Title of Invention

"A METHOD FOR PREPARATION OF MYCOLYTIC ENZYMES CONTAINING MAINLY CHITINASE"

Abstract

This invention relates to a method for preparation of mycolytic enzymes containing mainly chitinase. The said process uses a fungus Myrothecium verrucaria. This mycolytic enzyme complex is made up of mainly cell wall hydrolytic enzymes, such as chitinases, glucanases and proteinases in significant quantities and in different proportion. The process steps are: growing Myrothecium sp. characterized in that on bean pod till it reaches to speculation stage, growing in a conventional fermentation medium having carbon and nitrogen sources and minor ingredients such as herein described for a period of at least 7 days in a known manner, separating the mycelia by conventional methods to obtain a mixture of mycolytic enzymes in the separated liquid, separating the product by known methods.

Full Text

This invention relates to a method for preparation of mycolytic enzymes containing mainly chitinase. More particularly it relates to the said process using a fungus Myrothecium verrucaria. This mycolytic enzyme complex is made up of mainly cell wall hydrolytic enzymes, such as chitinases, glucanases and proteinases in significant quantities and in different proportion. The fungal protoplasts have received a great deal of attention to unravel variety of physiological functions and in a strain improvement programme too. Protoplasts have been used as an effective experimental tool for studying cell wall synthesis, enzyme production and secretion and steroid transformation. The fungal protoplast fusion and transformation system is useful to understand genetic compatibility between strains and species. Protoplasts can also be used for mutation towards strain improvement (Kelkar et al Enzyme Microb. Technol. 12: 510-514, 1990). Various commercial preparations used for isolating fungal protoplasts contain cell wall hydrolytic enzymes, such as chitinases, glucanases and proteinases in significant quantities and in different proportion (Hamlyn et al Enzyme Microb. Technol. 3:321-325, 1981). Other enzymes which also contribute in the isolation of protoplasts are mannanases, a-and (3- mannosidases and chitosanases. As fungi from different groups vary significantly in the chemical composition of their cell walls, all the mycolytic preparations available in the market are not equally efficient to release maximum number of protoplasts from all. Most of the commercial preparations such as Lysing enzyme (Sigma), Onozuka R-10 (Yakult Pharmaceuticals, Japan), Zymolyase, etc. contain mainly chitinase, glucanase and protease activities. Other activities such as chitosanase, mannanase, α- and ß-mannosidase are in low amount (Hamlyn et al Enzyme Microb. Technol. 3:321-325, 1981; Vyas and Deshpande J.Gen Appl. Microbiol. 35: 343-350, 1989). Therefore, these preparations are not useful for the wide range of fungi that have cell walls of diverse nature. Myrothecium verrucaria. NCIM 903, a deuteromycetous fungus with green elongated spores, produces extracellularly total mycolytic enzyme complex i.e chitinase, glucanase, protease extracellularly total mycolytic enzyme complex i.e chitinase, glucanase, protease chitosanase, mannanase, a- and ß-mannosidase activities when grown on chitin as a carbon source (Vyas and Deshpande, J.Gen. Appl. Microbiol, 35; 343-350, 1989). Therefore this enzyme complex can be used to make protoplasts from the fungi from different taxonomic groups, the representative examples are given in Table 3. The object of the present invention is to provide a method for the preparation of biomass in form of spores useful as an inoculums for enzyme production. Another object of the present invention is to produce high levels of extra cellular mycolytic enzyme complex which can degrade cell walls of different fungi. Another object of the present invention is to use the said enzyme preparation for isolation of protoplasts from taxonomically unrelated fungal species. Accordingly the present invention provides a process for the preparation of mycolytic enzymes containing chitinase, which comprises; growing Myrothecium sp. in bean pod till it reaches to speculation stage, growing in a conventional fermentation medium having carbon and nitrogen sources, minor ingredients and traced metal solution such as herein described for a period of at least 7 days in a known manner, separating the mycelia by conventional methods to obtain a mixture of mycolytic enzymes in the separated liquid, separating the product by known methods. In one of the embodiments of the present invention the carbon source (0.2-1.0%) used in the fermentation medium may be chitin, chitosan, glucosamine, N-acetylglucosamine, glucose and cellobiose to get mycolytic enzyme mixture comprising of chitinases, glucanases, proteinases, mannanases, α-and ß- mannosidases and chitosanases. In another embodiment the nitrogen source (0.14-0.7%) used may be ammonium sulfate, ammonium chloride, di-ammonium hydrogen phosphate, sodium nitrate, and potassium nitrate, peptone, and urea. In yet another embodiment other minor ingredients may be as follows given in Table 1 Table-1 (Table Removed) *Trace metal solution 0.1 ml contains (mg/ml): FeSO4, 5.0; ZnSO4, 1.4; MnSO4, 1.6; CoClz.2.0). In a feature of the present invention the sporulation of Myrothecium verrucaria, can be obtained on potato dextrose agar (PDA, dextrose 2%) slants, soil extract agar, which contains 1% soil extract and 2% agar. The maximum sporulation can be achieved on 5% bean pod. The bean pod used may be sterilized in solid or macerated form or supplemented with agar. The novelty of the process is the use of bean pods directly without extraction of the contents for sporulation, that saves the energy required for the extraction process of steaming for 15 min used for other media like PDA, soil extract agar, etc. Furthermore, the bean pods after sporulation can be directly used for the subculturing, and inoculation for the enzyme production experiment. 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 invention in any manner. Example 1 Myrothecium verrucaria was maintained on bean pod agar. For the preparation of inoculum, the inoculated bean pod agar slants were incubated for 7 days at 28° C and inoculated in the medium (Ml, composition as stated below) and incubated for 7 days at 28° C. The fungal biomass (5% v/v) grown in Ml medium was used to inoculate fresh Ml medium for the production of the mycolytic enzyme preparation of M verrucaria in shake flasks (500 ml capacity) containing 100 ml of the medium at 28°C for 7 days. The Ml medium contains chitin (0.5%) as a sole carbon source and ammonium sulfate (0.14%) as an inorganic nitrogen source. Other components of the production medium are: =(Table Removed) *Trace metal solution 0.1 ml contains (mg/ml). FeSO4, 5.0; ZnSO4, 1.4; MnSO4, 1.6; CoCl2.2.0). The pH of the medium was adjusted to 6.0 before autoclaving. The fungal cell wall degrading activities viz. chitinase, chitosanase, ß-l,3-glucanase, mannanase, α- and ß-mannosidase were measured in 0.2 M potassium phosphate buffer, pH 5.8 using conventional methods. The substrates used to measure activities were: phosphoric acid swollen chitin (ASC) or chitosan (ASCh) for total chitinase or chitosanase activities, respectively, for ß-l,3-glucanase activity laminarin, yeast mannan for mannanase activity and p-nitrophenyl-α-mannoside and p-nitrophenyl-ß-mannoside α- and ß-mannosidase activities , respectively. Proteinase activity was estimated using casein as a substrate in 0.2 M K- PO4 buffer, pH 7.0. Table 2 Mycolytic enzyme activities in M. verrucaria culture filtrate after 7 d. (Table Removed) The chitinase activity of the mycolytic enzyme preparation was also measured on different substrates to evaluate the nature of complete chitinase complex. The other substrates used were: Ethylene glycol chitin for chitinase that degrade chitin polymer randomly, /?-nitrophenyl-N-acetylglucosaminide for N-acetylglucosaminidase (an enzyme that degrade dimer of N-acetylglocusamine). The activities are given in Table 3. Table 3 (Table Removed) As a prerequisite for the isolation of protoplasts the efficiency of the mycolytic enzyme preparation was monitored using fungal mycelia degradation of three species, namely, Sclerotium rolfsii, Myrothecmm verrucaria and Fusarium species. The Table 3 shows the % hydrolysis of cell walls with the mycolytic enzymes. The % hydrolysis is calculated by estimating total sugars produced from 5 mg (dry weight) of cell walls using Somogyi's colorimetric method and N-acetylglucosamine as a standard: Amount of reducing sugars 203 %Hydrolysis= X X 100 Amount of substrate 221 (Vyas and Deshpande J.Gen Appl. Microbiol. 