Abstract:
This invention relates to a process for the production of carboxymethyl cellulase and xylanase which comprises inoculating, growing  Termitomyces clypeatus  which has been deposited in Indian Institute of Chemical Biology, Calcutta, one of the constituent laboratory of the applicant having accession no IICB-411 being deposited at ATCC having accession no ______ in a sterilized medium containing assimilable carbon source, assimilable nitrogen source and conventional micronutrients at a pH between 3 to 7 and incubating at temperature in the range of 25-30° C., separating the filtrate by known methods.

Description:
FIELD OF THE INVENTION  
         [0001]    This invention relates to a process for the production of an enzyme preparation containing xylanase and carboxymethyl cellulase, from  Termitomyces clypeatus  having accession no 11CB-411 and also being deposited in ATCC having accession no ______ The enzyme preparation is used in many industrial applications preferably, for the treatment of textile, agro-products and plant biomass.  
         BACKGROUND AND PRIOR ART REFERENCES  
         [0002]    Enzyme preparations containing xylanase and cellulase activities have many commercial uses as in, liquefaction of fruit, vegetables, clarification of must juices, extraction of fruit juices, treatment of oil seed for better oil recovery, improving digestibility of cattle feed, smoothening of denim cloths etc.  
           [0003]    Cellulose and hemicellulose are the two major components of plant biomass. The basic molecular structure of cellulose is a linear polymer of glucose linked together by 1,4 β-glucosidic linkages. In mature cellulose containing 8,000 to 12,000 glucose units, the molecules are arranged in fibrils consisting of several parallel chains held together by hydrogen bond. These fibrils are partly crystalline in nature. On the other hand, xylanse are the major polysaccharide present in the hemicellulosic fraction of the plant biomass. These polysaccharides have a common backbone of 1,4 linked β-D-xylopyranoside units substituted with α-arabinofuranosyl, α-4-O-methyl glucuronosyl or acetyl group to different extents depending on the nature and maturity of the plant.  
           [0004]    The microbial enzyme systems capable of hydrolysing cellulose and xylan are known as cellulolytic and xylanolytic enzymes respectively. Cellulolytic enzyme is mainly consisted of three types of enzyme;  
           [0005]    1. Endoglucanase (1,4-β-D-glucan 4 glucanohydrolase , Enzyme Commission number 3.2.1.4) which acts on cellodextrin, substituted cellulose like carboxymethyl cellulose, swollen cellulose  
           [0006]    2. Cellobiohydrolase (1,4β-D-glucan cellobiohydrolase, Enzyme Commission number 3.2.1.91) which acts on native cellulose releasing cellobiose from non reducing ends of native cellulose, but does not attack substituted cellulose  
           [0007]    3. β-Glucosidase (β-D-glucoside glucohydrolase, Enzyme Commission number 3.2.1.21) hydrolyses cellobiose into glucose.  
           [0008]    The endoglucanase activity, which is also known as carboxymethyl cellulase activity, is the major enzyme present in cellulolytic enzyme complex. Cellulase activity is generally measured in terms of carboxymethyl cellulase activity.  
           [0009]    The enzyme endo 1,4 β-xylanase (Enzyme Commission Number 3. 2. 1. 8) which catalyses random hydrolysis of 1,4 β-D-xylosidic backbone linkages of xylan is also the major enzyme on the xylanolytic enzyme complex. Endo-xylanase activity is similarly taken as the measure of xylanolytic activity.  
           [0010]    A large number of microorganisms including fungi, bacteria and actinomycetes produce cellulolytic enzyme which allow the them to grow on cellulose as the carbon source. But the extracellular enzyme activity liberated during growth of the cellulolytic microorganisms differs widely depending on the nature of the strain.  
           [0011]    Among fungi, good extracellular enzyme producers are  Tricoderma reesei, Tricoderma koningii, Aspergillus terrus  and  Pellicularia filamentosa.  Bacteria like Cellulomonas uda, Ruminococcus sp, bacteriodes sp,  Clostridium thermocellum  are moderate producers of cellulolytic enzyme. Actinomycetes like Thermoactinomycete sp, Streptomyces sp, Thermopolyspora sp, were also reported to produce cellulolytic enzyme. Among all the strains fungi are good producers of cellulolytic enzyme,  Tricoderma reesei  being the best so far reported. Fungi produce both cellulolytic and xylanolytic enzymes together in media supporting either cellulase or xylanase production. Coproduction of cellulase and xylanase by bacteria or actinomycetes is rarely observed  
           [0012]    Among the microorganisms, yeast, bacteria and actinomycetes are also producers of xylanase. But fungi produce xylanase in much higher quantities than those do by other microorganisms and bacteria and actiomycetes usually do not produce both xylanase and cellulase in a single fermentation Of the fungal xylanases, production of the extracellular enzyme by  Aspergillus niger, Aspergillus fumigatus, Penicillum funiculosum.  Reference may be made respectively to Frederick, M. M., Kiang, C. H., Frederick, J. R. and Reilly, P. J. Biotechnol. Bioeng. 27, 525-532, 1985, Flannigan, B. and Sellars, P. N. Trans. Br. Mycol. Soc. 71,353-358, 1978), and Mishra, C., Seeta, R. and Rao, M. Enz. Microb. Technol. 7, 295-299, 1985. Among the basidiomycete, the Common Split Gill  Schizophyllum conmume  is a high producer of xylanase. Reference may be made to Postrochers, M., Jurasek, I. and Paice, M. G. Development Industrial Microbiology. 22, 675, 1981. Most of these organisms are highly cellulolytic in character and produces much higher amounts of cellulase than xylanase in the same fermentation. The mycelial culture of basidiomycete  Termitomyces clypeatus  having accession number IICB-411, developed and propagated by us and deposited at one of the constituent laboratory of the applicant is a good producer of both cellulolytic and xylanolytic enzymes in the same medium. However, the amount of xylanase and carboxymethyl cellulase produced by the strain varies depending on the composition of the medium. Reference may be made regarding the producer strain to S Sengupta, A K Ghosh , M L Jana and A K Naskar: A process for the production of xylanase solution without any detectable carboxymethyl cellulase activity (unpublished Indian patent application 353/DEL/94), S Sengupta, D .Sengupta, A K Naskar, M L Jana, A K Ghosh: An improved process for the preparation of bright juices from non-citus fruits (Indian Patent application no 365/ DEL/99), S Sengupta, A K Ghosh, A K Naskar, D Sengupta, M L Jana: A process for the preparation of an enzyme compositon containing a mixture of pectinase and xylanase useful for clarification of non citrus fruit juice (Indian Patent application 686/DEL/99), S Sengupta, D Sengupta, A K Naskar, M L Jana : A process for the preparation of a novel enzymatic formulation useful for improved leavening of bakery products (Indian Patent filed NF/403/99).  
         OBJECTS OF THE INVENTION  
         [0013]    The main object of the present invention is to provide a process for the production of an enzyme preparation containing xylanase and carboxymethyl cellulase from  Termitomyces clypeatus  having accession no IICB-411 and also being deposited in ATCC having accession no ______  
           [0014]    Another object of the invention is use of enzyme preparation for many industrial applications preferably, for the treatment of textile, agro-products and plant biomass.  
         SUMMARY OF THE INVENTION  
         [0015]    Accordingly the present invention provides a process for the production of xylanase and carboxymethyl cellulase which comprises, inoculating and growing  Termitomyces clypeatus,  which has been deposited in Indian Institute of Chemical Biology, Calcutta, one of the constituent laboratory of the applicant having the accession No.IICB-411, and being deposited at ATCC having accession no _______, in a sterilized medium containing assimilable carbon source, assimilable nitrogen source and conventional micronutrients at a pH between 3-7 and incubating at a temperature in the range of 25-30° C., separating the filtrate by know methods.  
         DETAILED DESCRIPTION OF THE INVENTION  
         [0016]    The present invention provides an process for the production of carboxymethyl cellulase and xylanase, said process comprising innoculating , growing the strain  Termitomyces clypeatus  which was isolated and propagated by the applicants and deposited in Indian Institute of Chemical Biology, Calcutta, one of the constituent laboratory of the applicant having the accession No.IICB-411, and being deposited at ATCC having accession no ______  
           [0017]    In an embodiment of the present invention, the mycelial culture of  Termitomyces clypeatus  was characterized by different physical and biochemical properties.  
           [0018]    Physical properties: The strain produces silky white colony on solid medium, frequently producing aggression of mycelia inn areas to form strands and knots of mycelia. The hyphae produced are tubular undifferentiated and hyphae of subcuits with short branch are also present. The average diameter of the hyphae is between 5 to 10 micrometer and all are septed. The strain under microscope shows absence of any sporophoric structure or any basidiospore. A few clamp connections characteristic of mushroom culture is also visible within the hyphae Hyphae with both single and double nucleus are present. The culture remain white over a long period of growth, say 10 to 15 days without any coloration due to any sporulation. Biochemical properties: The mycelial culture is a brown-rot type and has no phenol oxidase or laccase activity. It grows well in defined medium containing carbohydrate and mineral salts. The growth is not well supported by glucose but highly stimulated by glucose polysaccharides. The strain has optimum C/N ratio at 5 and 10 for optimum growth in synthetic medium. Trace elements like Fe++, Mn++, Zn++, Ca++ and Mo++ have much influence on growth. The dry mycelia obtained in liquid medium at optimum growth phase has composition as: protein 31.76%, carbohydrate 52.0%, fat 1.0%, fiber 10.5% and ash 2.7%. The strain in suitable medium gives positive tests for carboxymethyl cellulase, cellobiase, invertase, amylglucosidase, amylase, endo-xylanse, arabinofuranosidase, acety esterase and xylosidase activities but negative tests for laccase, phenol oxidase, chitinase and mannanase.  
           [0019]    The strain was cultivated on a medium containing an assimilable carbon source, an assimilable nitrogen source, inorganic salts and organic nutrients. The assimilable carbon source include carbohydrate such as glucose, xylose, mannose, arabinose, starch, dextrin, cellulose and xylan, agro-residue such as starch free wheat bran, corn cob powder saw-dust and amino acid such as aspartate, glutamate, serine and alanine. Assimilable nitrogen sources or ammonium salts like ammonium chloride, ammonium dihydrogen phosphate, ammonium nitrate, ammonium chloride, organic nitrogen as soyapeptone and bactopeptone organic nutrient include malt extract, yeast extract, wheat bran and corn steep liquor. The fungus is maintained in agar-slant containing (w/v), malt extract 5%, potato extract 20%, glucose 5%, KH 2 PO 4 0.0874% pH 5.0. In the production of the enzyme composition, cultivation was carried out at temperature between 25-30° C., preferably 29±1° C. under aerobic condition in shake flask in liquid medium on rotary shaker 200-300 strokes per minute. Among the assimilable carbon sources, xylan is the best carbon source with the concentration range from 0.5-2% (w/v), preferably 1% (w/v).  
           [0020]    Among the assimilable nitrogen sources, bactopeptone, or ammonium sulphate or ammonium dihydrogen phosphate is preferred. Addition of complex nutrients yeast extract preferably from 0.5-1% w/v improves enzyme production by 2 folds, corn steep liquor preferably 0.1-0.5 w/v Presence of Tween-80 in an amount from 0.1 to 0.2% w/v improves enzyme production by 25-50%. Initial pH of the medium may be maintained from 3 to 7.0, preferably 3.5. Inoculum is developed in shake flask in a medium preferably containing (w/v) malt extract 5%, potato extract 20%, glucose 5%, KH 2 PO 4  0.0874%, 5.0 for 4 days. The mycelia obtained was suspended in sterile water and blended aseptically in a waring blender. Blended culture obtained from the volume of the inoculum medium was inoculated to its ten volume of fermentation medium (1:10 v/v). Optimum enzyme production in the culture filtrate took place in between 4 th  to 5 th  days after inoculation. The enzyme produced in the culture filtrate was freed from fungal mycelia by filtration or centrifugation and used as the source of the enzyme.  
           [0021]    In an embodiment of the present invention, the assimilable carbon source used is selected from carbohydrates selected from glucose, xylose, mannose, arabinose, starch, dextrin, cellulose, xylan, amino acids selected from the group comprising glutamate, aspartate, alanine and serine and agro residue selected from corncob powder, starch free wheat bran powder and any other sources.  
           [0022]    In another embodiment of the present invention, the assimilable nitrogen source used is selected from ammonium salts selected from chloride, nitrate, dihydrogenphosphate and organic nitrogen selected from bacto-peptone, soya-peptone, malt extract, yeast extract wheat bran, corn steep liquor.  
           [0023]    In still another embodiment of the present invention, the cellulose or agro residue employed ranges from 0.5 to 2.0%.  
           [0024]    In yet another embodiment of the present invention, the optimum enzyme production by the strain takes place on 4 to 5 days of fermentation.  
           [0025]    In yet another embodiment of the present invention, carboxymethyl cellulase and xylanase in the culture filtrate are optimally active between temperature range of 50° to 60° C.  
           [0026]    In yet another embodiment of the present invention, carboxymethyl cellulase and xylanase activities of the culture filtrate are optimally active at a pH ranging from 5 to 6.  
           [0027]    In yet another embodiment of the present invention, carboxymethylcellulase and xylanase are stable up to the temperature of 60° C.  
           [0028]    Carboxymethylcellulase and xylanase activities present in the culture filtrate were assayed by Dinitro-salicylic acid method using caboxymethyl cellulose (soluble, sodium salt) and larch wood xylan as the substrates respectively. Reference may be made to Sumner, J. B. and Somers, G. F., 1949 in Laboratory experiments in biological chemistry, Academic Press, New York, pp.38-39 
       
    
    
       [0029]    The invention is further illustrated with the following example, which should not be construed to limit the scope of the invention.  
       EXAMPLE 1  
       [0030]    A culture medium consisting of (w/v): Cellulose powder 2, yeast extract 0.5, peptone 0.5, Tween-80, 0.2%, sucrose 2 , CaCl 2 ,  4 H 2 O, 0.0036%, NaMoO 4 , 0.0032%, FeSO 4 , 7H 2 O-0.027%, boric acid 0.057% and NH4H2PO4-2.4%, pH 3.5 was prepared. One liter of medium was distributed as 50 mL of medium per 500 mL conical shaking flask and autoclaved at 15 pound per square inch pressure for 20 minutes. The medium was inoculated with aspetically blended mycelia of  Termitomyces clypeatus  having the accession No.IICB-41 previously grown in shake flasks for 5 days in a medium containing (w/v), malt extract 5%, potato extract 20%, glucose 5% and KH 2 PO 4− 0.0874% for 5 days at 29±1° C. with shaking. The culture broth was found to contain 200 units of xylanase and 190 units of carboxymethyl cellulase per mL as determined by dinitrosalicylic acid method. About 900 mL of culture filtrate was recovered by filtration of the culture filtrate under suction.  
       EXAMPLE 2  
       [0031]    A culture medium containing (w/v) % Starch free wheat bran powder 2, yeast extract 0.5, peptone 0.5, Tween-80-0.1, wheat bran 0.5, CaCl 2 , 2H2O, 0.037, MgSO 4 , 7H 2 O, 0.052, MnCl 2 , 4H 2 O, 0.0036, NaMoO4 0 0032, boric acid 0 057%, NH 4 H 2 PO 4− 2.4, pH 5.0 was prepared Starch free wheat bran is prepared by removing contaminated starch by boiling with water, followed by digestion with amylase One-liter medium was distributed as 50 mL per 500 mL conical shaking flask and autoclaved at 15 pounds per square inch pressure for 20 minutes. The flask was inoculated asceptically with 10% (v/v) blended mycelia of  Termitomyces clypeatus  having the accession No. IICB-411 previously grown in shake flasks for 5 days in a medium containing (w/v) % malt extract 5, potato extract 20, glucose 5, and KH 2 PO 4  0.0874% for 4 days at 29±1° C. in a rotary shaker (200-250 rpm). Growth was continued for 4 days. The culture filtrate was filtered by suction. The units of xylanase and carboxymethyl cellulase activities that determined by dinitrosalicylic acid method was found to be 300 units and 150 units per mL respectively. About 900 mL of the culture filtrate was recovered.  
         [0032]    The Main Advantage of the Present Invention  
         [0033]    1. The producer organism is an edible fungus and use of the enzyme preparation in the processing of any food preparation is not restricted.  
         [0034]    2. The producer organism is an asporogenous mycelial culture and possibility for the development of fungal spore allergy is absent.  
         [0035]    3. The optimum enzyme production by the strain takes place on 4 to 5 days of fermentation compared to other fungal fermentation, which takes minimum 7 days of fermentation.  
         [0036]    4. Both carboxymethyl cellulase and xylanase in the culture filtrate are optimally active between same temperature range of 50° to 60° C.  
         [0037]    5. Both carboxymethylcellulase and xylanase aactivities of the culture filtrate are optimally active between same pH range of 5 to 6  
         [0038]    6. Both carboxymethylcellulase and xylanase are stable upto the temperature of 60° C.