Title of Invention

"AN IMPROVED PROCESS FOR THE PRODUCTION OF IMMOBILIZED MILK CLOTTING PROTEASE"

Abstract

An improved process for the production of immobilized milk clotting protease useful for the preparation of cheese, which comprises suspending crosslinked macroporous glycidyl methacrylate - pentaerythritol triacrylate copolymers, in acetate buffer, adding the milk clotting protease solution obtained in a manner as herein described, agitating the immobilization reaction mixture in the range of 90 to 150 rpm, maintaining a temperature in the range of 25 to 28°C for a period ranging from 16 to 20 hours, isolating the immobilized milk clotting protease by filtration.

Full Text

This invention relates to an improved process for the production of immobilized milk clotting protease. The invention particularly relates to a process for the immobilization of milk clotting protease on novel crosslinked macroporous glycidyl methacrylate - pentaerythritol triacrylate copolymers useful for the preparation of cheese. k-Casein stabilizes the casein micelles in the milk via protecting against flocculation by Ca++. Milk clotting protease, also referred as rennet, hydrolyses selectively the peptide bond between alanine and methionine at position 105-106 in k-casein to form para-k-casein and a glycopeptide. Thus, the action of milk clotting protease destabilizes the casein micelles and leads to its gelation followed by formation of cheese. A variety of microorganisms such as Rhizomucor miehei, Rhizomucor pusillus. Endothia parasitica, Irpex lacteus, Aspergillus niqer. Kluyveromyces lactis and Escherichia coli are developed for production of milk clotting protease as described in Advances in Applied Microbiology, vol. 20, p. 39-73, 1976 and Biotechnology, vol. 9, 2nd Edition, p. 353-384, 1995. These milk clotting proteases are used instead of calf chymosin. However, microbial milk clotting protease preparations contain nonspecific aspartic proteases at varying degree which causes excessive degradation of cheese proteins. In our copending Application No. 251/BOM/96, we have described and claimed a process for production of milk clotting protease by fermentation of Aspergillus niqer strain MC4 . The enzyme preparation contains very low amount of aspartic proteases. Currently, in the process for the production of cheese, milk is incubated with a solution of milk clotting protease at a given temperature for a certain period and the cheese formed is separated by filtration and processed for fortification as described in Biotechnology, vol. 9, 2nd Edition, p.353-384, 1995. Major disadvantage with this process is that the aspartic proteases can not be separated and remain entrapped in the cheese. During storage, the aspartic proteases hydrolyse protein molecules present in the cheese which results in altered flavor and bitter taste thereby reduces the shelf life of cheese. Further, other metabolites secreted by the microorganism are at times, present in the milk clotting protease and contaminate the cheese. To overcome these factors, use of an immobilized milk clotting protease are explored. Milk clotting proteases are immobilized on a number of different types of solid supports such as DEAE-cellulose, AE- cellulose, porous glass, succinic amido propylated glass, photo-crosslinkable resin polymer, paraffin wax, Sepharose 4B, Agarose and alkyl amide sand as described in World Journal of Microbiology and Biotechnology, vol. 9, p.139-144, 1993. However, these supports are not dedicated to the milk clotting protease and casein micelles system. Choice of right matrix for immobilization of an enzyme depends on the appropriate pore size and pore size distribution, enzyme properties, interference or hindrance in the catalytic reaction and mechanical stability at the temperature, pH and sheer during operation. In our copending Application No.2937/D/97, we have described and claimed a process for the preparation of novel crosslinked macroporous glycidyl methacrylate - pentaerythritol triacrylate copolymers which are designed to encounter the above mentioned factors and hence are useful for the immobilization of milk clotting protease. The said protease is an enzyme which does not have any nutritional value and hence it is not used as food. The main object of the present invention is to provide a process for the production of the immobilized milk clotting protease using crosslinked macroporous glycidyl methacrylate - pentaerythritol triacrylate copolymers as the polymer matrix. The other object of the present invention is to obtain the immobilized milk clotting protease free from the metabolites secreted during the fermentation of Aspergillus niger strain MC4 Accordingly, the present invention provides an improved process for the production of immobilized milk