37: 267-275, 1991) Table 4 (Table Removed) To isolate protoplasts from taxonomically different fungi, namely Saccharomyces cerevisiae, Sclerotium rolfsii and Myrothecium verrucaria, the fungal biomass grown in MGYP (malt extract, 0.3%; glucose, 1%; yeast extract, 0.3%; peptone, 0.5%) medium was harvested after 20 h, washed twice with distilled water and then with 0.2 M K- PC>4 buffer, pH 5.8 containing 0.6 M sorbitol forM verrucaria, 0.6 M KC1 for S. rolfsii, 0.8 M NaCl for S. cerevisiae. The water was removed by pressing the biomass in between the filter papers. The damped fungal biomass (250 mg) was incubated with 5 ml of crude enzyme preparation on a reciprocating shaker (75 strokes/ min) at 28°C for 4 h. Subsequently, the protoplast suspension was filtered through sintered glass filter (Jena, porosity Gl, pre-saturated with the stabilised buffer) to remove cell debris. The protoplasts were then collected by centrifugation at 500 x g for 30 min, gently re- suspended and washed twice with stabilised buffer. The viability of protoplasts was tested by eosin staining. The fungi contain chitin, N-acetylglucosarnine polymer, as a main structural component in their cell walls. The concentration of chitin varies significantly in different fungi. The fore mentioned organisms have different cell wall compositions. For instance, cell wall of S. rolfsii is predominantly chitinous, while that of S. cerevisiae contains more percentage of glucan and mannan. M verntcaria was used as the third candidate to evaluate autolytic (hydrolysing own cell wall) potential of the enzyme (Table 5). Table 5 Protoplast isolation using M verntcaria mycolytic enzymes in 4 h. (Table Removed) Advantages I. The method is useful for highest biomass production of deuteromycetous fungus, as fungus sporulates more on bean pods. The bean pods can be used directly without extraction of the contents that saves the energy required for the extraction process. Furthermore, the bean pods after sporulation can be directly used for the subculturing, and inoculation for the enzyme production experiment. 2. The invention also provides enhanced extracellular enzyme production, and hence cost effective for production of total mycolytic enzymes. 3. The total mycolytic enzymes produced by the method that include chitinase, chitosanase, mannanase, α- and ß-mannosidase in addition to other enzymes reported earlier is effective for fungal mycelia degradation and for isolation of protoplasts from taxonomically different fungi. We Claim: 1. A process for the preparation of mycolytic enzymes containing chitinase which comprises; growing Myrothecium sp. in bean pod till it reaches to speculation stage, growing in a conventional fermentation medium having carbon and nitrogen sources, minor ingredients and traced metal solution such as herein described for a period of at least 7 days in a known manner, separating the mycelia by conventional methods to obtain a mixture of mycolytic enzymes in the separated liquid, separating the product by known methods. 2. A process as claimed in claim 1 wherein, the bean pods used is sterilized in solid or macerated form or supplemented with agar. 3. A process as claimed in claims 1 to 2 wherein, carbon source used is selected from chitin, chitosan, glucosamine, glucose or cellulose in concentration ranging from 0.2-1.0%. 4. A process as claimed in claims 1 to 3 wherein, the nitrogen source is ammonium sulfate, peptone, or urea or mixtures thereof and concentration used as in the range of 0.14-0.7%. 5. A process as claimed in claims 1 to 4 wherein, minor ingredients used are (g/100ml) KH2PO4 0.2-0.5, K2HPO4 0.05-0.2, MgSO4 0.035-0.08, NaCl 0.05-0.1, CaCl2 0.04- 0.08, Yeast extract 0.05-0.1, Oxgall 0.05-0.1, Urea 0.01-0.05, Peptone 0.02- 1.0,trace metal solution 0.1 ml. 6. A process as claimed in claims 1 to 5 wherein, trace metal solution used consists of (mg/ml): FeSO4, 5.0; ZnSO4,1.4 MnSO4, 1.6; CoCl2, 2.0). 7. A process for the preparation of mycolytic enzymes containing chitinase as substantially described herein before with reference to the examples contained therein.

Documents:

110-del-2001-abstract.pdf

110-del-2001-claims.pdf

110-del-2001-correspondence-others.pdf

110-del-2001-correspondence-po.pdf

110-del-2001-description (complete).pdf

110-del-2001-form-1.pdf

110-del-2001-form-19.pdf

110-del-2001-form-2.pdf

110-del-2001-form-3.pdf