CS205306B1 - Manufacturing process of alpha-amylase enzyme - Google Patents

Description

The invention aa relates to an epo-method of A-amylase enzyme production.

For the industrial production of the " t-amylase " enzyme, production methods that utilize certain types of bacteria e.g. Streptomycee aureofacies resp. Bacillus subtilis, or some species of Aspergillus and Penicillium. A disadvantage of the production of α-amylase in said strains is the disadvantageous ratio of maltose-type oligosaccharides to D-glucose as reaction products of the amylolytic reaction, respectively. difficulties in separating biomass from the culture medium.

These disadvantages are overcome by a process for the production of et-amylase on a liquid nutrient medium containing sources of assimilable carbon and nitrogen and mild nutrient salts, which is based on the fact that the strains

Trichoderma harianum CCM P - 340 « koningii CCM P - 10 w longibrachiatum CCM F - 342 « sp. CCM P-521 harzianum CCM P - 470 » sp. CCM P-522 • hamatum CCM P - 541 » vlride CCM F - 486 • viride CCM P - 534

205 306

208 300

Trichoderma sp. CCM F - 556 * aureoviride CCM F - 559 β longibrachlatum CCM P - 560 saturniaporum CCM P-561 w sp. CCM P-571 piluliforum CCM P - 573 polysporum CCM P - 542 N polysporum CCM F - 572 N sp. CCM P-591 * sp. CCM P - 592 » sp. CCM F - 593 W ps eudokoningii CCM F - 563 . * koningii CCM F - 341 H koningii CCM P - 544

they are cultivated singly or in a mixture on a nutrient medium containing 0.1 to 3 wt. % soluble starch at 20 to 40 ° C for 48 to 120 hours at pH 4.2 to 6.5, wherein the α-amylase is obtained by evaporating the growth soil or by precipitation with ammonium sulfate or by affinity purification on a cross-linked e6-1,4- <glucan.

The mentioned strains are stored in MS. Collection of Microorganisms, University of J. S, Purkyne, Brno, Obranov Peace 10.

The advantages of said α-amylase production method are the simple separation from the culture soil, the health safety of biomass over fungal fermentations and the relatively high specific production activity, which is up to 2.3 U / mg biomass. An advantage is also the use of biomass soperated from the production medium e.g. in the production of antibiotics.

Example 1

From the washed amylose gel / AO no. 186 059/1978 // cut triangular profiles of 4 cm side, place in a Petrl dish and water with a solution of Czepek-Dox cultivation soil with amylose gel as the only carbon source without agar and sucrose so that half of the gel protrudes above the surface of the culture solution. After sterilization at 100 ° C, a culture of Trichoderma harzianum CCM F-470 is inoculated on the gel surface and cultured at 37 ° C. The amylose gel liquefied over 16 hours together with the multiplied Trichoderma harzianum biomass (0.38 g) was used as a seed pellet (2 L) containing 30 g of corn starch, 2.3 g of ammonium sulfate and 2.0 g of potassium dihydrogen phosphate, adjusted to pH 5.3. Cultivation of Trichoderma harzianum was carried out for 116 hours at 18 ° C. After the cultivation process was complete, the Trichoderma harzianum biomass was filtered off and the filtrate was concentrated by evaporation. A crude enzyme preparation was obtained with an oC-amylase activity of 46 U and a specific activity of 0.116 U / mg.

Example 2

As in Example 1, except that the culture soil contains 1 g starch per liter of nutrient soil and is cultured at 40 ° C for 48 hours at pH ,5 6.5 and the filtrate obtained after separation of the Trichoderma harzianum biomass. CCM P - 340 is processed using

205 300 fused starch by affinity method. The purified enzyme etamylase obtained had a specific activity of 29.2 U / mg.

Example 3

The procedure was as in Example 1 except that a mixed culture of trichoderma viride CCM F-486, Trichoderma hamatum CCM F-541 and Trichoderma koningii CCM F-341 was cultured at 25 ° C for 96 hours at pH 5.0 s. the enzyme et-amylase was obtained from the production addition by precipitation with ammonium sulfate (700 g). The obtained crude enzyme preparation had a specific activity of 0.25 U / mg.

Claims (1)

OBJECT OF THE INVENTION A method for producing an α-amylase enzyme on a liquid nutrient broth containing sources of assimilable carbon and a mineral nutrient sol, characterized in that the strains harzianum CCM - 340 koningii CCM F-10 longibrachiatum CCM F - 342 sp. CCM F - 521 harzianum CCM F - 470 sp. CCM F - 522 hamatum CCM F - 541 viride CCM F - 486 viride CCM F - 534 sp. CCM F - 556 aureoviride CCM F - 559 longibrachiatum CCM F - 560 saturnisporum CCM F - 561 sp. CCM F - 571 piluliforum CCM F - 573 polysporum CCM F - 542 polysporum CCM ø - 572 sp. CCM F - 591 sp. CCM F - 592 ' sp. CCM F - 593 pseudokoningii CCM F - 563 koningii CCM F - 341 koningii CCM F-544 aa are cultivated singly or in a mixture on a nutrient supplement containing 0.1 to 3 wt. % soluble starch at 20 to 40 ° C for 48 to 120 hours at pH 4.2 to 6.5, wherein the etamylase is obtained by evaporating the growth soil or by precipitation with ammonium sulfate or by affinity purification not with crosslinked et-1,4 glucan.