CS230890B1 - A method for producing ε-glucosidase enzyme by microscopic fungi of the genus Blastobotrys - Google Patents

Abstract

Method for producing /J-glucosidase using microscopic fungi of the genus Blastobotrys. The invention relates to the field of microbiology, chemistry and biochemistry of carbohydrates. The purpose of the invention is to describe the production of the enzyme jS-iglucosidase, as one representative of the cellulolytic complex, or in a mixture with /?-1,4-glucan glucanohydrolase (cellulose Cx) and cellobiohydrolase (cellulose Ci). The stated purpose is achieved using microscopic fungi of the genus Blastobotrys, namely Blastobotrys proliferans, Blastobotrys aristata and Blastobotris nivea individually or in a mixture thereof on suitable nutrient media. jS-glucohydrolase can be obtained with a relatively high specific activity of up to 8 nkat/mg, including with the help of a native cellulose analogue of tamarind polysaccharide as a waste product (Cuba, Caribbean region). The invention has wide application in microbiology, chemistry and biochemistry of carbohydrates, but also in analytical chemistry and wherever a simple method of processing wood and agricultural waste is involved.

Description

The invention relates to a method of producing an enzyme (S-glucoside glucohydrolase ( _{i (} 3-glucosidase, EC 3.2.1.21)) by microscopic fungi of the genus Blastobotrys grown on suitable nutrient media.

The following microorganisms are most commonly used for industrial production of .gamma.-glucosidase: Aspergillus oryzae, Merulius lachrymans (G. de Stevens in Methods in Enzymology, Volume 1, SP Collowick and NO Kaplan, Acad. Press. New York 1955, pp. 173-178). ). However, there are other microorganisms which have not been known to date to produce the .beta.-glucosidase enzyme. Using the gel method (A.O. No. 183 169), we found significant β-glucosidase production in the 3 species of Blastobotrys described, namely Blastobotrys aristata, Blastobotrys proliferans and Blastobotrys nivea.

The species Blastobotrys proliferans is a newly described species of the genus Blastobotrys isolated from berry nuts (Bertholletia excelsa) from a damaged kernel, while the species Blastobotrys aristata was isolated from a sample of moldy plaster supplied by the welfare staff. from Bítov Castle [both new species are described by Marvanová L .: Two new Blastobotrys species. Transactions ot the British Mycological Society 66, 217 (1976). The species Blastobotrys nivea isolated from compost was described by von Klopotek A .: Blastobotrys nivea gen. nov., sp. nov., Archives of Microbiology 58, 92 (1967). The genus Blastobotrys belongs to the family Moniliaceae, genus Moniliales, class Denteromycetes. There is nothing known in the literature about the production of polysaccharide hydrolase by said species of the genus Blastobotrys.

The principle of the invention is that by culturing the following three strains of the genus Blastobotrys:

Blastobotrys proliferans. Blastobotrys aristata Blastobotrys nivea

CCMF - 493 CCMF - 410 CCMF - 433 individually or mixtures thereof on a culture medium containing cellobiose, cellulose or its hydroxyethyl, hydroxypropyl, carboxymethyl derivatives or native cellulose derivative of tamarind polysaccharide at 25 to 37 ° C and pH 4,8 for 72 For up to 120 hours, the enzyme .gamma.-glucosidase, which is obtained in the raw state by evaporation of the growth soil, is optionally partially tempered by precipitation with ammonium sulfate or organic solvents, preferably ethanol and acetone.

To further purify the crude β-glucosidase enzyme after dialysis, for example, adsorption of 6-glucosidase from a mixture of proteins to aluminum metahydroxyd and subsequent enzyme liberation with Na 2 CO 3 as described by G. de Stevens in the above reference can be used. The advantage of this procedure is that the preparation of (S-glucosidase is very simple and unpurified native substrates readily available as various types of agricultural waste can be used as part of the culture medium. In case the whole enzyme preparation is to contain the whole complex cellulolytic enzymes, combinations with Blastobotrys proliferans are preferred, since said strain also produces, in addition to β-glucosidase activity, cellulases C ^x and G β (β-1,4-glucan glucanohydrolase EC 3.2.1.4 and cellobiohydrolase EC 3.2.1.- ).

Another advantage is the high yield of β-glucosidase activity. On the other hand, when there is a requirement for pure β-glucosidase enzyme activity without concomitant cellulolytic enzymes, said preparation can be obtained simply and without concomitant cellulolytic enzymes by the proposed new process using the strains Blastobotrya aristata and Blastobotrys nivea individually or in a mixture thereof. In addition to the β-glucosidase activity and the enzymes capable of cleaving xylcza, said strains of the genus Blastobotrys also produce a new enzyme preparation obtained by concentrating the culture medium after cultivation of said representatives of the genus Blastobotrys can also be used to saccharify tamarind polysaccharide (Tamarindus indica).

Example 1

Blastobotrys proliferans CCMF-493, Blastobotrys nivea CCMF-433 and Blastobotrys aristata CCMF-410 are propagated on a growth medium containing 2 g of gel (prepared by cross-linking hydroxyethylcellulose according to AO No. 183 169) immersed in half-height in 20 ml of Yaest Nitrogen Base (0.6 g / 100 ml) at pH 4.8 and cultured on the surface for 3 days at 30 ° C, when the gel is run off The accumulated biomass of Blastobotrys is used as a mixed soil inoculum containing 20 g of corn steep liquor 5 g of carboxymethylcellulose (pH 4.8j per l of water and cultivation is continued for a further 2 days. Then the Blastobotrys biomass is filtered off and the crude enzyme / β-glucosidase is obtained by evaporating the culture medium on a vacuum evaporator (total activity 33 U, ie 0). 55, tick, specific activity of 12 nkat / mg protein.

Example 2

As inoculum, Blastobotrys aristata CCMF-410 and Blastobotrys nivea CCMF-433 are used which are propagated on soil containing 2 g of ammonium sulfate, 1 g of KH2PO4 and 5 g of cellobiose per liter (pH 4.8). After 120 hours of culture (25 ° C), the biomass is filtered off and the remaining culture broth is precipitated with ethanol (2 volumes, parts of ethanol per volume, part of culture broth).

Β-glucosidase 0.6 <at is precipitated in the sediment with a specific activity of 2.0 nkat / mg, which preparation does not contain cellulolytic activity ((3-1,4-glucan glucanohydrolase and cellobiohydrolase)).

Example 3

As described in Example 1, except that tamarind polysaccharide and Blastobotrys proliferans CCMF-493 pure culture were used instead of hydroxyethyl and carboxymethylcellulose. Cultivation is carried out at 37 ° C for 3 days. The crude thickened enzyme preparation, β-glu The invention has use in a basic microcosidase comprising 0.48 µm and a specific activity of 8 µg / mg containing 0.25 µg of β-1,4-glucan glucanohydrolase activity and 3 nkat of celluloid hydrolase activity.

The invention has utility in basic microbiological biochemical research and in the production of β-glucosidase as one of the important enzymes in the saccharification of polysaccharides of various wood and agricultural wastes.

Claims (1)

Example 3 As shown in Example 1, except that polysaccharide-tamarind is used instead of hydroxyethyl and carboxymethylcellulose and pure culture of Blas-tobotrys proliferans CCMF-493 is used as inoculum. Cultivation takes place at 37 ° C for 3 days.Surface thickened enzyme preparation, β-glu- The invention is intended to be used in the basic microcididase containing 0.48, ukat and the specific activity of the yukat / mg contains 0.25, ukat / 3 -1,4-glucan glucanohydrolase activity and 3-nitrocellohydrolase activity. The invention has utility in the basic micro-biological biochemical research and the production of β-glucosidase as one of the important enzymes in the saccharification of polysaccharides of various wood and agricultural wastes. OBJECT OF THE INVENTION A method of producing the enzyme β-glucosidase by the use of microscopic fungi of the genus Blasto-botrys, characterized in that the following strains of the genus Blastobotrys are cultivated: Blastobotrys proliferansBlastobotrys aristataBlastobotrys nivea individually or mixtures thereof CCMF-493CCMF-410CCMF-433 on a nutrient-containing cellulose, cellulose or its hydroxyethyl, hydroxypropyl, carboxymethyl derivative, or native cellulose derivative, tamarind at 25-37 ° C, and a pH of 4.8 for 72-120 hours, and the γ-glucosidase enzyme is recovered in the crude by evaporation of the growth stage, optionally in part ammonium or organic solvent, preferably ethanol or acetone.