CS230889B1 - Method of producing alpha amylase enzyme - Google Patents

Abstract

Method for producing alpha amylase enzyme. The invention relates to the field of microbiology, chemistry and biochemistry of carbohydrates, food and pharmaceutical chemistry. The aim of the invention is to obtain a new alpha amylase enzyme capable of hydrolytically splitting alpha-1,4-glucans of starch into maltose oligosaccharides with a high proportion of maltose and maltotriose. The above purpose is achieved by using the microscopic fungus Blaštobotrys proliferans from the genus Blastobotrys Klopotek, order Moniliaceae, genus Moniiiales, triad Deuteromycates, cultivated on suitable nutrient media. The alpha amylase enzyme can be obtained with a relatively high specific activity of up to 4.0 nkat/mg protein and suitable properties, especially for a high proportion of maltose and maltotriose in starch hydrolysate. The invention has application in microbiology, biochemistry and chemistry of carbohydrates, food and pharmaceutical industries.

Description

230889 3

The present invention relates to a process for the production of alpha amylase enzyme (alpha-1,4-glucan glucan thyridolase EC 3.2.1.1) by means of the microscopic Blastobotrys proliferans fungus by culturing on suitable stages.

For industrial production. alpha-amylases are most commonly used by microorganisms: Bacillus subtilis, Bacillus amylo-liqueficiens, Streptomyces aureofaciens, Aspergillus niger, Aspergillus oryzae, and the like. There are, however, other microorganisms in which the ability to produce alpha amylase has not been known to date. Using the gel method (A.No. 18-059) to test for microalgae amylolytic activity, we have found significant production of alpha amylolytic activity by microscopic blastobotrys proliferans.

Blastobotrys proliferans is a newly written species of the genus Blastobotrys Klopotek of the family Mo-niliaceae of the genus Moniliales, class Deuteromyces cetes [Marvan L .: Two new Blastobotrysspecies. Transactions of the British Mycological Society 68, 217 (1976)] isolated from parsley nuts (Bertholletia excelsa) from a damaged core. The production of polysaccharide hydrolases by said species of Blastobotrysproliferans is unknown.

SUMMARY OF THE INVENTION It is an object of the present invention to cultivate the strain Blastobotrys proliferans CCMF-493 deposited as CBS 522-75 in the Netherlands and ATCC 34,216 in a collection of US type lumps on a nutrient containing 1-> 4. -D-glucan as an enzyme inducer at 37 ° C and a pH of 4.8 for 48-120 hours produces an alpha amylase enzyme, which blacks in crude state by evaporation of the growth method in a hardened state by precipitation with ammonium sulfate or organic solvents , preferably ethanol and acetone. For example, affinity methods can be used to further purify the novel enzyme.

Alternatively, said Blastobotrys proliferan strain is used in processes for the production of alpha amylase, for example, the charification of starch polysaccharides. The alpha amylase obtained, especially in the pure state, is useful in the preparation of alternative sweeteners, with respect to the high proportion of maltose and maltotriose in the enzymatic hydrolyzate (up to 55%). This fact favors the use of the enzyme product where high glucose content in the hydrolyzate is not desirable (diet diet for phenylketonurics, bombonian syrup). The preferred hydrolyzate composition predetermines enzymatic hydrolysis using the Blastobotrys proliferans alpha-amylase to prepare the maltose-type rare oligosaccharides. On the other hand, the feminine preparation also contains alpha glucosidase (alpha-glucoside glucohydrolase EC 3.2.1.20) and therefore uncleaned enzyme can be utilized for saccharification [1 -> 4) -? - D-glucans starch to glucose. Example 1

Blastobotrys proliferans CCMF-493 is grown on a growth pod containing 2 g of gel-prepared amylase (AO number 186 059) immersed in half-height into the Nitrogen Base Growth Substrate (Oxoid), pH 4.8, 0.6 g / 100 ml and culturing the surface for 48 hours at 37 [deg.] C., when gel flow occurs. The accumulated bio-meat (0.07 g) was used to ferment the base containing 22 g of corn extract and 2 g of potato starch and 0.5 g of KH 2 PO 4 per liter of water (pH 4.8) for 72 hours. Po72. hours, the Blastobotrys proliferate biomass is filtered off and the crude alpha amylase enzyme is recovered by evaporation of the fermentation batch in a vacuum evaporator (total activity of 1.8nkat / l, specific activity 2.6 nkat / 1 mg protein). Example 2

The procedure of Example 1 except that gluten baking (18%), 3 g starch and 0.5 g KH 2 PO 4 in 1 L water (pH 4.8) are used as the fermentation stage, with the fermentation running for 120 hours. The crude alpha amylase enzyme has a total activity of 2.2, ukat / l and a specific activity of 3.5 nkat / 1 mg protein. Example 3

The procedure of Example 1 except that the obtained fermentation is treated by ethanol precipitation in a volume ratio of ethanol to fermentation point 2: 1. After drying, the obtained protiate is processed by affinity method (AO No. 157 955) and the refined alpha amylase has activity 1 , 6, ukat and a specific activity of 4.0 nkat / mg, with no more than 0.05 nkat α-glucosidase activity. Example 4

The procedure of Example 3 except that acetone is used to precipitate the enzyme. The recovered alpha amylase obtained has an activity as described in Example 3. Example 5

The procedure of Example 3, except that ammonium sulphate is used to precipitate at a saturation of 0.7. The recovered alpha amylase has activity and specific activity as described in Example 3.

The present invention has utility in the preparation of amylase specific properties for baseline, precious sugars, and / or precious sugars. preparation of mixed sugars for food and pharmaceutical purposes.

Claims (1)

230889 5 6 Blastobotrysproliferans fungus producing enzyme amylase by microscopy, characterized in that the culture is Blastobotrys proliferans CCMF-493 naive, containing a (1- * 4) -αD-glucane enzyme inducer at 37 ° C and pH value 4.8 for 48 to 120 hours and enamase of alpha amylase is recovered in the crude stock by evaporation of the growth soil, optionally in pre-purified state by precipitation with ammonium sulfate, or organic solvents, preferably with ethanol or acetone, and with pure affinity methods.