Title of Invention	"A PROCESS FOR THE PREPARATION OF PROTEASE INHIBITOR USING NOVEL ALKALO THERMOPHILIC BACILLUS SP"
Abstract	This invention relates to a process for the preparation of protease inhibitor using novel alkalothermophilic bacillus sp. and in particular relates to the process for the preparation of a low molecular weight peptide aspartic acid protease inhibitor using alkalothermophilic Bacillus (AT Bacillus sp.) The alkalothermophilic Bacillus sp. was isolated from the soil sample of hot spring of Vajreshwari, Maharashtra, India. The alkalothermophiclic Bacillus sp. is distinctively different from other species of Bacillus in its growth, temperature and pH. The organism is found to be an obligatory alkalophile, gram positive, aerobic, motile, spore forming rods with the cells growing together and forming filament. On alkaline nutrient agar at 50°C, the colonies of the organism are butyrous, glistering and pale cream coloured.

Title of Invention

"A PROCESS FOR THE PREPARATION OF PROTEASE INHIBITOR USING NOVEL ALKALO THERMOPHILIC BACILLUS SP"

Abstract

This invention relates to a process for the preparation of protease inhibitor using novel alkalothermophilic bacillus sp. and in particular relates to the process for the preparation of a low molecular weight peptide aspartic acid protease inhibitor using alkalothermophilic Bacillus (AT Bacillus sp.) The alkalothermophilic Bacillus sp. was isolated from the soil sample of hot spring of Vajreshwari, Maharashtra, India. The alkalothermophiclic Bacillus sp. is distinctively different from other species of Bacillus in its growth, temperature and pH. The organism is found to be an obligatory alkalophile, gram positive, aerobic, motile, spore forming rods with the cells growing together and forming filament. On alkaline nutrient agar at 50°C, the colonies of the organism are butyrous, glistering and pale cream coloured.

