CS230888B1 - Method of producing 3-mannanase using microscopic fungi of the genus Lemonniera - Google Patents

Abstract

Method for producing /3-1,4-mannan mannan hydrolase using microscopic fungi of the genus Lemonniera. The invention relates to the field of mycobiology, chemistry and biochemistry of carbohydrates and environmental protection. The purpose of the invention is to obtain /3-1,4-mannan mannan hydrolase (^-mannanase FC 3.2.1,78) capable of hydrolytically splitting ,0-1,4-mannan and /1-mannanase components of hemicelluloses. The above effect is achieved using microscopic fungi from the group of aquatic fungi of the genus Lemonniera, namely Lemonniera aquatica, Lemonniera centrosphaera, Lemonniera cornuta and Lemonniera terrestris cultivated on suitable nutrient media. ι/3-mannanase can be obtained with a relatively high specific activity (about 10 nkat/mg protein) and the ability to split not only pure 0-1,4-mannan but also the 0-mannan portion of hemicelluloses. The invention has applications in technical microbiology, carbohydrate biochemistry and wherever a simple method of processing wood waste is required.

Description

The invention relates to a process for the production of [beta] -1,4-mannan mannanase hydrolase ([beta] -mannanase, EC 3.2.1.78) by means of microscopic slides of the genus Lemonniera grown on suitable nutrient media.

The production of the β-mannanase enzyme is rarely described, although some microorganisms are known to have the ability to produce this enzyme. For example, the β-mannanase component is also contained in a complex hemicellulase enzyme system produced by microscopic fungi of the genus Rhizopus (product of Sigma), Aspergillus niger [Yamazaki, N, Sinner M, Dietrichs Η. H.: Holzforschung (1976, 30, 101) by Xanthomonas campestris (Dekker, R.F.H., Canda, G.P .: Arch. Microbiol. (1979 122, 297) and mucosa Physarum polycephalum (Fr. patent 2385 730)].

However, the ability to produce this enzyme is substantially more widespread than would correspond to a small number of publications restricted to a few microorganisms. Using gel. methods (A.O. No. 186 059) testing microorganisms for polysaccharide hydrolase endothelialising during culture, we found significant production of β-mannanase activity in aqueous hyphomycetes of the genus Lemooniera.

The genus Lemonniera belongs to the family Moniliaceae of the genus Moniliales, class Deuteromycetes. Representatives of this genus include. between water hyphomyces.

The essence of the invention is that

the following four Lemonnier, namely: strain the family Lemonniera aquatica CCMF - - 124 Lemonniera centrosphaera CCMF - - 149 Lemonniera cornuta CCMF - - 325 Lemonniera terrestris CCMF - - 125

individually or mixtures thereof on a broth containing pure, β-1,4-mannan or an insoluble derivative thereof prepared in a cross-linking reaction with 2-chloromethyl oxirane or a hemicellulose mixture of β-1,4-mannan-containing polysaccharides at a temperature of 15 to 25 ° C and pH 5.0-6.0, preferably pH 5.5 for 96-144 hours, produces the enzyme β-mannanase, which is obtained in the raw state by evaporation of the growth medium, optionally in a concentrated state by precipitation with ammonium sulfate or organic solvents, preferably ethanol and acetone.

An affinity method using fused mannan (ex Phytelephas macrocarpe) as an affinity ligand can be used to further purify the crude β-mannanase enzyme after dialysis.

The advantage of the new process is, in particular, that the preparation of β-mannanase is very simple and unpurified native galactomannan substrates readily available as various types of agricultural and wood waste can be used as part of the culture pod. Another advantage is the relatively high yield of β-mannanase activity, even at lower fermentation temperatures (20 ° C). Since these strains of the genus Lemonniera produce other hemicellulose and cellulolytic enzymes, as well as enzymes capable of oxidatively degrading lignin, in addition to the β-ananasase activity, these strains can also be used for complex utilization of wood mass, however under optimum conditions for their reproduction.

Example 1

Lemonniera aquatica CCMF-124, Lemonniera centrosphaera CCMF-149, Lemonniera cornuta CCMF-325, Lemonniera terrestris CCMF-125 are propagated on a growth gel containing 1.6 gels (prepared by splicing / 3-1,4-mannan of ivory plant (Phytelaphas macrocarpe) No. 186 059] immersed in a half-height in Yeast Nitrogen Base growth pod (0.6 g / 100 ml]] at pH 5.5 and cultured on the surface for 3 days at 20 ° C, when the gel spills The accumulated biomass of the Lemonniera species is used as a mixed pod inoculum containing 20 g of corn steep liquor, 5 g / 3-1,4-mannan (pH 6.0) per liter of water, and cultivated for a further 3 days at 20 ° C. The Lemonniera biomass is filtered off and the crude β-mannanase enzyme is obtained by evaporating the culture medium on a vacuum evaporator (total β-mannanase activity of 52 nkat, with a specific activity of 8.6 nkat / mg protein) Example 2. The Lemonniera strain is used as inoculum. aquatica CGMF-124 The powder is administered containing 1.6 g of gelled [beta] - 1,4 * mannan (A. O. č. 186 059] immersed in a half-height in a Yaest Nitrogen Base growth pod (0.6 g / 100 mL), pH 5, and cultured for 2 days at 15 ° C, when the gel spills. The accumulated biomass of Lemonniera aquatica is used as an inoculum of fodder containing corn extract (20 g) and 5 g guar galactomannan (Cyamoqsis tetragonoloba L., Fam. Leguminosae) per liter of water (pH 5.5) and cultivated for a further 2 days at temperature 20 ° C. Thereafter, the Lemonniera aquastica biomass is filtered off and the β-mannanase enzyme is obtained in a partially refined state by precipitation with ammonium sulfate (saturation degree 0.65) Total activity (3-mannanase 65 nkat with specific activity 9.2 nkat) mg protein Example 3

As described in Example 1 except that the strain Lemonniera terrestris CCMF-125 is used instead of Lemonniera aquatica. Cultivation is carried out for 3 days on a growth plate with corn steep liquor and β-mannan (pH 5.5, temperature 20 ° C). total

The β-mannanase activity was 58 nkat with a specific activity of 10.2 nkat per 1 mg protein.

The invention has utility in basic microbiological and biochemical research and in the production of β-mannanase as one of the important enzymes in the saccharification of polysaccharides of various wood wastes. The invention can also be used in the treatment of water containing extractable wood material components as waste.

Claims (1)

OBJECT OF INVENTION Method of production of β-mannanase using microscopic fungi of the genus Lemonniera, characterized by culturing as follows strains of the genus Lemonniera: Lemonniera aquatica Lemonniera corros Lemonniera terrnuta Lemonniera terrestris CCMF-124 CCMF-149 CCMF-325 CCMF-125 individually, or mixtures thereof on a nutrient medium containing [2-1,4-mannan or a 3-1,4-mannan synthetic derivative at 15 to 25 ° C and pH 5] 0 to 6.0 for 120 to 144 hours and the .beta.-mannanase enzyme is obtained in the raw state by evaporation of the growth soil, optionally in a quenched state, by precipitation with ammonium sulfate or organic solvents, preferably with ethanol or acetone.