CS230887B1 - The method of producing ,41,4-glucan hydrolase using the microscopic fungus Blastobotrys proliferans - Google Patents

Abstract

Method for producing β-1,4-glucan glucan hydrolase using the microscopic fungus Blastobotrys proliferans. The invention relates to the field of microbiology, chemistry and biochemistry of carbohydrates. The purpose of the invention is to obtain β-1,4-glucan glucan hydrolase (cellulase) capable of hydrolytically cleaving not only cellulose and its synthetic derivatives, but also the native derivative tamarind. The above effect is achieved using the microscopic fungus Blastobotrys proliferans of the genus Blastobotrys Klopotek of the order Moniliaceae, of the genus Moniliales of the class Deuteromycetes cultivated on suitable nutrient media. β-1,4-glucan glucan hydrolase can be obtained with a relatively high specific activity of 11 to 15 nkat/mg biomass and suitable properties, especially with regard to the high presence of the rare oligosaccharide cellotriose in the enzymatic hydrolysate (up to 15%). The invention has applications in microbiology, biochemistry and carbohydrate chemistry, wherever a simple method of processing agricultural waste is involved.

Description

The invention relates to a method for producing the enzyme β-glucan glucanohydrolase (EC 3.2.1.4) by means of a microscopic fungus Blastobotrys proliferans grown on suitable nutrient platforms.

The following microorganisms are most commonly used for industrial production of [beta] -glucan glucanohydrolase: Trichoderma viride, Trichoderma coningia, Neurospora crassa and others.

However, there are other microorganisms which have not previously been known to have the ability to produce β-glucan glucanohydrolase. Using gel meitode (A.O. No. 133 169) testing microorganisms for cellulase activity during culture of microorganisms, we found significant production of cellulytic activity by microscopic fungus Blastobotrys proliferans.

Blastobotrys proliferans is a newly described species of the genus Blastobotrys Klopotek of Moniliaceae, of the genus Moniliales, of the Deuteromycetes class [Marvanová L .: Two new Blastobotrys species. Transactions of the British Mycological Society 66, 217 (1976)], isolated from a damaged nucleus (Bertholletia excelsa), the production of polysaccharide hydrolase by said Blastobotrys proliferans is unknown.

It is an object of the present invention to cultivate the strain Blastobotrys proliferans CCMF-493 deposited as CBS 522.75 in the Netherlands, respectively. ATCC 34 216 - Collection of US type cultures on cellulose containing cellulose or its derivatives (hydroxyethyl, carboxymethyl, hydroxypropyl, tamarind), at 37 ° C for 48 to 120 hours and pH 4.8 producing the enzyme The 1,4-glucan glucanohydrolase (EC 3.2.1.4), which is obtained in the raw state by evaporation of the growth stage, is purified in the purified state by precipitation with ammonium sulfate (67% saturation), ethanol or acetone (1: 1).

The advantage of the process is, in particular, that the preparation of β-1,4-glucan glucanohydrolase is very simple, it is possible to use a part of the cultivation stage and unpurified native substrates containing besides cellulose also other polysaccharides (hemicelluloses, acidic polysaccharides, lichenan etc.). in addition to the enzymes of the cellulolytic complex (0-1,4-glucan glucanohydrolase, β-glucosidase, celobiohydrolase EC 3.2.1-), it also produces onzyme systems capable of cleaving the above polysaccharides. A further advantage is the high yield of -1-1,4-glucan glucanhydrolase activity as well as the properties of the isolated enzyme, in particular its ability to produce cellulose during the hydrolysis of the cellulosic material (up to 15% of the product mixture). Important property that differs said / 3-1.4-glucan glucanohydrolases from other heretofore described ¹ / ϊ-1,4-glucan glucanohydrolase is preferred nešpecifita - native ability to hydrolyze cellulose analogs, tamarind polysaccharide, isolated from nuts tamarinds (Tamarindus indica) as a result of which said polysaccharide can be used for nutritional purposes.

Example 1

Blastobotrys proliferans CCMF-493 is propagated on a growth broth containing 2 g of a hydroxyethylcellulose gel prepared by fusing (AO No. 183 169) immersed halfway in 20 ml Yeast Nitrogen Base (pH 4.8) growth broth (0.6 g / 100 ml) ) and cultured superficially for 3 days at 37 ° C, when the gel is liquefied. The accumulated biomass of Blastobotrys proliferans (0.18 gj is used as soil inoculum containing 20 g of corn steep liquor, 10 g of hydroxyethylcellulose per liter of soil (pH 4.8) and cultivated for 120 hours. After 120 hours of cultivation, Blastobotrys proliferans biomass The resulting enzyme preparation has a β 1,4-glucan glucanohydrolase activity of 0.33 µz at a production specific activity of Blastobotrys proliferans 11 nkat / mg biomass.

Example 2

The procedure of Example 1, except that a gel prepared from a cross-linked tamarind polysaccharide (AO No. 186 059) is used instead of a gel prepared from hydroxyethylcellulose and the culture pod (11) contains 25 g of a tamarind polysaccharide suspension in place of the corn leach and carboxymethyl cellulose. 2 g of ammonium sulfate and 1 g of potassium dihydrophosphate. Cultivation is carried out for 48 hours at 37 ° C. After the cultivation was complete, the biomass was filtered off and partially purified [1,1,4-glucan glucanohydrolase was obtained by precipitation with ammonium sulfate to a saturation degree of 67%. The obtained enzyme preparation had an activity of 0.29 µ cat and a specific activity of 13 nkat / mg protein.

Example 3

The procedure of Example 1 except that β-1,4-glucan glucanohydrolase was obtained from the culture stage after removal of the biomass by precipitation with ethanol in a volume ratio of 1.1 to the remainder of the culture stage. (3-1,4-Glucan glucanohydrolase activity) of 0.42 µl was obtained with a specific activity of 15 nkat / mg protein.

Example 4

The procedure of Example 1, except that a cross-linked hydroxypropyl cellulose gel was prepared instead of a cross-linked hydroxyethyl cellulose gel for inoculum preparation. The activity of the obtained enzyme preparation was as described in Example 1.

WITH

The invention has utility both in the preparation of 0-1,4-glucan glucanohydrolase having advantageous properties (high cellulose content in cellulose hydrolyzate), in the processing of tamarind polysaccharide, respectively. in the recovery of various wood and agricultural products containing polysaccharides.

Claims (1)

The present invention has utility in the preparation of ,41,4-glucan glucanolase with advantageous properties (high proportion of celotriosis in cellulosic hydrolyzate), in the treatment of 6 tamarind polysaccharides, respectively. for utilizing various wood and agricultural products containing polysaccharides. SUBJECT A method for producing the 1,4-glucanglucanohydrolase enzyme by Blasitobotrys proliferans microscopy, which cultures a Blastobotrys prodersans CCMF-493 strain on cellulose-containing cellulose or hydroxyethyl, carboxymethyl, hydroxypropyl derivatives or native derivative thereof cellulose polysaccharide tamorind, DISCHARGE at 37 ° C and pH 4.8 for 48-120 hours, and the enzyme β-1,4-glucan glucan hydrolase is recovered in the crude by evaporation of the growth soil, optionally in precipitated sulfate. ammonium or organic solvents, preferably ethanol or acetone.