Full Text	This invention relates to a process for the preparation of protease inhibitor using novel alkalothermophilic bacillus sp. More particularly it relates to the process for the preparation of a low molecular weight peptide aspartic acid protease inhibitor using alkalothermophilic Bacillus (AT Bacillus sp.) deposited at National Collection of Industrial Microorganism(NCIM),Pune designated as NCIM 59. The alkalothermophilic Bacillus sp. NCIM 59 was isolated from the soil sample of hot spring of Vajreshwari, Maharashtra, India. The alkalothermophiclic Bacillus sp. is distinctively different from other species of Bacillus in its growth, temperature and pH. The organism is found to be an obligatory alkalophile, gram positive, aerobic, motile, spore forming rods with the cells growing together and forming filament. On alkaline nutrient agar at 50°C, the colonies of the organism are butyrous, glistering and pale cream coloured. Proteases are responsible either directly or indirectly for all bodily functions including cell growth, nutrition, differentiation and apoptosis. They also play a significant role in intracellular and extracellular protein turn over (house keeping and repair), cell migration and invasion, fertilization and implantation (Protease inhibitors, novel therapeutic application and development, Tony E Hugli, TIBTECH, 14, 409-412, 1996). Since proteases are necessary for normal and abnormal body functions, their effective regulatory counterparts i.e., protease inhibitors, are tremendously essential for physiological regulations. Protease inhibitors have been the source of attention in many disciplines. Due to their presence in valuable plant feeds and involvement in nutritive properties they have evoked the interest of nutritionists. Inhibitor proteins have been studied for the elucidation of mechanism of inhibition of proteases, as well as for the studies on protein-protein interactions and associations. Due to their unique pharmacological properties, protease inhibitors are also used as valuable tools in medical research. Protease inhibitors are classified into Synthetic and Naturally occuring inhibitors. They occur in numerous animal tissues and fluids, in many plant tissues (particularly in legume seeds and other storage organs) and in microorganisms. (Protease inhibitors, Yehudith Birk, Hydrolytic enzymes, A Neuberger and K. Brocklehurst (Eds), Elsevier Science Publishers B. V. (Biomedical Division), 257, 1987). The most abundant source of the inhibitors in plants is the seeds, but their location is not necessarily restricted to this part of the plant. They are also found in leaves, tubers, etc,. As for the intracellular localization of the inhibitors, they appear to be associated primarily with the cytosol, but in some instances they have been localized in protein bodies. The inhibitors of animal origin are found both in tissues and in secretions of organs. The pancreatic trypsin inhibitor has been found as an intracellular component in various bovine organs: in the pancreas, lung, liver, spleen, parotoid gland and also in pituitary gland. In addition to the thoroughly studied pancreatic trypsin inhibitors, a large number of protease inhibitors from different animal sources have been isolated. Many of them are secretory proteins, such as trypsin inhibitors of blood plasma, milk colustrum, seminal plasma and submandibular glands. The plasma protease inhibitors constitute a major group of the functional proteins of the blood plasma. Most of them inhibit serine proteases but their mechanism of interaction is still being actively pursued by many investigators. The presence of protease inhibitors in microorganisms came into existence from the studies on antibiotics as they act as inhibitors of the enzymes which are involved in growth and multiplication. Proteolytic enzymes outside of microbial cells hydrolyze organic nitrogen compounds in the medium, so they are thought to be harmful to cells. The production of inhibitors of the proteolytic enzymes by microorganisms is probably a mechanism to provide cell protection. In contrast to the inhibitors of proteolytic enzymes obtained from animals and plants, the inhibitors from microorganisms are of smaller molecular nature. Specific inhibitors of microbial origin have been used as useful tools in biochemical analysis of biological functions and diseases. (Enzyme inhibitors of microbial origin, Hamao Umezawa, University Park Press). Few of the inhibitors of microbial origin of therapeutic interest are given below: Leupeptin- from Streptomyces, is the inhibitor of trypsin, plasmin, kalikrein and papain. Chymostatin- from Streptomyces, is the inhibitor of chymotrypsin. Dopastin- from Pseudomonas, Oosponol- from Oospora, Oudenone- from Oudemansialla radicata and Fusaric acid- from Fusarium are the inhibitors of dopamine hydroxylase. Pepstatin A- from Streptomyces, is the inhibitor of pepsin, an aspartic acid protease. It inhibits the HIV-1 protease, which is also an aspartic protease and the key enzyme for the propagation of the HIV. The expanding Acquired Immuno Deficiency Syndrome (AIDS) epidemic and the relentless nature of the disease have intensified the search for effective antiviral therapies, to control the replication of the HIV, the causative agent of AIDS. The HIV -1 protease is the key enzyme for the propagation of the virus. Thus specific inhibition of the HIV-1 protease by inhibitors is useful in preventing the infection. HIV-1 protease is structurally and mechanistically related to mammalian and microbial aspartic proteases such as pepsin, cathepsin, renin, and endothiopepsin. The classification of HIV-1 protease in the aspartyl family was also predicted from its primary sequence analysis. A highly conserved sequence Asp-Thr-Gly (D-T-G) in retroviral proteases, is also conserved in the active site of the cellular and fungal proteases. Molecular modeling studies have also confirmed the functional and structural similarities of the retroviral proteases to other aspartyl proteases. Various synthetic peptidic and non peptidic compounds have been shown to inhibit HIV-1 protease. Well documented examples of isolation of compounds by microbial screening represented by the discovery of potent compounds such as cyclosporin, movionolin, and avermycin, etc,. An antifungal antibiotic cerulenin from Cephalosporium and pepstatin A, a pepsin inhibitor from Streptomyces, have been well characterized as HIV-1 protease inhibitors. (C. Debouck, AIDS Research and Human Retroviruses, 8, 153-164, 1992). Extensive evidence suggests that, the degradation of hemoglobin is necessary for the growth of erythrocytic malarial parasite, apparently to provide free amino acids for parasitic protein synthesis. On the basis of the data available, the aspartic acid proteases are thought to be responsible for the initial cleavages of hemoglobin. Both aspartic acid and cystein proteases have synergistic effects in inhibiting the growth of the cultural malarial parasite and also these proteases act synergistically to degrade hemoglobin. Therefore, the combination of inhibitors of malarial cystein and aspartic acid proteases, may provide a most effective chemotherapeutic regimen and best limit the development of parasitic resistant to protease inhibitors. Pepstatin, the inhibitor of aspartic acid proteases, along with the cystein protease inhibitor E-64, blocks the Plasmodium falciparum development. (Proteases of Malarial Parasite: New