incubated at 60 °C for 20 min. The pink colour developed is read at 557 nm against blank containing distilled water instead of enzyme solution. Protease does not exhibit action towards collagen substrate.
The substrate specificity of the protease is evaluated using various substrates such as bovine hemoglobin by a method as reported by Anson (Journal of General Physiology, 22: 79-89, 1938), azocasein by a method as reported by Brock et al. (Applied and

Environmental Microbiology, 44:561-569, 1982), azocoll by a method as reported by Chavira et al. (Analytical Biochemistry, 136: 446-450, 1984), hide powder azure by a method as reported by Mozersky and David (Journal of American Leather Chemists Association, 87: 287-294, 1992) and keratin azure by a method as reported by Santo et al. (Current Microbiology, 33: 364-370, 1996), This enzyme is active against casein, hemoglobin, azocasein, azocoll, hide powder azure and keratin azure suggesting that it has a broad specificity towards various substrates. It also has an elastase activity as it hydrolyzes elastin-congo red. However, the protease does not have collagenolytic activity as it does not liberate hydroxy proline from collagen.
Table 1 provides a comparative data relating to the protease of the present invention and that produced by Bacillus subtilis FP-133 strain and also by a different strain of Bacillus clausii I-52, considered to be the closest prior art.
(Table Removed)
Table 1. Comparative account of protease from Bacillus subtilis of the present invention with that of the closest prior art with respect to properties and applications.
(Table Removed)

The following examples are given by way of illustration only and therefore should not be construed to limit the scope of the present invention.
Example 1
A culture medium [pH - 6.4] was prepared by mixing the following ingredients:

(Table Removed)

pH - 6.4
750 ml medium containing the ingredients was taken in a 2.8 L Fernbach flask and cells of Bacillus subtilis MTCC 5333 were inoculated in the medium. The flask was fitted to a shaker moving at 125 rpm. The temperature was maintained at 30 °C and shaking was continued. After a period of 24 hrs, the shaker was stopped and 600 ml of the medium was transferred to a 20 L fermentor with working volume of 14 L. Polypropylene glycol 2000 was used as antifoam agent. Air was fed to the fermentor at the rate of 0.5 vvm and the temperature was maintained at 30 °C. The liquid was subjected to stirring with an agitator rotating at a speed of 200 rpm. After a period of 30 hrs, the culture medium was harvested and subjected to centrifugation to collect the crude enzyme in liquid form.
Using casein substrate, the activity of the crude enzyme was found to be 25 units/ml. This broth containing crude liquid enzyme was subjected to ultrafiltration using 10-kDa membrane to obtain liquid enzyme concentrate. The protease activity of the ultrafiltrate was found to be 150 units/ml. This ultrafiltrate was subjected to salting out employing 60% ammonium sulphate saturation and the resulting precipitate was subjected to freeze drying with 2% maltodextrin to obtain 110 g of dry protease powder. It was stored at 4 °C. The characteristics of the protease were studied and they are mentioned below.

Activity with casein : 850 units/g Molecular weight : 78 kDa
The concentrate obtained by ultrafiltration and ammonium sulphate saturation was subjected to gel-filtration and ion exchange chromatographic methods using Sephadex G200 and QAE-Dowex an ion exchange matrix respectively to purify the protease and the molecular weight was determined by 10% SDS-PAGE. Halotolerance
The effect of NaCI on protease stability was studied by incubating 1 g protease having activity of 850 units each with 50 ml solution of 1, 2.5, 5 and 6 M NaCI solution for 24 hrs at 30 °C and the residual activity was measured. Also, the effect of NaCI concentration on the activity was evaluated by incubating 0.1 ml of enzyme suspension having an activity of 8.5 units/ml with 1.9 ml of assay mixture containing 1% casein and 1-6 M NaCI and the assay was carried out. The stability and activity of protease were about 80 and 70% respectively in the presence of 5 M NaCI.
Oxidant stability
The stability of protease in the presence of oxidant was examined by incubating 1 g
protease each with 50 ml solution of 0.5, 1, 2 and 4 M hydrogen peroxide for 4 hrs at 30
°C and the residual activity was estimated. About 70% of the original activity was
retained with 2 M oxidant whereas 45% of the activity was retained with 4 M hydrogen
peroxide.
Detergent stability
The stability of protease in the presence of different commercial detergents was studied
by incubating 1 g protease each with 50 ml solution containing commercial detergents
at the concentration of 10 mg/ml for 4 hrs at 30 °C and the residual activity was
determined. 80-90% of the protease activity was retained in the presence of commercial
detergents.