clotting protease useful for the preparation of cheese, which comprises suspending crosslinked macroporous glycidyl methacrylate - pentaerythritol triacrylate copolymers, in acetate buffer, adding the milk clotting protease solution obtained in a manner as herein described agitating the immobilization reaction mixture in the range of 90 to 150 rpm, maintaining a temperature in the range of 25 to 28°C for a period ranging from 16 to 20 hours, isolating the immobilized milk clotting protease by fi'tratinn In an embodiment of the invention, the glycidyl methacrylate-pentaerythritol * triacrylate copolymer is prepared as described and claimed in our copending Application No.2937/D/97. In yet another embodiment of the invention, the acetate buffer used for suspending the crosslinked macroporous glycidyl methacrylate - pentaerythritol triacrylate copolymers may have a molarity from 0.05 to 0.1 having a pH in the range of 3.8 to 4.4. In still another embodiment of the invention, the acetate buffer used for suspending the immobilized enzyme may be from 0.05 to 0.1 molar and a pH in the range of 3.8 to 4.4. The activity of milk clotting protease was measured by adding 0.5 ml of the enzyme or 0.5 ml of settled immobilized enzyme beads to 5.0 ml of 10% w/v solution of skim milk powder containing 0.01 M CaCI2. The reaction mixture was incubated at 35°C with with shaking and the time lapsed between the addition and first appearance of solid material was measured. The amount of the enzyme required to clot the milk in one minute is considered to contain 400 u of milk clotting protease as defined in Methods in Enzymology, vol. 19, p. 446-459, 1970. The process of the present invention is described by following Examples are illustrative only and should not be construed to limit the scope of the invention, in any manner. Examples to describe the preparation of novel crosslinked macroporous glycidyl methacrylate - pentaerythritol triacrylate copolymers. Example 1: In an inert atmosphere of nitrogen, 1.5 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 20.2 ml of glycidyl methacrylate, 12.5 ml of pentaerythritol triacrylate and 43.5 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.6 gram of azo bis isobutyronitrile was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 70°C for 3 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by methanol and dried in a vacuum oven at 4 0°C. The yield of copolymer obtained was 31.25 grams. Example 2.: In an inert atmosphere of argon, 1.5 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 14.6 ml of glycidyl methacrylate, 18.1 ml of pentaerythritol triacrylate and 43.5 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.9 gram of benzoyl peroxide was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 70°C for 4 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by ethanol and dried in a vacuum oven at 40°C. The yield of copolymer obtained was 3 0.30 grams. Example 3_: In an inert atmosphere of argon, 1.5 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 9.4 ml of glycidyl methacrylate, 23.3 ml of pentaerythritol triacrylate and 43.5 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.6 gram of azo bis isobutyronitrile was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 80°C for 3 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by methanol and dried in a vacuum oven at 4 0°C. The yield of copolymer obtained was 33.50 grams. Example 4.: In an inert atmosphere of nitrogen, 1.0 gram of poly vinyl pyrrolidone was dissolved in 100 ml of distilled water in a 250 ml capacity glass reactor. 11.8 ml of glycidyl methacrylate, 29.2 ml of pentaerythritol triacrylate and 43.5 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.8 gram of methyl ethyl ketone peroxide was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 80°C for 3 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by ethanol and dried in a vacuum oven at 4 0°C. The yield of copolymer obtained was 32.80 grams. Example J5: In an inert atmosphere of nitrogen, 1.5 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 10.7 ml of glycidyl methacrylate, 22.0 ml of pentaerythritol triacrylate and 16.4 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.6 gram of azo bis isobutyronitrile was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 70°C for 4 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by ethanol and dried in a vacuum oven at 40°C. The yield of copolymer obtained was 31.65 grams. Example 6.: In an inert atmosphere of nitrogen, 1.5 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 10.7 ml of glycidyl methacrylate, 22.0 ml of pentaerythritol triacrylate and 54.6 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.6 gram of azo bis isobutyronitrile was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 70°C for 4 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by ethanol and dried in a vacuum oven at 4 0°C. The yield of copolymer obtained was 34.10 grams. Example 1 In an inert atmosphere of nitrogen, 1.5 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 10.7 ml of glycidyl methacrylate, 22.0 ml of pentaerythritol triacrylate and 65.4 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 3 00 rpm. 0.6 gram of azo bis isobutyronitrile was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 70°C for 3 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by methanol and dried in a vacuum oven at 40°C. The yield of copolymer obtained was 34.60 grams. Examples to describe the process for the production of milk clotting protease by Aspergillus niger strain MC4 under submerged fermentation. Example 8. 3 gram each of dextrose, casein and agar were suspended in 100 ml of potato extract prepared by boiling 50 gram of potatoes. The suspension was heated to 80°C to dissolve the contents and sterilised by autoclaving at a temperature of 121°C and a pressure of 15 lbs for a period upto 20 minutes. The solution was transferred asceptically to sterile petriplates and cooled to room temperature. A loop of spores of Aspergillus niger strain MC4 from the slants was transferred asceptically to potato dextrose agar plates and the plates were incubated at a temperature of 28°C for 6 days. The growth thus obtained served as inoculum for production of enzyme. Example S> : The growth medium was prepared by dissolving 2 gram of meat peptone, 2 gram of starch, 0.03 gram of MgSO4 7H20, 0.5 mg of FeS04 7H2O, 0.75 mg of MnS04 7H2O, 0.15 mg of ZnS04 7H20 and 0.2 mg of CoCl2 in 100 ml of water and pH made to 6.5. 100 ml of the growth medium was transferred to 500 ml capacity Erlenmeyer flask, plugged with cotton and sterilised by autoclaving at 121°C and 15 lbs for 20 minutes. After cooling to room temperature, 1 squire cm growth of Aspergillus niger strain MC4 obtained as described in Example 8 was transferred asceptically to the sterilised growth medium. The flask was incubated on a rotary shaker at 220 rpm and 28°C for 96 hours. The mycelium was separated by the filtration through nylon cloth. The pH of culture filtrate was adjusted to 4.0 and again filtered through 0.2 micron membrane and the filtrate thus obtained served as the enzyme solution. The culture filtrate contained 100 u/ml of milk clotting protease activity and 0.04 u/ml of aspartic protease activity. Example 10: The growth medium was prepared by dissolving 1 gram of skim milk powder, 0.1 gram of CaCl2, 50 mg of Brij 35, 2 gram of meat peptone, 2 gram of starch, 0.03 gram of MgS04 7H20, 0.5 mg of FeS04 7H20, 0.75 mg of MnS04 7H20, 0.15 mg of ZnS04 7H20 and 0.2 mg of CoCl2 in 100 ml of water and pH made to 6.5. 100 ml of the growth medium was transferred to 500 ml capacity Erlenmeyer flask, plugged with cotton and sterilised by autoclaving at 121°C and 15 lbs for 20 minutes. After cooling to room temperature, 1 squire cm growth of Aspergillus niger strain MC4 obtained as described in Example 8 was transferred asceptically to the sterilised growth medium. The flask was incubated on a rotary shaker at 220 rpm and 28°C for 96 hours. The mycelium was separated by the filtration through nylon cloth. The pH of culture filtrate was adjusted to 4.0 and again filtered through 0.2 micron membrane and the filtrate thus obtained served as the enzyme solution. The culture filtrate contained 400 u/ml of milk clotting protease activity and 0.01 u/ml of aspartic protease activity. Examples to describe the process for the production of immobilized milk clotting protease using novel crosslinked macroporous glycidyl methacryate - pentaerythritol triacrylate copolymers. Example 11: 1 gram of copolymers prepared as described in Examples 1 to 4 were suspended in 20 ml of acetate buffer having molarity of 0.05, pH 4.0 in a 50 ml capacity Erlenmeyer flask. To this suspension the enzyme solution containing 1000 u of milk clotting protease prepared as described in Examples 8 to 10 was added and the immobilization mixture was agitated at an rpm of 110 and maintained at a temperature of 28°C for 18 hours. The immobilized milk clotting protease was collected by filtration. The milk clotting activity of the immobilized enzyme was determined. The results are summarised in Table-1 below: Table-1 (Table Removed) The activity of immobilized milk clotting protease prepared using copolymer having 167% cross link density prepared as described in Example 3 was 1000 u/g which was more when compared to copolymers prepared as described in Examples 1, 2 and 4. 1 gram of copolymers prepared as described in Examples 3, 5, 6 and 7 were suspended in 20 ml of acetate buffer having molarity of 0.05, pH 4.0 in a 50 ml capacity Erlenmeyer flask. To this suspension the enzyme solution containing 4000 u of milk clotting protease prepared as described in Examples 8 to 10 was added and the immobilization mixture was agitated at an rpm of 110 and maintained at a temperature of 28°C for 18 hours. The immobilized milk clotting protease was collected by filtration. The milk clotting activity of the immobilized enzyme was determined. The results are summarised in Table-2 below : Table-2 (Table Removed) The activity of immobilized milk clotting protease prepared using the copolymer prepared with 32.7 ml of cyclohexanol as described in Example 7 was 4000 u/g which was more when compared to the copolymers prepared as described in Examples 3, 5 and 6. Example 13: 1 gram of copolymer prepared as described in Example 7 was suspended in 20 ml of acetate buffer having molarity of 0.05, pH 4.0 in a 50 ml capacity Erlenmeyer flask. To this suspension the enzyme solution containing 6000 u of milk clotting protease prepared as described in Examples 8 to 10 was added and the immobilization mixture was agitated at an rpm of 110 and maintained at a temperature of 28°C for 18 hours. The immobilized milk clotting protease was collected by filtration. The milk clotting activity of the immobilized enzyme was 6000 u/g. Example 14: 50 grams of the copolymer prepared as described in Example 7 was suspended in 1500 ml of acetate buffer having molarity of 0.05 and pH 4.0 in a glass container and the beads were kept in suspension by overhead stirrer (150 rpm) . To this suspension 300000 u of milk clotting protease prepared as described in Examples 8 to 10 were added. The immobilization mixture was agitated (150 rpm) at 28°C for 18 hours. The immobilized milk clotting proteases was collected by filtration and stored as suspension in acetate buffer having molarity of 0.05 and pH 4.0 at 4°C containing 0.02% w/v of sodium azide. The activity of the immobilized milk clotting protease was 6000 u/g. Example 15: 1.0 gram of immobilized milk clotting protease having activity of 6000 u was suspended in 10 ml of acetate buffer having molarity of 0.05 and pH 5.8 containing 0.2% w/v of sodium azide and incubated at 37°C for 100 days. The residual activity was 5900 u. The advantages of the invention are : 1. The copolymers prepared as described in Examples 1 to 7 do not need any treatment or derivatisation prior to immobilization of milk clotting protease. 2. Immobilization of milk clotting protease to the copolymer prepared according to Example 7 is achieved to the extent of 100%. 3 . The pore size and pore size distribution of the copolymer prepared according to the Example 7 is such that casein micelles present in milk are translocated freely with out any hindrance. 4. The milk clotting protease immobilized inside the pores of the copolymer prepared according to Example 7 is available for the reaction in the preparation of cheese. 5. The immobilized milk clotting protease is free from the metabolites secreted during the fermentation of Aspergillus niqer strain MC4. In our copending Application No. NF - 100/97, we have described and claimed a process for the production of cheese using the immobilized milk clotting protease prepared by the process described and claimed in the present Application. We claim : 1. An improved process for the production of immobilized milk clotting protease useful for the preparation of cheese, which comprises suspending crosslinked macroporous glycidyl methacrylate - pentaerythritol triacrylate copolymers, in acetate buffer, adding the milk clotting protease solution obtained in a manner as herein described agitating the immobilization reaction mixture in the range of 90 to 150 rpm, maintaining a temperature in the range of 25 to 28°C for a period ranging from 16 to 20 hours, isolating the immobilized milk clotting protease by filtration. 2. An improved process as claimed in claim 1 wherein the acetate buffer used for suspending the crosslinked macroporous glycidyl methacrylate -pentaerythritol triacrylate copolymers used is of a molarity from 0.05 to 0.1 having a pH in the range of 3.8 to 4.4. 3. An improved process as claimed in claims 1-2 wherein the concentration of the acetate buffer used for suspending the immobilized enzyme is from 0.05 to 0.1 molar and a pH in the range of 3.8 to 4.4. 4. An improved process for the production of immobilized milk clotting protease useful for the preparation of cheese Substantially as herein described with reference to the examples nos. 11 to 14 .

Documents:

2936-del-1997-abstract.pdf

2936-del-1997-claims.pdf

2936-del-1997-complete specification (granted).pdf

2936-del-1997-correspondence-others.pdf

2936-del-1997-correspondence-po.pdf

2936-del-1997-description (complete).pdf

2936-del-1997-form-1.pdf

2936-del-1997-form-2.pdf