Targets for Chemotherapy, Philip J. Rosenthal, Emerging Infectious Diseases, 4 (1), 49-57, 1998). So far no report is available for the preparation of the protease inhibitor using alkalothermophilic Bacillus sp. Based on the fact that the aspartic acid protease plays a significant role in the development of the malarial parasite, the inventors of the present invention believe that the inhibitor produced as per the procedure of the present invention using AT Bacillus NCTM59, could be a potent inhibitor for the protease particularly the aspartic acid protease more particularly for protease of malarial parasite. The inhibitor described in the present invention inhibits pepsin, an aspartic protease. Pepsin present in the gastric secretion is responsible for the degradation (digestion) of proteinaceous food. Excess secretion of pepsin has harmful effects on the stomach as it damages the digestive tract and causes stomach ulcer or duodenal ulcer. Considering the fact that the inhibitor is an active inhibitor of pepsin, it has potential application as a therapeutic agent against stomach or duodenal ulcers. Pepstatin A, a pepsin inhibitor has been reported to inhibit HIV-1 protease which is also an aspartic protease. The inventors of present invention have observed that the inhibitor also inhibits other enzymes having aspartic acid in the active site. So we strongly feel that the microbial protease inhibitor could inhibit the HIV-1 protease. The main objective of the present invention is to provide a process for the preparation of low molecular weight peptidic aspartic acid protease inhibitor using Alkalothermophilic Bacillus sp. NCIM 59. Accordingly, the present invention provides a process for the preparation of low molecular weight peptidic aspartic acid protease inhibitor using alkalothermophilic Bacillus sp. NCIM59, which involves following steps: subculturing the said species onto malt extract, glucose, yeast extract, and peptone MGYP agar slant to obtain freshly grown AT Bacillus, inoculating this Bacillus in a conventional fermentation medium at a temperature in the range of 37-50 (C under aseptic conditions for a period ranging between 24 to 48 hrs,, harvesting the culture broth, separating the solids by conventional methods to obtain cell free liquid containing the protease inhibitor, treating the cell free liquid with a decolourising agent for a period ranging between 2 to 24 hrs., to obtain the colourless liquid, subjecting this liquid to ultrafiltration, concentrating the ultrafiltered liquid to obtain the low molecular weigh protease inhibitor. Accordingly, the present invention provides a process for the preparation of protease inhibitor using novel alkalothermophilic Bacillus species, which comprises; a) subculturing the alkalothermophilic Bacillus sp. in a fermentation medium containing assimilable carbon and nitrogen sources at a temperature in the range of 37-50 deg.C under aseptic conditions for a period ranging between 24 to 48 hrs., b) harvesting the culture broth by known methods, c) separating the solids by conventional methods to obtain cell free liquid containing the protease inhibitor, d) treating the cell free liquid with a decolorising agent for a period ranging between 2 to 24 hrs. to obtain a colorless liquid, e) separating the decolorising agent and recovering the protease inhibitor by conventional methods such as herein described In one of the embodiments of the present invention the agar slant such as MGYP (Malt extract, Glucose, Yeast extract, and Peptone) may be used to grow Bacillus to get fresh inoculum. In one of the embodiments of the present invention the AT Bacillus sp. was isolated from the hot spring from Vajreshwari, India, and which has been deposited in National Collection for Industrial Micro-organisms, NCL, Pune 411 008, having accession No. NCIM 59. The characteristics of this strain are i) aerobic ii) gram positive iii) motile iv) spore forming v) capable of growing in a alkaline medium at Ph 8-10 and v) showing negative reaction towards production of indole, hydrogen sulfide,ammonia and urease and positive reaction for hydrolysis of starch, production of catalase, hydrolysis of casein and reduction of nitrate. In another embodiment the decolourising agent used may be activated charcoal or cellulose. In a feature of the present invention the composition of the MGYP medium containing the carbon, nitrogen source and other micro ingredients may be: I. glucose - 0.5% - 1% II. beef extract - 0.5% - 0.75% III. sodium chloride -0.1%- 0.3% IV. magnesium sulfate - 0.05%- 0.1% V. dipotassium hydrogen phosphate -0.05% - 0.1% VI. soyameal - 1 % - 2%. Production of the inhibitor and its activity is also dependent upon the nature and concentration of the media ingredients, inoculum development and parameters such as aeration, agitation, etc. In shake flasks maximum production obtained after 24-48 hours, when a 5-20 % v/v vegetative inoculum in the actively growing phase is transferred to the production media. Post fermentation processing of the broth for the isolation of the inhibitor includes, centrifugation or filtration of the broth. Cell free supernatant is treated with activated charcoal to remove the colour material. The charcoal is removed by centrifugation or filtration. The resulting supernatant is subjected to ultrafiltration using filtration membranes. In a feature of the present invention the pepsin inhibitor was selectively purified by removing the high molecular weight compounds from the culture broth by treating with activated charcoal. The charcoal treated broth was filtered through ultrafiltration membrane and concentrated by lyophilization. The concentrated inhibitor sample was loaded onto a reverse phase-high performance liquid chromatography column using a linear gradient of acetonitrile and triflouroacetate, and the peaks were checked for the inhibitor activity. Among the two compounds detected in the HPLC analysis, the compound having less retention time showed the inhibition against pepsin. This compound absorbs strongly at 210 nm. The compound was collected and checked as a single purified peptide after loading onto the HPLC column. One unit of inhibitor is defined as the amount of inhibitor which inhibits the protease activity expressed in terms of decrease in optical density of 0.001 per minute. The process of the present invention is described herein below with examples which are illustrative only and should not be construed to limit the scope of the present invention in any manner. Example-1 Eighteen hour grown AT Bacillus NCIM 59 on an agar slant, at 50(C, was inoculated into a medium containing glucose-1%, peptone-0.75%, beef extract-0.75%, sodium chloride-0.3%, dipotassium hydrogen phosphate-0.1%, magnesium sulfate-0.1% and soyameal-2.0%, and this fermentation medium was incubated on a rotary shaker at 50(C for 12 hours. 