CS205311B1 - Manufacturing process of enzymes of cellulose breaking - Google Patents

Description

The invention relates to a method for producing cellulose-cleaving enzymes by yeast organisms.

In particular, microscopic fungi such as Trichoderma viride and Trichoderma koningii are used as producers of the cellulase enzyme system. 3398055 /. Although in some isolates, especially Trichoderma viride, the specific activity of cellulase enzyme system production is relatively high (up to 0.4 U / mg), the disadvantage is the long lag phase prior to the cellulase enzyme system production, wide variability of properties, mycotoxin formation in some isolates and loss of production ability during preservation of production strains.

These disadvantages are overcome by the method of producing cellulose-cleaving enzymes, which is based on the fact that the strains

Leucosporidium scotii CCY 64-1-1

Leucosporidium capsuligenum CCY 64-2-1

CCY 64-2-2 CCY 64-5-1 are pre-cultured individually or in a mixture on a mineral nutrient broth containing β / 1 → 4 / glucan as enzyme inducer at 28 ° C for 72 hours at pH 5.9 and subsequent Inoculate to the production stage with cellulose, ammonium salt nitrogen and phosphate, pH 4,2 to 6,5, while, 205 311

₀ 205 311 cellulase was produced for 120 hours at a temperature of 20 to 40 C a growth p8dy isolated by evaporation, or by precipitation eiran ammonium or an organic solvent, preferably ethyl alcohol.

Leucosporidium strains used for cellulase production are deposited in MS. collection of yeasts, Institute of Chemical Technology 3AV, Bratislava, Dubravska cesta.

The advantage of this process is the high specific activity of the produced cellulosic enzyme complex, in total up to 0.54 U / mg, the stability of the mixed culture during cultivation and the possibility of utilizing nutritionally valuable yeast biomass for feeding.

Example 1

From the washed gel prepared from cross-linked hydroxyethylcellulose AO no. 183 169, cut 4 cm triangular profiles, and place them in a Petri dish and embed with a solution of Yeast Nitrogen Base (Oxoid) (0.6 g / L) with hydroxyethylcellulose gel as the sole carbon source. After sterilization at 100 ° C / 3 times for 1 hour /, a culture of Leucosporidium scottii CCY 64-1-1 is inoculated on the gel surface and cultured at 28 ° C. Cellulose gel liquefied over 72 hours together with multiplied L. scottii biomass (0.23 g) was used as a partially clarified inoculum on a production stage (2 L) from hydroxyethyl cellulose wadding waste (150 g) and 4 g ammonium sulfate and 2 g dihydrophosphate potassium (pH 5.9). Cultivation was carried out for 120 hours at 28 ° C. After the cultivation was complete, the biomass was centrifuged (2000 g, 5 minutes) and the supernatant was concentrated to a residue. A crude enzyme preparation was obtained with a cellulase activity of 58 U and a specific activity of 0.43 U / mg.

Example 2

As in Example 1, except that Leucosporidium capsuligenum CCY 64-2-2 was used instead of Leucosporidium scottii. The resulting crude enzyme preparation had an activity of 21 U and a specific activity of 0.53 U / mg.

Example 3

As in Example 1, except that 0.2% carboxymethylcellulose and 0.8% corn steep liquor are used in the production soil instead of hydroxyethylcellulose. The resulting european enzyme preparation had an activity of 62 U and a specific activity of 0.11 U / mg.

Example 4 as in Example 1, except that an enzyme preparation with a cellulose activity of 55 U and a specific activity of 0.92 U / mg was obtained by precipitating the enzyme 655 g of ammonium sulfate per 1 liter.

Example 5

As in Example 1, except that 4 strains of the genus were used for inoculation

Leucosporidium in the mixture, namely: Leucosporidium scottii CCY 64-1-1 and Leucosporidium capsuligenum CCY 64-2-1, CCY 64-2-2 and CCY 64-5-1. The crude enzyme preparation obtained had a 89 U cellulase activity and a specific production activity of 0.68 U / mg biomass.

205 311

Claims (1)

OF THE INVENTION Method for the production of cellulose-cleaving enzymes, characterized in that the strains Leucosporidium acottii CCY 64-1-1 capsuligenum CCY 64-2-1 CCY 64-2-2 CCY 64-5-1 are pre-cultured individually or in a mixture on a mineral nutrient broth containing (3/1 - * - 4) glucan as enzyme inducer at 28 ° C for 72 hours at pH 5.9 and subsequently inoculated to a production soil with cellulose, ammonium salt nitrogen and phosphate, pH 4.2 to 6.5, the cellulase being produced for 120 hours at 20 to 40 ° C. Isolated by evaporation of the growth soil, possibly by precipitation with sulfate with an ammonium or organic solvent, preferably ethyl alcohol.