Specificity of protease on other substrates:
Activity on elastin : 45 units/g
Collagenolytic activity using bovine collagen : Nil Activity using hemoglobin substrate: 400 units/g Activity using Azocasein substrate: 230 units/g Activity using Azocoll substrate: 20 units/g Activity using Hide powder azure substrate: 15 units/g Activity using Keratin azure substrate: 12 units/g
Example 2
A culture medium was prepared by mixing the following ingredients.

(Table Removed)

pH-6.5
2.1 L medium containing the ingredients was taken in a 3 L Fermentor and 50 ml of 24 hrs seed inoculum of Bacillus subtilis MTCC 5333 developed in shake flask culture was transferred to the medium. The fermentation was carried out at 30°C and agitation of 250 rpm for 20 hrs after which, 1.5 L of this culture was transferred to 20 L fermentor with a medium volume of 13.5 L. Air was fed at a flow rate of 1 vvm and the temperature was maintained at 30 °C. The medium in the fermentor was subjected to stirring with an agitator rotating at a speed of 250 rpm. After a period of 28 hrs, the culture medium was harvested and subjected to centrifugation to collect the crude enzyme in liquid form.
Using casein substrate, the activity of the crude enzyme was found to be 22 units/ml. This broth containing crude liquid enzyme was subjected to ultrafiltration using 10-kDa membrane to obtain liquid enzyme concentrate having activity of 140 units/ml. This
ultrafiltrate was subjected to spray drying with inlet and outlet temperatures of 115 °C and 70 °C respectively along with 20% maltodextrin and 5% lactose to obtain about 200 g of dry protease powder. It was stored at 4 °C. The characteristics of the protease were studied and they are mentioned below. Activity : 400 units/g Halotolerance
The effect of NaCI on protease stability was studied by incubating 1 g protease having activity of 400 units each with 50 ml solution of 1, 2.5, 5 and 6 M NaCI solution for 24 hrs at 30 °C and the residual activity was measured.
Also, the effect of NaCI concentration on the activity was evaluated by incubating 0.1 ml of enzyme suspension having an activity of 4 units/ml with 1.9 ml of assay mixture containing 1% casein and 1-6 M NaCI and the assay was carried out. The stability and activity of protease were about 75 and 65% respectively in the presence of 6 M NaCI.
Oxidant stability
The stability of protease in the presence of oxidant was examined by incubating 1 g protease each with 50 ml solution of 1, 2.5, 5 and 10% sodium perborate for 4 hrs at 30 °C and the residual activity was estimated. About 80% of the original activity was retained with 5% oxidant whereas 50% of the activity was retained with 10% perborate.
Detergent stability
The stability of protease in the presence of different cationic, anionic and nonionic surfactants was studied by incubating 1 g protease each with 50 ml solution containing surfactants at the concentration of 10 mg/ml for 4 hrs at 30 °C and the residual activity was determined. 60-70% protease activity was retained with cationic surfactants and 80-90% protease activity was retained in the presence of anionic surfactants. However, the activity was enhanced by 10-40% in the case of nonionic surfactants.

Specificity of protease on other substrates:
Activity on elastin : 18units/g
Collagenolytic activity using bovine collagen : Nil Activity using hemoglobin substrate: 200 units/g Activity using Azocasein substrate: 125 units/g Activity using Azocoll substrate: 12 units/g Activity using Hide powder azure substrate: 7 units/g Activity using Keratin azure substrate: 4 units/g
The protease was used in dehairing of salted goat skins. 5 Nos. of wet salted goatskins, weighing 10 kg were suspended in a 100% dehairing water float containing 2.5% protease having 1000 units (based on casein assay) and 0.4% Tween 80 on the salted weight of skins for 8 hrs at room temperature. The pH of the float was adjusted to 9.0 using 0.4 % sodium carbonate. Dehairing was carried out in a usual manner.
Example 3
A culture medium was prepared by mixing the following ingredients.

(Table Removed)

pH-6.0
14 L medium containing the ingredients was taken in a 20 L fermentor and 700 ml of 24 hrs culture of Bacillus subtilis MTCC 5333 developed in Fernbach flask culture was transferred to the medium. The fermentation was carried out at 30 °C and agitation of 200 rpm for 20 hrs after which, 10 L of this culture was transferred to 300 L fermentor

with a medium volume of 200 L. Air was fed at a flow rate of 1 vvm and the temperature was maintained at 30 °C. The medium in the fermentor was subjected to stirring with an agitator rotating at a speed of 150 rpm. After a period of 28 hrs, the culture medium was harvested and subjected to continuous centrifugation to collect the crude enzyme in liquid form.
Using casein substrate, the activity of the crude enzyme was found to be 20 units/ml.
This broth containing crude liquid enzyme was subjected to ultrafiltration using 10-kDa
membrane to obtain liquid enzyme concentrate having activity of 130 units/ml. This
ultrafiltrate was stored at 4 °C. The characteristics of the protease were studied and
they are mentioned below.
Activity with casein : 130 units/ml
Halotolerance
The effect of NaCI on protease stability was studied by incubating 20 ml protease concentrate having activity of 2500 units each with 30 ml solution of 2, 4 and 8 M NaCI for 24 hrs at 30 °C and the residual activity was measured. Also, the effect of NaCI concentration on the activity was evaluated by incubating 0.1 ml of diluted enzyme suspension having an activity of 6.5 units/ml with 1.9 ml of assay mixture containing 1% casein and 1-5 M NaCI and the assay was carried out. The stability and activity of protease were about 80 and 70% respectively in the presence of 5 M NaCI.
Oxidant stability
The stability of protease in the presence of oxidant was examined by incubating 25 ml
protease each with 25 ml solution of 1, 2, 4 and 8 M hydrogen peroxide for 4 hrs at 30
°C and the residual activity was estimated. About 65% of the original activity was
retained with 2 M oxidant whereas 40% of the activity was retained with 4 M hydrogen
peroxide.
Detergent stability

The stability of protease in the presence of commercial detergents was studied by incubating 25 ml protease concentrate with 25 ml solution containing detergents at the concentration of 40 mg/ml for 4 hrs at 30 °C and the residual activity was determined. 60-80% of the protease activity was retained in the presence of commercial detergents.
Specificity of protease on other substrates: Activity on elastin : 15 units/ml Collagenolytic activity using bovine collagen : Nil Activity using hemoglobin substrate: 55 units/g Activity using Azocasein substrate: 60 units/g Activity using Azocoll substrate: 2 units/g Activity using Hide powder azure substrate: 3 units/g Activity using Keratin azure substrate: Nil
Example 4
A culture medium was prepared by mixing the following ingredients.

(Table Removed)

pH-6.4
1.5 L medium containing the ingredients except casein was taken in a 3 L fermentor and 100 ml of 24 hrs culture of Bacillus subtilis MTCC 5333 developed in Erlenmeyer conical flask was transferred to the medium. The fermentation was carried out at 30 °C and agitation of 250 rpm for 10 hrs after which, 500 ml of 4% casein solution was fed at a flow rate of 0.7 ml/min continuously for 12 hrs using peristaltic pump. Air was fed at a flow rate of 0.5 vvm and the temperature was maintained at 30 °C. The medium in the

fermentor was subjected to stirring with an agitator rotating at a speed of 300 rpm. After a period of 12 hrs, the culture medium was harvested and subjected to continuous centrifugation to collect the crude enzyme in liquid form.
The activity of the crude enzyme was found to be 32 units/ml using casein substrate.
This broth containing crude liquid enzyme was subjected to ultrafiltration using 10-kDa
membrane to obtain liquid enzyme concentrate having activity of 180 units/ml. This
ultrafiltrate was spray dried employing inlet and outlet temperatures of 115 °C and 75 °C
respectively with 20 % maltodextrin and 5% lactose to get 50 g protease powder having
activity of 450 units/g and it was stored at 4 °C. The characteristics of the protease were
studied and they are mentioned below.
Activity with casein : 450 units/g
Halotolerance :
Stability with 6 M NaCI for 24 hrs at 30°C: 70%
Activity in the presence of 6 M NaCI: 55%
Oxidant stability:
The following stability studies with oxidants were carried out for 4 hrs at 30 °C
Stability with 2 M H2O2: 85%
Stability with 4 M H2O2: 40%
Stability with 5% perborate : 70%
Stability with 10% perborate : 30%
Specificity of protease on other substrates: Activity on elastin : 30 units/g Collagenolytic activity using bovine collagen : Nil Activity using hemoglobin substrate: 180 units/g Activity using Azocasein substrate: 125 units/g Activity using Azocoll substrate: 13 units/g Activity using Hide powder azure substrate: 10 units/g Activity using Keratin azure substrate: 8 units/g

The protease was used in dehairing of salted hides.
4 Nos. of dry salted cow hides, weighing approximately 48 kg were immersed in dehairing suspension comprising 150% water, 3.0% protease having 1350 units (based on casein assay) and 1% Tween 80 on the salted weight of hides for 10 hrs at room temperature. The pH of the float was adjusted to 8.0 using 0.5 % sodium carbonate. Dehairing is carried out in a usual manner.
Example 5
A culture medium was prepared by mixing the following ingredients.

(Table Removed)

pH-6.7
600 ml medium containing the ingredients was taken in a 2.8 L Fernbach flask and cells of Bacillus subtilis MTCC 5333 were inoculated in the medium. The flask was kept for incubation in a shaker at 150 rpm. The temperature was maintained at 30 °C and shaking was continued. Olive oil was used as antifoam agent. After a period of 24 hrs, the shaker was stopped and 500 ml of the medium was transferred to a 20 L fermentor with working volume of 12 L. Air was fed to the fermentor at the rate of 1 vvm and the temperature was maintained at 30 °C. The liquid in the fermentor was subjected to stirring with an agitator rotating at a speed of 200 rpm. After a period of 30 hrs, the culture medium was harvested and subjected to centrifugation to collect the crude enzyme in liquid form.
The activity of the crude enzyme was found to be 20 units/ml using casein substrate. This broth containing crude liquid enzyme was subjected to ultrafiltration using 10-kDa

membrane to obtain liquid enzyme concentrate. The protease activity of the ultrafiltrate
was found to be 120 units/ml. This ultrafiltrate was subjected to salting out employing
60% ammonium sulphate saturation and the resulting precipitate was subjected to
freeze drying with 2% lactose to obtain 90 g of dry protease powder. It was stored at 4
°C. The characteristics of the protease were studied and they are mentioned below.
Activity with casein : 700 units/g
Halotolerance
Stability with 4 M NaCI for 24 hrs at 30 °C: 85%
Activity in the presence of 4 M NaCI: 75%
Oxidant stability
The following stability studies with oxidants were carried out for 4 hrs at 30 °C
Stability with 2 M H2O2: 70%
Stability with 4 M H2O2: 50%
Stability with 5% perborate : 75%
Stability with 10% perborate : 35%
Specificity of protease on other substrates:
Activity on elastin : 45 units/g
Collagenolytic activity with bovine collagen : Nil
Activity with hemoglobin substrate: 280 units/g
Activity with Azocasein substrate: 170 units/g
Activity with Azocoll substrate: 12 units/g
Activity with Hide powder azure substrate: 18 units/g
Activity with Keratin azure substrate: 10 units/g
The protease was used in dehairing of salted and soaked hides.
(a) 4 Nos. of wet salted buff hides, weighing approximately 60 kg were immersed in dehairing suspension comprising 200% water, 3.0% enzyme having 2000 units (based