10% v/v of the freshly grown inoculum was added to the fermentation flasks. After 48 hours of growth on a rotary shaker at 50(C the cells and the residual soyameal was removed by centrifugation. To 100 ml of centrifuged supernatant 10 grams of activated charcoal was added and incubated overnight at 4(C. This charcoal treated broth was filtered through filter paper 2-3 times to ensure there was no trace of charcoal remained. The maximum production of the inhibitor obtained was 100-112 U/ml in the charcoal treated broth. Example-2 The medium was formulated for the production flasks using lactose, sorbitol, xylan, fructose, maltose and sucrose, all at a concentration of 1% and fermentation process was carried out for 48 hrs. After 48 hours, the production of inhibitor obtained was maximum of 98 U/ml in presence of sucrose. Example-3 In this example the fermentation medium used in example-1 was supplimented with amino acids for checking the effect on the production of the inhibitor. The amino acids used in the production flasks were alanine, arginine, asparagine, cystein, glutamic acid, glycine, histidine, proline and serine, all at a final concentration of 0.5%. After 48 hours, the product was isolated and the results are tabulated in Table-1 given herein below. It is observed that maximum production of the inhibitor of 105 U/ml was obtained in presence of asparagine. Table-1 (Table Removed) Example-4 The medium was formulated for the production of the inhibitor using 1% w/v of following nitrogen sources in the fermentation flasks. Soyameal, casein, casamino acids, urea, tryptone, peptone, beef extract, skimmed milk and yeast extract were among the nitrogen sources tested. Although, after 48 hours a maximum production of 70 U/ml of the inhibitor was obtained in presence of beef extract, the production was considerably high i.e., 90 U/ml -100 U/ml, when beef extract and soyameal were used together in the medium. Example-5 In this example the medium was supplimented with various inducers like soyameal, casein, yeast extract, tryptone, skimmed milk, urea and casamino acids, at a concentration of 1% w/v in the production flasks. After 48 hours, maximum production of 140 U/ml of the inhibitor was obtained in presence of casamino acids. Example-6 The charcoal treated supernatant was passed through an Amicon UM-02 membrane and concentrated by lyophilization. The activity of the inhibitor was also increased correspondingly. The concentrated inhibitor sample was injected into an RP-HPLC column in a linear gradient of 0 % acetonitrile- 50 % acetonitrile and 0.05 % trifluoroacetate. The compound having less retention time, showed the inhibition against pepsins this compound was related on RP-High peyemeric liquid chromatography column using the same solvent system and found to be homogenous and pure. The purified inhibitor was analysed for the amino acid sequences and for the determination of the molecular mass. Examplc-7 The fungal strains Trichoderma reesei (NC1M 992,1051,1052,1186), Fusariumoxysponim (NCIM 1008,1043,1072), Aspergillus flaws (NCIM 535,536,538,542), Aspergillus oryzae (NCIM 637,643,649,1032), Mtsarium moniliforme (NCIM 1099,1100), Alternaria solmri (NCIM 887), Claviceps purpurea (NCIM 1046), Curvularia fallax (NCIM 714), Cunmlaria hmaia (NCIM 716), Curvularia cynibopogonis (NCIM 695) and Penicillium fellatanum ( NCIM 1227) obtained from National Collection of Industrial Microorganisms (NCIM), Punc, were grown on potato dextrose agar slants for 7- 8 days. Fungal mycelium from the freshly grown culture was inoculated at the centre of petri plates containing potato dextrose agar medium and incubated till they form small circular growth. The time period for the vegetative growth of different fungus is different. On the periphery of the advancing fungal mycelia, for filter paper discs were impregnated at equal distances, with four different concentrations of the inhibitor sample. The plates were incubated at room temperature for 24-48 h to check the crescent zone of the retarded mycelial growth. For sporulating fungus we also had checked the inhibition by spore suspension method. In this method the agar slants were incubated at room temperature till they sporulate. 4 ml of sterile distilled water was added aseptically to the agar slant and the spores were scrapped by using an inoculation loop. 1 ml of this suspension was mixed with 4 ml of malt extract, glucose, yeast extract, peptone media containing 0.4% agarose and poured onto the potato dextrose agar plate. This plate was incubated at room temperature for 4-10 h. Filter paper discs were impregnated and different concentrations of the inhibitor was added onto it. The plates were further incubated at room temperature for 24-48 h and the inhibition zone against the growth was checked. The low molecular weight aspartic acid protease inhibitor was found to strongly inhibit the mycelial growth of Aspergillus flaws (NCIM 535,542), Aspergillus oryzae (NCIM 637,643), Curvularia fallax (NCIM 714), Curvularia lunata (NCIM 716)., Curvularia cymbopogonis (NCIM 695), Claviceps purpurea (NCIM 1046), Trichoderma reesei (NCIM 992,1051,1052,1186), Fusarium oxysporum (NCIM 1008,1072), and Alternaria solani (NCIM 887). The advantages of the invention are: The bioactive microbial protease inhibitor reported in the present invention shows the inhibition against pepsin, an aspartic acid protease and other enzymes having aspartic acid in the active site. Hence the inhibitor has potential to inactivate pepsin, HIV protease, as well as the aspartic acid protease of the malarial parasite. On the basis of the structure of the bioactive inhibitor as the lead compound, structurally related novel bioactive molecules can be designed. Protease inhibitors inhibit proteases that are common in animals and microorganisms. They are known to function as a natural phytochemical defense against predators, since they inhibit the proteases that occur in many species of herbivorous insects and plant pathogen. The low molecular weight protease inhibitor reported in this invention has antifungal property against phytopathogenic fungi. This is the first report of a low molecular peptidic aspartic acid protease inhibitor having antifungal property. So it can be used as an antifungal agent to control the fungal diseases of the plants. As a biomolecule it is biodegradable and does not have any hazardous effect on the environment. We Claim: 1. A process for the preparation of protease inhibitor using novel alkalothermophilic Bacillus species, which comprises; a) subculturing the alkalothermophilic Bacillus sp. in a fermentation medium containing assimilable carbon and nitrogen sources at a temperature in the range of 37-50 deg.C under aseptic conditions for a period ranging between 24 to 48 hrs., b) harvesting the culture broth by known methods, c) separating the solids by conventional methods to obtain cell free liquid containing the protease inhibitor, d) treating the cell free liquid with a decolorising agent for a period ranging between 2 to 24 hrs. to obtain a colorless liquid, e) separating the decolorising agent and recovering the protease inhibitor by conventional methods such as herein described. 2. A process as claimed in claim 1 wherein assimilable carbon sources used are selected from lactose, sorbitol, xylan, fructose, maltose, and sucrose and the nitrogen sources used are selected from soyameal, casein, casamino acids, urea, tryptone, beef extract, skimmed milk and yeast extract. 3. A process as claimed in claim 1 wherein, the decolorising agent used is selected from charcoal or cellulose. 4. A process as claimed in claim 1 wherein the decolorising agent is separated by centrifugation or filtration and the protease inhibitor is recovered by concentrating the filtrate by lyophilization and purifying the inhibitor by reverse phase high performance liquid chromatography. 5. A process for the preparation of protease inhibitor using novel alkalothermophilic Bacillus species as substantially described hereinbefore with reference to examples 1-7.

Full Text

This invention relates to a process for the preparation of protease inhibitor using novel alkalothermophilic bacillus sp. More particularly it relates to the process for the preparation of a low molecular weight peptide aspartic acid protease inhibitor using alkalothermophilic Bacillus (AT Bacillus sp.) deposited at National Collection of Industrial Microorganism(NCIM),Pune designated as NCIM 59.
The alkalothermophilic Bacillus sp. NCIM 59 was isolated from the soil sample of hot spring of Vajreshwari, Maharashtra, India. The alkalothermophiclic Bacillus sp. is distinctively different from other species of Bacillus in its growth, temperature and pH. The organism is found to be an obligatory alkalophile, gram positive, aerobic, motile, spore forming rods with the cells growing together and forming filament. On alkaline nutrient agar at 50°C, the colonies of the organism are butyrous, glistering and pale cream coloured.
Proteases are responsible either directly or indirectly for all bodily functions including cell growth, nutrition, differentiation and apoptosis. They also play a significant role in intracellular and extracellular protein turn over (house keeping and repair), cell migration and invasion, fertilization and implantation (Protease inhibitors, novel therapeutic application and development, Tony E Hugli, TIBTECH, 14, 409-412, 1996). Since proteases are necessary for normal and abnormal body functions, their effective regulatory counterparts i.e., protease inhibitors, are tremendously essential for physiological regulations. Protease inhibitors have been the source of attention in many disciplines. Due to their presence in valuable plant feeds and involvement in nutritive properties they have evoked the interest of nutritionists. Inhibitor proteins have been studied for the elucidation of mechanism of inhibition of proteases, as well as for the
studies on protein-protein interactions and associations. Due to their unique pharmacological properties, protease inhibitors are also used as valuable tools in medical research.
Protease inhibitors are classified into Synthetic and Naturally occuring inhibitors. They occur in numerous animal tissues and fluids, in many plant tissues (particularly in legume seeds and other storage organs) and in microorganisms. (Protease inhibitors, Yehudith Birk, Hydrolytic enzymes, A Neuberger and K. Brocklehurst (Eds), Elsevier
Science Publishers B. V. (Biomedical Division), 257, 1987). The most abundant source of the inhibitors in plants is the seeds, but their location is not necessarily restricted to this part of the plant. They are also found in leaves, tubers, etc,. As for the intracellular localization of the inhibitors, they appear to be associated primarily with the cytosol, but in some instances they have been localized in protein bodies. The inhibitors of animal origin are found both in tissues and in secretions of organs. The pancreatic trypsin inhibitor has been found as an intracellular component in various bovine organs: in the pancreas, lung, liver, spleen, parotoid gland and also in pituitary gland. In addition to the thoroughly studied pancreatic trypsin inhibitors, a large number of protease inhibitors from different animal sources have been isolated. Many of them are secretory proteins, such as trypsin inhibitors of blood plasma, milk colustrum, seminal plasma and submandibular glands. The plasma protease inhibitors constitute a major group of the functional proteins of the blood plasma. Most of them inhibit serine proteases but their mechanism of interaction is still being actively pursued by many investigators.
The presence of protease inhibitors in microorganisms came into existence from the studies on antibiotics as they act as inhibitors of the enzymes which are involved in
growth and multiplication. Proteolytic enzymes outside of microbial cells hydrolyze organic nitrogen compounds in the medium, so they are thought to be harmful to cells. The production of inhibitors of the proteolytic enzymes by microorganisms is probably a mechanism to provide cell protection. In contrast to the inhibitors of proteolytic enzymes obtained from animals and plants, the inhibitors from microorganisms are of smaller molecular nature. Specific inhibitors of microbial origin have been used as useful tools in biochemical analysis of biological functions and diseases. (Enzyme inhibitors of microbial origin, Hamao Umezawa, University Park Press).
Few of the inhibitors of microbial origin of therapeutic interest are given below: Leupeptin- from Streptomyces, is the inhibitor of trypsin, plasmin, kalikrein and papain. Chymostatin- from Streptomyces, is the inhibitor of chymotrypsin. Dopastin- from Pseudomonas, Oosponol- from Oospora, Oudenone- from
Oudemansialla radicata and Fusaric acid- from Fusarium are the inhibitors of dopamine hydroxylase.
Pepstatin A- from Streptomyces, is the inhibitor of pepsin, an aspartic acid protease. It inhibits the HIV-1 protease, which is also an aspartic protease and the key enzyme for the propagation of the HIV.
The expanding Acquired Immuno Deficiency Syndrome (AIDS) epidemic and the relentless nature of the disease have intensified the search for effective antiviral therapies, to control the replication of the HIV, the causative agent of AIDS. The HIV -1 protease is the key enzyme for the propagation of the virus. Thus specific inhibition of the HIV-1
protease by inhibitors is useful in preventing the infection. HIV-1 protease is structurally and mechanistically related to mammalian and microbial aspartic proteases such as pepsin, cathepsin, renin, and endothiopepsin. The classification of HIV-1 protease in the aspartyl family was also predicted from its primary sequence analysis. A highly conserved sequence Asp-Thr-Gly (D-T-G) in retroviral proteases, is also conserved in the active site of the cellular and fungal proteases. Molecular modeling studies have also confirmed the functional and structural similarities of the retroviral proteases to other aspartyl proteases. Various synthetic peptidic and non peptidic compounds have been shown to inhibit HIV-1 protease. Well documented examples of isolation of compounds by microbial screening represented by the discovery of potent compounds such as cyclosporin, movionolin, and avermycin, etc,. An antifungal antibiotic cerulenin from Cephalosporium and pepstatin A, a pepsin inhibitor from Streptomyces, have been well characterized as HIV-1 protease inhibitors. (C. Debouck, AIDS Research and Human Retroviruses, 8, 153-164, 1992).
Extensive evidence suggests that, the degradation of hemoglobin is necessary for the growth of erythrocytic malarial parasite, apparently to provide free amino acids for parasitic protein synthesis. On the basis of the data available, the aspartic acid proteases are thought to be responsible for the initial cleavages of hemoglobin. Both aspartic acid and cystein proteases have synergistic effects in inhibiting the growth of the cultural malarial parasite and also these proteases act synergistically to degrade hemoglobin. Therefore, the combination of inhibitors of malarial cystein and aspartic acid proteases, may provide a most effective chemotherapeutic regimen and best limit the development of parasitic resistant to protease inhibitors. Pepstatin, the inhibitor of aspartic acid proteases, along with the cystein protease inhibitor E-64, blocks the Plasmodium falciparum development. (Proteases of Malarial Parasite: New Targets for Chemotherapy, Philip J.
Rosenthal, Emerging Infectious Diseases, 4 (1), 49-57, 1998). So far no report is available for the preparation of the protease inhibitor using alkalothermophilic Bacillus sp.
Based on the fact that the aspartic acid protease plays a significant role in the development of the malarial parasite, the inventors of the present invention believe that the inhibitor produced as per the procedure of the present invention using AT Bacillus NCTM59, could be a potent inhibitor for the protease particularly the aspartic acid protease more particularly for protease of malarial parasite. The inhibitor described in the present invention inhibits pepsin, an aspartic protease. Pepsin present in the gastric secretion is responsible for the degradation (digestion) of proteinaceous food. Excess secretion of pepsin has harmful effects on the stomach as it damages the digestive tract and causes stomach ulcer or duodenal ulcer. Considering the fact that the inhibitor is an active inhibitor of pepsin, it has potential application as a therapeutic agent against stomach or duodenal ulcers. Pepstatin A, a pepsin inhibitor has been reported to inhibit HIV-1 protease which is also an aspartic protease. The inventors of present invention have observed that the inhibitor also inhibits other enzymes having aspartic acid in the active site. So we strongly feel that the microbial protease inhibitor could inhibit the
HIV-1 protease.
The main objective of the present invention is to provide a process for the preparation of low molecular weight peptidic aspartic acid protease inhibitor using Alkalothermophilic Bacillus sp. NCIM 59.
Accordingly, the present invention provides a process for the preparation of low molecular weight peptidic aspartic acid protease inhibitor using alkalothermophilic Bacillus sp. NCIM59, which involves following steps: subculturing the said species onto
malt extract, glucose, yeast extract, and peptone MGYP agar slant to obtain freshly grown AT Bacillus, inoculating this Bacillus in a conventional fermentation medium at a temperature in the range of 37-50 (C under aseptic conditions for a period ranging between 24 to 48 hrs,, harvesting the culture broth, separating the solids by conventional methods to obtain cell free liquid containing the protease inhibitor, treating the cell free liquid with a decolourising agent for a period ranging between 2 to 24 hrs., to obtain the colourless liquid, subjecting this liquid to ultrafiltration, concentrating the ultrafiltered liquid to obtain the low molecular weigh protease inhibitor.
Accordingly, the present invention provides a process for the preparation of protease inhibitor using novel alkalothermophilic Bacillus species, which comprises; a) subculturing the alkalothermophilic Bacillus sp. in a fermentation medium containing assimilable carbon and nitrogen sources at a temperature in the range of 37-50 deg.C under aseptic conditions for a period ranging between 24 to 48 hrs., b) harvesting the culture broth by known methods, c) separating the solids by conventional methods to obtain cell free liquid containing the protease inhibitor, d) treating the cell free liquid with a decolorising agent for a period ranging between 2 to 24 hrs. to obtain a colorless liquid, e) separating the decolorising agent and recovering the protease inhibitor by conventional methods such as herein described
In one of the embodiments of the present invention the agar slant such as MGYP (Malt extract, Glucose, Yeast extract, and Peptone) may be used to grow Bacillus to get fresh inoculum.
In one of the embodiments of the present invention the AT Bacillus sp. was isolated from the hot spring from Vajreshwari, India, and which has been deposited in National Collection for Industrial Micro-organisms, NCL, Pune 411 008, having accession No. NCIM 59. The characteristics of this strain are i) aerobic ii) gram positive iii) motile iv) spore forming v) capable of growing in a alkaline medium at Ph 8-10 and v) showing negative reaction towards production of indole, hydrogen sulfide,ammonia and urease and positive reaction for hydrolysis of starch, production of catalase, hydrolysis of casein and reduction of nitrate.
In another embodiment the decolourising agent used may be activated charcoal or cellulose.
In a feature of the present invention the composition of the MGYP medium containing the carbon, nitrogen source and other micro ingredients may be:
I. glucose - 0.5% - 1%
II. beef extract - 0.5% - 0.75%
III. sodium chloride -0.1%- 0.3%
IV. magnesium sulfate - 0.05%- 0.1%
V. dipotassium hydrogen phosphate -0.05% - 0.1%
VI. soyameal - 1 % - 2%.
Production of the inhibitor and its activity is also dependent upon the nature and concentration of the media ingredients, inoculum development and parameters such as aeration, agitation, etc. In shake flasks maximum production obtained after 24-48 hours, when a 5-20 % v/v vegetative inoculum in the actively growing phase is transferred to the production media. Post fermentation processing of the broth for the isolation of the inhibitor includes, centrifugation or filtration of the broth. Cell free supernatant is treated with activated charcoal to remove the colour material. The charcoal is removed by centrifugation or filtration. The resulting supernatant is subjected to ultrafiltration using filtration membranes.
In a feature of the present invention the pepsin inhibitor was selectively purified by removing the high molecular weight compounds from the culture broth by treating with activated charcoal. The charcoal treated broth was filtered through ultrafiltration membrane and concentrated by lyophilization. The concentrated inhibitor sample was
loaded onto a reverse phase-high performance liquid chromatography column using a linear gradient of acetonitrile and triflouroacetate, and the peaks were checked for the inhibitor activity. Among the two compounds detected in the HPLC analysis, the compound having less retention time showed the inhibition against pepsin. This compound absorbs strongly at 210 nm. The compound was collected and checked as a single purified peptide after loading onto the HPLC column. One unit of inhibitor is defined as the amount of inhibitor which inhibits the protease activity expressed in terms of decrease in optical density of 0.001 per minute.
The process of the present invention is described herein below with examples which are illustrative only and should not be construed to limit the scope of the present invention in any manner.
Example-1
Eighteen hour grown AT Bacillus NCIM 59 on an agar slant, at 50(C, was inoculated into a medium containing glucose-1%, peptone-0.75%, beef extract-0.75%, sodium chloride-0.3%, dipotassium hydrogen phosphate-0.1%, magnesium sulfate-0.1% and soyameal-2.0%, and this fermentation medium was incubated on a rotary shaker at 50(C for 12 hours. 10% v/v of the freshly grown inoculum was added to the fermentation flasks. After 48 hours of growth on a rotary shaker at 50(C the cells and the residual soyameal was removed by centrifugation. To 100 ml of centrifuged supernatant 10 grams of activated charcoal was added and incubated overnight at 4(C. This charcoal treated broth was filtered through filter paper 2-3 times to ensure there was no trace of charcoal remained. The maximum production of the inhibitor obtained was 100-112 U/ml in the charcoal treated broth.
Example-2
The medium was formulated for the production flasks using lactose, sorbitol, xylan, fructose, maltose and sucrose, all at a concentration of 1% and fermentation process was carried out for 48 hrs. After 48 hours, the production of inhibitor obtained was maximum of 98 U/ml in presence of sucrose.
Example-3
In this example the fermentation medium used in example-1 was supplimented with amino acids for checking the effect on the production of the inhibitor. The amino acids used in the production flasks were alanine, arginine, asparagine, cystein, glutamic acid, glycine, histidine, proline and serine, all at a final concentration of 0.5%. After 48 hours, the product was isolated and the results are tabulated in Table-1 given herein below. It is observed that maximum production of the inhibitor of 105 U/ml was obtained in presence of asparagine. Table-1
(Table Removed)

Example-4
The medium was formulated for the production of the inhibitor using 1% w/v of following nitrogen sources in the fermentation flasks. Soyameal, casein, casamino acids, urea, tryptone, peptone, beef extract, skimmed milk and yeast extract were among the nitrogen sources tested. Although, after 48 hours a maximum production of 70 U/ml of the inhibitor was obtained in presence of beef extract, the production was considerably high i.e., 90 U/ml -100 U/ml, when beef extract and soyameal were used together in the medium.
Example-5
In this example the medium was supplimented with various inducers like soyameal, casein, yeast extract, tryptone, skimmed milk, urea and casamino acids, at a concentration of 1% w/v in the production flasks. After 48 hours, maximum production of 140 U/ml of the inhibitor was obtained in presence of casamino acids.
Example-6
The charcoal treated supernatant was passed through an Amicon UM-02 membrane and concentrated by lyophilization. The activity of the inhibitor was also increased correspondingly. The concentrated inhibitor sample was injected into an RP-HPLC column in a linear gradient of 0 % acetonitrile- 50 % acetonitrile and 0.05 % trifluoroacetate. The compound having less retention time, showed the inhibition against
pepsins this compound was related on RP-High peyemeric liquid chromatography column using the same solvent system and found to be
homogenous and pure. The purified inhibitor was analysed for the amino acid sequences and for the determination of the molecular mass.
Examplc-7
The fungal strains Trichoderma reesei (NC1M 992,1051,1052,1186), Fusariumoxysponim (NCIM 1008,1043,1072), Aspergillus flaws (NCIM
535,536,538,542), Aspergillus oryzae (NCIM 637,643,649,1032), Mtsarium
moniliforme (NCIM 1099,1100), Alternaria solmri (NCIM 887), Claviceps
purpurea (NCIM 1046), Curvularia fallax (NCIM 714), Cunmlaria hmaia
(NCIM 716), Curvularia cynibopogonis (NCIM 695) and Penicillium
fellatanum ( NCIM 1227) obtained from National Collection of Industrial
Microorganisms (NCIM), Punc, were grown on potato dextrose agar slants for 7-
8 days. Fungal mycelium from the freshly grown culture was inoculated at the
centre of petri plates containing potato dextrose agar medium and incubated till
they form small circular growth. The time period for the vegetative growth of
different fungus is different. On the periphery of the advancing fungal mycelia,
for filter paper discs were impregnated at equal distances, with four different
concentrations of the inhibitor sample. The plates were incubated at room
temperature for 24-48 h to check the crescent zone of the retarded mycelial
growth. For sporulating fungus we also had checked the inhibition by spore suspension method. In this method the agar slants were incubated at room temperature till they sporulate. 4 ml of sterile distilled water was added aseptically to the agar slant and the spores were scrapped by using an inoculation loop. 1 ml of this suspension was mixed with 4 ml of malt extract, glucose, yeast extract, peptone media containing 0.4% agarose and poured onto the potato dextrose agar plate. This plate was incubated at room temperature for 4-10 h. Filter paper discs were impregnated and different concentrations of the inhibitor was added onto it. The plates were further incubated at room temperature for 24-48 h and the inhibition zone against the growth was checked. The low molecular weight aspartic acid protease inhibitor was found to strongly inhibit the mycelial
growth of Aspergillus flaws (NCIM 535,542), Aspergillus oryzae (NCIM 637,643), Curvularia fallax (NCIM 714), Curvularia lunata (NCIM 716)., Curvularia cymbopogonis (NCIM 695), Claviceps purpurea (NCIM 1046), Trichoderma reesei (NCIM 992,1051,1052,1186), Fusarium oxysporum (NCIM 1008,1072), and Alternaria solani (NCIM 887).
The advantages of the invention are:
The bioactive microbial protease inhibitor reported in the present invention shows the inhibition against pepsin, an aspartic acid protease and other enzymes having aspartic acid in the active site. Hence the inhibitor has potential to inactivate pepsin, HIV protease, as well as the aspartic acid protease of the malarial parasite. On the basis of the structure of the bioactive inhibitor as the lead compound, structurally related novel bioactive molecules can be designed. Protease inhibitors inhibit proteases that are common in animals and microorganisms. They are known to function as a natural phytochemical defense against predators, since they inhibit the proteases that occur in many species of herbivorous insects and plant pathogen. The low molecular weight protease inhibitor reported in this invention has antifungal property against phytopathogenic fungi. This is the first report of a low molecular peptidic aspartic acid protease inhibitor having antifungal property. So it can be used as an antifungal agent to control the fungal diseases of the plants. As a biomolecule it is biodegradable and does not have any hazardous effect on the environment.

We Claim:
1. A process for the preparation of protease inhibitor using novel alkalothermophilic
Bacillus species, which comprises; a) subculturing the alkalothermophilic
Bacillus sp. in a fermentation medium containing assimilable carbon and nitrogen
sources at a temperature in the range of 37-50 deg.C under aseptic conditions for
a period ranging between 24 to 48 hrs., b) harvesting the culture broth by known
methods, c) separating the solids by conventional methods to obtain cell free
liquid containing the protease inhibitor, d) treating the cell free liquid with a
decolorising agent for a period ranging between 2 to 24 hrs. to obtain a colorless
liquid, e) separating the decolorising agent and recovering the protease
inhibitor by conventional methods such as herein described.
2. A process as claimed in claim 1 wherein assimilable carbon sources used are
selected from lactose, sorbitol, xylan, fructose, maltose, and sucrose and the
nitrogen sources used are selected from soyameal, casein, casamino acids, urea,
tryptone, beef extract, skimmed milk and yeast extract.
3. A process as claimed in claim 1 wherein, the decolorising agent used is selected
from charcoal or cellulose.
4. A process as claimed in claim 1 wherein the decolorising agent is separated by
centrifugation or filtration and the protease inhibitor is recovered by
concentrating the filtrate by lyophilization and purifying the inhibitor by reverse
phase high performance liquid chromatography.
5. A process for the preparation of protease inhibitor using novel alkalothermophilic
Bacillus species as substantially described hereinbefore with reference to
examples 1-7.

Documents:

3560-del-1998-abstract.pdf

3560-del-1998-claims.pdf

3560-del-1998-correspondence-others.pdf

3560-del-1998-correspondence-po.pdf

3560-del-1998-description (complete).pdf

3560-del-1998-form-1.pdf

3560-del-1998-form-19.pdf

3560-del-1998-form-2.pdf

3560-del-1998-form-3.pdf

3560-del-1998-petition-138.pdf

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Patent Number

215711

Indian Patent Application Number

3560/DEL/1998

PG Journal Number

12/2008

Publication Date

21-Mar-2008

Grant Date

03-Mar-2008

Date of Filing

27-Nov-1998

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110 001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	CHANDRAVANU DASH	NATIONAL CHEMICAL LABORATORY, PUNE 411 008, MAHARASHTRA, INDIA.
2	SANGITA UDAY PHADTARE	NATIONAL CHEMICAL LABORATORY, PUNE 411 008, MAHARASHTRA, INDIA.
3	ABSAR AHMAD	NATIONAL CHEMICAL LABORATORY, PUNE 411 008, MAHARASHTRA, INDIA.
4	VASANTI VISHNU DESHPANDE	NATIONAL CHEMICAL LABORATORY, PUNE 411 008, MAHARASHTRA, INDIA.
5	MALA BALCHANDRA RAO	NATIONAL CHEMICAL LABORATORY, PUNE 411 008, MAHARASHTRA, INDIA.

PCT International Classification Number

C12N 009/50

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA