CS208239B1 - Method for producing cellulose-cleaving enzymes - Google Patents

Abstract

The invention relates to the production of cellulose-digesting enzymes by some fungi of the genus Trichoderma. The essence of the invention lies in the fact that strains of the genus Trichoderma are individually or in a mixture pre-cultivated on a mineral nutrient medium containing ^(1->4) glucan as an enzyme inducer at a temperature of 30 °C, for 24 hours at pH = 5 and subsequently inoculated on a production medium with cellulose, nitrogen in the form of ammonium salts and phosphate pH 4.2 to 6.5, while cellulose is produced for 120 hours at a temperature of 20 to 40 °C and isolated by evaporation of the growth medium, or by precipitation with ammonium sulfate or an organic solvent. Cellulase-type enzymes are useful in the process of cellulose saccharification for the study of protoplasts of bacteria and plants and for the hydrolytic decomposition of wood waste.

Description

The invention relates to a method for producing cellulose-digesting enzymes using certain fungi of the genus Trichoderma.

As producers of cellulase, 3 species are used within the genus Trichoderma, in addition to Trichoderma viride, Trichoderma koningii and Trichoderma lignorum (US patent δ. 3398055} Brigitte Norkvans Physiol. Plantarum 16 (1963) 1; T. Iwaeaki, K. Hayaahi, M. Funatsu (1964), J. Biochem. (Tokyo) 2%, 209-12} Lonanok A. G., Shklyar B. Kh. (1970) Veetei Akad. Nauk, Balarua SSR, Ser. Biyal. Navuk £, 58-61). Although in some isolates, especially Trichoderma viride, the specific activity of cellulase production is relatively high (up to 0.4 U/mg), the disadvantage is the relatively long lag phase preceding the formation of cellulase, wide variability of properties and the decrease in production capabilities during storage of productive strains.

The above-mentioned shortcomings are eliminated by the process according to the invention, the essence of which lies in the fact that strains of the genus Trichoderma:

Trichoderma harzianum CCM F-340 H long arms atum CCM F-342 M harzianum CCM F-470 M etc. CCM F-521 M sp. CCM F-522 M hamatum CCM F-541 M sp. CCM F-556 H aureovirus CCM F-559 M longibrechiates CCM F-56© saturnisporum CCM F-561 M sp. CCM F-571 H pilliferum CCM F-573 W polysporum CCM F-542 N polysporum CCM F-572 N sp. CCM F-591 W sp. CCM F-592 w sp. CCM F-593 M pseudokoningii CCM F-563

are individually pre-cultured weakly in a mixture on a mineral nutrient medium containing β(1—»4) glucan as an enzyme inducer at a temperature of 30 °C, for 24 hours at pH 5 and subsequently inoculated onto a production medium with cellulose, nitrogen in the form of ammonium salts and phosphate pH 4.2 to 6.5, whereby the cellulase is produced for 120 hours at a temperature of 20 to 40 °C and isolated by evaporation of the nutrient medium, or by precipitation with ammonium sulfate or an organic solvent and preferably ethanol.

The mentioned strains of the genus Trichoderma used for the production of cellulase are stored in the Czechoslovak Collection of Microorganisms, University <J. B. Purkyně, Brno, Obránců míru 10.

The advantage of the above-mentioned cellulase production method is the high specific activity of the produced cellulase enzyme system, in total up to 0.74 U/mg, and the stability of the mixed culture after 208 239

6a· storage· in a lyophilized state.

The practical use of the above procedure lies in the fact that the cellulase system produced into the culture medium can be isolated by a simple method. From the macromolecular substrate, oligosaccharides of the cellobiose type are formed. Cellulase type enzymes are usable in the isolated state in the process of cellulose acharification and for the study of bacterial and yeast protoplasts. Another possibility of using this property of the above strains of the genus Trichoderma is in the hydrolytic decomposition of wood waste to form an enzymatic hydrolysate that can be utilized in the next stage in the accumulation of nutritionally valuable protein by cultivating yeasts, yeast-like microorganisms.

Example 1

From the washed gel of cross-linked hydroxyethylcellulose according to the author's patent no.

183 169 triangular profiles with a side of 4 cm are cut, placed in a Pstri dish and covered with a solution of Czapek - Dox culture medium; pH « 5 (with hydroxyethyl cellulose gel as the only carbon source) without agar and sucrose so that half the thickness of the gel protrudes above the surface of the culture medium. After sterilization at 100 °C, Trichoderma harzianum CCM F - 340 cultures are inoculated on the surface of the gel and cultivated at 30 °C. Cellulose gel liquefied within 24 hours, together with 8 propagated cultured strains of Trichoderma harzianum CCM F - 340 (0.24 g) was used as inoculum for production feed (2 l) from waste hydroxyethyl cellulose wadding (150 g) with 4 g ammonium sulfate and 2 g potassium dihydrogen phosphate (pH = 4.5). Cultivation was carried out for 120 hours at 20 °C. After the end of the cultivation, the biomass was filtered off and the filtrate was concentrated on an evaporator. A crude enzyme preparation with a cellulase activity of 37 U and a specific activity of 0.62 U/mg was obtained.

Example 2

-The procedure was as in Example 1, except that the strain Trichoderma polyaporum CCM F - 542 was used, the pH used according to Example 1 was adjusted to pH 6.0 and the cultivation was carried out at 30 °C for 120 hours. The enzyme preparation had an activity of 42 U and a specific activity of 0.29 U/mg.

Example 3

The procedure was the same as in Example 1, except that instead of the strains mentioned, a mixed culture of the strains Trichoderma karzanium CCM F - 470, Trichoderma hamatum CCM F - 541 and Trichoderma piluliferum CCM F - 573 was used. The pH used according to Example 1 was adjusted to pH 6.5 and the cultivation was carried out at 40 °C for 120 hours. The enzyme preparation had an activity of 42 U and a specific activity of 0.74 U/mg.

Claims (1)

It is practiced that the cellulase system produced by the culture medium is isolated by a simple method. Cellobiose-type oligosaccharides are formed from the macromolecular substrate. Cellulase enzymes can be used in the isolated cellulose saccharification process to study bacterial protoplasts and celluloses. A further possibility to use this property of these Trichoderma strains is to hydrolyze the wood waste to form an enzymatic hydrolyzate utilizable at the third stage of protein buildup. yeast cultivation, respectively. kva-sinkovýeh microorganisms. EXAMPLE 1 Triangular profiles of 4 cm are cut out of the washed crosslinked hydroxyethylcellulose gel according to the certificate No. 183 169, placed in a Petri dish and glaze with a solution of the culture medium Czapek-Dox; pH 5 (with a hydroxyethylcellulose gel and a single carbon source) without agar and sucrose, so that the half-pitch of the gel extends above the culture medium. After sterilization at 100 ° C, the Trichemerma harzianum CCM F-340 is inoculated on the surface of the culture gel and cultured at 30 ° C. A cellulosic gel flowing over a period of 24 hours, together with an expanded bred strain of Trichoderma harzianum CCM F-340 (0.24 g), was used as inoculum for the production batch (2 1) from the hydroxyethylcellulose waste (150 g) with 4 g of ammonium sulfate and 2 g of potassium dihydrogen phosphate (pH = 4.5). Cultivation was carried out for 120 hours at 20 ° C. Upon completion of the culture, the biomass was filtered off and the filtrate was concentrated to an evaporator. A crude enzyme preparation was obtained with a cellulase activity of 37 U and a specific activity of 0.62 U / mg. Example 2 - The procedure of Example 1 was followed except that Trichoderma poly-sporum strain CCM F-542 was used as described in Example 1 and was adjusted to pH 6.0 and cultured at 30 ° C for 120 hours. The enzyme preparation had an activity of 42 U and a specific activity of 0.29 U / mg. EXAMPLE 3 The procedure of Example 1 was followed except that a mixed culture of Trichoderma karzanium CCM F-470, Trichoderma hamatumCCM F-541 and Trichoderma piluliferum CCM F-573 was used in place of the above strains. adjusted to pH 6.5 and cultured at 40 ° C for 120 hours. The enzyme preparation had an activity of 42 U and a specific activity of 0.74 U / mg. SUMMARY OF THE INVENTION A process for producing cellulose-cleaving enzymes, characterized in that the Trichoderma strain: 208,239 3 Trichoderma harzianum CCM F-340 M CCM F-342 M harzianum CCM F-470 M ep. CCU F - 521 M sp. CCU F - 522 M hamatum CCM F - 541 H sp. CCM F - 556 M aureoviride CCM F - 559 N longibrachiatum CCM F - 560 M saturnisporum CCM F - 561 M sp. CCU F - 571 N pipuliferum CCM F - 573 M polysporum CCM F - 542 M polysporum CCM F - 572 M sp. CCM F - 591 M sp. CCM F - 592 M sp. CCM F - 593 M peeudoconjugation CCM F - 563 is pre - cultured individually or on a mineral nutrient feed containing (J - 1 - 4) glucan as an enzyme inducer at 30 ° C for 24 hours, at pH 5 - 0 and are subsequently transferred to the cellulose and cellulose, ammonium salt and phosphate binder, pH 4.2 to 6.5, wherein the cellulase is produced for 120 hours at 20 to 40 ° C and isolated by evaporation of the growth medium, optionally by precipitation with ammonium sulfate or organic solvents. preferably ethanol. Printed by Jihočeské tiskárny, n. P., České Budějoviceprovoz 11 Český Krumlov Price: 2,40 Kčs