on casein assay), and 1.5% Tween 80 on the salted weight of hides and the hides were dehaired after 12 hrs by the usual procedure.
(b) 5 Nos. of wet salted buff halves, weighing approximately 30 kg were initially soaked using 300% water for 3 hrs and the wet weight was noted. The presoaked halves were subjected to main soaking comprising 300% water, 0.01% wetting agent, 0.01% preservative on the wet weight and the pH was adjusted to 8.0 using sodium carbonate and left overnight (18 hrs) at ambient temperature.
The soaked hides were green-fleshed using conventional method and the soaked defleshed weight was noted. The defleshed hides were immersed in dehairing suspension after green fleshing comprising 100% water, 2.0% enzyme and 1% Triton X 100. The pH of dehairing float was adjusted to 9.0 to 10.0 using approximately 0.4% sodium carbonate and handled for three or four times and left overnight. The hides were dehaired after 12 hrs by the usual procedure.
Example 6
A culture medium was prepared by mixing the following ingredients.

(Table Removed)

pH-6.5
The 14 L medium containing the above ingredients was taken in 20 L fermentor and it was fed with 5% of 20 hrs inoculum developed in Fernbach flasks. Air was flown to the fermentor at the rate of 1 vvm and the temperature was maintained at 30 °C. The liquid in the fermentor was subjected to stirring with an agitator rotating at a speed of 200 rpm.

After a period of 30 hrs, the culture medium was harvested and subjected to centrifugation to collect the crude enzyme in liquid form.
The activity of the crude enzyme was found to be 20 units/ml. This broth containing
crude liquid enzyme was subjected to ultrafiltration using 10-kDa membrane to obtain
liquid enzyme concentrate having activity of 100 units/ml. This ultrafiltrate was subjected
to spray drying along with 20% maltodextrin and 5% lactose to obtain about 200 g of dry
protease powder. The characteristics of the protease were studied and they are
mentioned below.
Activity with casein : 350 units/g
Halotolerance
Stability with 5 M NaCI for 24 hrs at 30°C: 70%
Activity in the presence of 5 M NaCI: 65%
Oxidant stability
The following stability studies with oxidants were carried out for 4 hrs at 30°C
Stability with 2 M H2O2: 70%
Stability with 4 M H2O2: 40%
Stability with 5% perborate : 70%
Stability with 10% perborate : 45%
Specificity of protease on other substrates: Activity on elastin : 15 units/g Collagenolytic activity with bovine collagen : Nil Activity with hemoglobin substrate: 125 units/g Activity with Azocasein substrate: 80 units/g Activity with Azocoll substrate: 5 units/g Activity with Hide powder azure substrate: 8 units/g Activity with Keratin azure substrate: 5 units/g

The protease was used as an additive in laundry detergent formulation. For use as a detergent additive, the washing efficiency of the protease was determined by washing tests on 100 Nos. of 10 x 5 cm cotton fabric pieces that were heavily soiled with blood/milk/egg yolk. The stained fabric pieces were washed with 1 liter of water containing 0.2 g (75 units based on casein assay) enzyme and 6 g of commercially available detergent base formulation for 20 min. pH of the washing solution was 10.0. Washing was carried out at 25 °C. After the washing process, the test fabric was rinsed twice with tap water. The washing efficiency was determined by measuring the protein content in the wash solution. The protease was found to be efficient in the removal of protein stains. There was a rise of 20% protein removal when the protease was added along with detergent compared to detergent alone wash.
The main advantages of the present invention are the following:
• Production of protease from a fast growing industrial Bacillus subtilis MTCC 5333
strain is beneficial in terms of scalability and high yield and the process of
production is safer as the bacterium is "Generally Regarded as Safe" (GRAS)
microbe.
• The production medium in which the strain Bacillus subtilis MTCC 5333 is grown,
requires commercially available nutrients and does not require alkali or salt to
produce high yields of halotolerant alkaline protease.
• The protease is having halotolerance and it is exhibiting its activity and stability in
the presence of 6 M salt concentration. Hence the protease can find its potential
applications in industry where high saline conditions are employed.
• The protease is having oxidant stability and it is stable in the presence of
oxidants such as hydrogen peroxide and perborate. It is also stable in the
presence of variety of surfactants and detergent components.
•
• Properties such as halotolerance, stability in oxidants, surfactants and alkaline
conditions in addition to broad substrate specificity enable the protease to be
used as an effective additive in laundry and heavy duty detergent formulations.
• The halotolerant property and stability towards surfactants enable the protease to
be used in the soaking bath itself, thereby contributing to the productivity
enhancement in dehairing operations of leather manufacture.
• The protease exhibits elastase activity but it has no collagenolytic activity. This
feature enables the protease to be used in ecobenign lime-sulfide free enzymatic
dehairing of skins and hides in leather processing.
• The protease possesses halotolerance, stability in surfactants, detergent
components, oxidants and alkaline conditions and broad substrate specificity
including activity towards elastin and absence of collagenolytic activity and
combination of all the properties enables the protease to be used in leather,
detergent and other industries.

We Claim:
1. A novel alkaline protease produced from a bacterial strain of Bacillus subtilis,
deposited at MTCC, IMTECH, Chandigarh, India and designated as MTCC 5333,
wherein the said protease exhibits the following characteristics:
[a] molecular weight in the range of 76 to 84 kDa;
[b] specificity against substrates such as non-collagenous fibrous proteins,
globular proteins, casein, egg albumin, proteoglycans and elastin;
[c] stability in pH range of 5.0 to 12.0 and temperature ranging between 10 to
60 degree C;
[d] activity in pH range of 6.0 to 11.0 and temperature ranging between 10 to
70 degree C;
[e] activity and stability in the presence of salt, surfactants and oxidants;
[f] action towards elastin but inert to collagen.

2. A novel alkaline protease as claimed in claim 1, wherein the oxidants against
which the protease exhibits stability are such as hydrogen peroxide, sodium
perborate.
3. A novel alkaline protease as claimed in claim 1, wherein the protease exhibits
stability and activity in NaCI solution of strength not more than 6 M.
4. A novel alkaline protease as claimed in claim 1, wherein the protease exhibits
stability and activity in hydrogen peroxide solution of strength not more than 4 M.
5. A novel alkaline protease as claimed in claim 1, wherein the protease exhibits
stability and activity in sodium perborate solution of strength not more than 10%.

6. A novel alkaline protease as claimed in claim 1, wherein it is useful for dehairing
of skins, hides and surfaces as well as in removing proteinaceous stains.
7. A biologically pure bacterial strain of Bacillus subtilis, deposited in MTCC,
IMTECH, Chandigarh and designated as MTCC 5333, wherein the said isolate
exhibits the following characteristics:
[a] gram positive, obligate aerobe, spore forming, motile, and rod shaped
bacterium;
[b] colony morphology having round configuration, wavy margin, convex
elevations, rough surface and opaque density;
[c] biochemical characteristics: positive for Voges Proskauer test, citrate
utilization, casein hydrolysis, starch hydrolysis, gelatin hydrolysis, nitrate
reduction and catalase test but negative for indole test, methyl red, urea
hydrolysis, nitrite reduction and H2S production; produces acid from the
carbohydrates such as dextrose, fructose, cellobiose, galactose, inositol,
inulin, maltose, mannitol, mannose, salicin, sorbitol and sucrose but not from
lactose, arabinose and rhamnose;
[d] grows well at initial pH values between 5.0-11.0 and over a range of
temperatures from 15 to 60 degree C.
8. A biologically pure bacterial strain as claimed in claim 7, wherein it is isolated
from an effluent sample having pH 11.0, collected from effluent treatment plant of
KAR tannery, Dindigul, Tamilnadu, India.
9. A biologically pure bacterial strain as claimed in claim 7, wherein the said strain
is stable at a pH ranging from 5.0 to 11.0.

10. A biologically pure bacterial strain as claimed in claim 7, wherein the said strain
is stable at a temperature ranging from 15 to 60 degree C.
11. A biologically pure bacterial strain as claimed in claim 7, wherein the said strain
is tolerant to salt concentrations upto 6M.
12. A process for the preparation of a novel alkaline protease as claimed in claim 1,
from bacterial source, wherein the process steps comprise:
[i] culturing Bacillus subtilis, deposited in MTCC, IMTECH, Chandigarh and designated as MTCC 5333 in a culture medium containing essentially a carbon source, nitrogenous source, inorganic salt and preferably an antifoaming agent, maintained at a pH in the range of 6.0 to 7.0, under aerobic submerged culture conditions, at temperature ranging from 25 to 35 degree C, preferably under shaking conditions;
[ii] harvesting the culture medium obtained in step [i] after a period of 24 to 36 hrs to obtain the enzyme alkaline protease in the extracellular medium;
[iii] separating the enzyme from the culture medium as obtained in step [ii] by known methods followed by purification to obtain the novel alkaline protease in liquid form;
[iv] optionally drying the novel alkaline protease in liquid form as obtained in step [iii] to obtain the powdered alkaline protease.
13. A process as claimed in claim 12, wherein the bacterial strain used is isolated
from an effluent treatment plant of KAR Tannery, Dindigul, Tamilnadu, India.
14. A process as claimed in claim 12, wherein the carbon source used for the culture
medium is selected from glucose, sucrose, starch, glycerol, lactose.

15. A process as claimed in claim 12, wherein the nitrogenous source used for the
culture medium is selected from soya bean meal, casein, corn steep liquor,
casamino acids, yeast extract, either individually or in different combination.
16. A process as claimed in claim 12, wherein the inorganic salt used is selected
from CaCI2, MgSO4, KH2PO4, either individually or in different combination.
17. A process as claimed in claim 12, wherein the antifoaming agent used is selected
from olive oil, polypropylene glycol 2000, silicone oil, soyabean oil, cotton seed
oil.
18. A process as claimed in claim 12, wherein the production medium for the
protease is prepared without using either alkali or common salt.
19. A process as claimed in claim 12, wherein the method of separation used is such
as microfiltration, centrifugation.
20. A process as claimed in claim 12, wherein the method of purification is such as
ultrafiltration, salting out method, gel-filtration, ion exchange chromatographic
methods in any combination and order.
21. A process as claimed in claim 12, wherein the method of drying the liquid
protease is such as freeze drying, spray drying, air drying, vacuum drying.
22. A novel alkaline protease and a process for the preparation thereof substantially
as herein described with reference to the foregoing examples.

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=6RFNPtprxyH6/PFd7jiv1A==&loc=+mN2fYxnTC4l0fUd8W4CAA==

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Patent Number

271983

Indian Patent Application Number

2375/DEL/2007

PG Journal Number

12/2016

Publication Date

18-Mar-2016

Grant Date

12-Mar-2016

Date of Filing

13-Nov-2007

Name of Patentee

1. COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH; 2. INDIAN INSTITUTE OF SCIENCE

Applicant Address

1. ANUSANDHAN BHAWAN, RAFI MARG, NEW DELHI-110 001,INDIA; 2. BANGALORE

Inventors:

#	Inventor's Name	Inventor's Address
1	SRINIVASAN SIVASUBRAMANIAN	CENTRAL LEATHER RESEARCH INSTITUTE, CHENNAI
2	RAMACHANDRA BOOPATHY NAIDU	CENTRAL LEATHER RESEARCH INSTITUTE, CHENNAI
3	NUMBI RAMUDU KAMINI	CENTRAL LEATHER RESEARCH INSTITUTE, CHENNAI
4	MARICHETTI KUPPUSWAMY GOWTHAMAN	CENTRAL LEATHER RESEARCH INSTITUTE, CHENNAI
5	NARASIMHAN KANNAN CHANDRABABU	CENTRAL LEATHER RESEARCH INSTITUTE, CHENNAI
6	RENGARAJULU PUVANAKRISHNAN	CENTRAL LEATHER RESEARCH INSTITUTE, CHENNAI
7	11-YEGNISETTIPALLI KRISHNAIAH SAIKUMARI	IISC BANGALORE
8	PADMANABHAN BALARAM	IISC BANGALORE
9	PALANIVEL SARAVANAN	CENTRAL LEATHER RESEARCH INSTITUTE, CHENNAI
10	SAMAYAVARAM RAMALINGAM	CENTRAL LEATHER RESEARCH INSTITUTE, CHENNAI
11	RAMAKRISHNAN RAMESH	CENTRAL LEATHER RESEARCH INSTITUTE, CHENNAI
12	JAMES KANAGARAJ	CENTRAL LEATHER RESEARCH INSTITUTE, CHENNAI

PCT International Classification Number

C12N9/58; A23L1/30

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA