Process for the production of an enzyme preparation...

Details Process for the production of an enzyme preparation... Process for the production of an enzyme preparation...

2001-02-23

2003-05-27

Lilling, Herbert J. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Using a micro-organism to make a protein or polypeptide

C435S183000, C435S200000, C435S201000, C435S209000

Reexamination Certificate

active

06569646

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a process for the production of an enzyme preparation containing xylanase and carboxymethyl cellulase, from
Termitomyces clypeatus
having accession no IICB-411 at the Indian Institute of Chemical Biology, Calcutta, India. The strain of
Termitomyces clypeatus
also has been deposited in the Microbial Type Culture Collection (MTCC) at Chandigarh, India, which has the status of an International Depository under the Budapest Treaty, under the accession no. MTCC 5091. 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
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, smoothing of denim cloths, etc.
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 &bgr;-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 bonds. These fibrils are partly crystalline in nature. On the other hand, xylans are the major polysaccharide present in the hemicellulosic fraction of the plant biomass. These polysaccharides have a common backbone of 1,4 linked &bgr;-D-xylopyranoside units substituted with &agr;-arabinofuranosyl, &agr;-4-O-methyl glucuronosyl or acetyl group to different extents depending on the nature and maturity of the plant.
The microbial enzyme systems capable of hydrolysing cellulose and xylan are known as cellulolytic and xylanolytic enzymes respectively. Cellulolytic enzymes mainly consist of three types of enzyme;
1. Endoglucanase (1,4-&bgr;-D-glucan 4 glucanohydrolase, Enzyme Commission number 3.2.1.4) which acts on cellodextrin, substituted cellulose like carboxymethyl cellulose, swollen cellulose;
2. Cellobiohydrolase (1,4&bgr;-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;
3.&bgr;-Glucosidase (&bgr;-D-glucoside glucohydrolase, Enzyme Commission number 3.2.2.21) hydrolyses cellobiose into glucose.
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.
The enzyme endo 1,4&bgr;-xylanase (Enzyme Commission Number 3.2.1.8) which catalyses random hydrolysis of 1,4&bgr;-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.
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.
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
Among the microorganisms, yeast, bacteria and actinomycetes are also producers of xylanase. But fungi produce xylanase in much higher quantities than 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 , Penicillium 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 produce 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 laboratories 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/DELI99), S Sengupta, A K Ghosh, A K Naskar, D Sengupta, M L Jana: A process for the preparation of an enzyme composition containing a mixture of pectinase and xylanase useful for clarification of non citrus fruit juice (Indian Patent application 686/DELI99), 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
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 at the Indian Institute of Chemical Biology, Calcutta, India and accession no. MTCC 5091 at the Microbial Type Culture Collection (MTCC) at Chandigarh, India, an International Depository under the Budapest Treaty.
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
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 the Indian Institute of Chemical Biology, Calcutta, one of the constituent laboratories of the applicant having the accession No.IICB-411, and at the Microbial Type Culture Collection (MTCC) at Chandigarh, India, an International Depository under the Budapest Treaty, under the accession no. MTCC 5091, 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 known methods.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a process for the production of carboxymethyl cellulase and xylanase, said process comprising innoculating and growing the strain
Termitomyces cly

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