CS256501B1 - Microbial multlenzyme agent - Google Patents

Abstract

Microbial multienzyme agent degrading ester bonds of triglycerides and carbon chains of fatty alcohols, acids and hydrocarbons and their derivatives of organic and synthetic origin, contains lipases with a minimum activity of 2 units per mg of protein, oxidoreductase β with an activity of alkauhydroxylase min. 0.5 nkat per mg of protein and alcohol dehydrogenase with an activity of min. 0.5 units per mg of protein. The method of preparation consists in that microorganisms of the genus Candida lipolytica β, preferably the mutant strain Candida lipolytica OBM 358, are cultivated on a medium containing alkanes, hardened fat, starch autolysate, sulfate, phosphate, magnesium and ammonium ions at a temperature of 28 to 37 °C and pH 7 to 9, and an enzyme complex is extracted from the obtained biomass, which is stabilized by freeze sublimation or precipitation methods.

Description

The invention relates to a microbial multienzyme complex degrading lipids and carbon chains and a method for its preparation.

It is known that a number of microorganisms, bacteria, fungi and yeasts can use hydrocarbons and their derivatives as the sole source of energy. The most easily assimilated hydrocarbons are alkanes with a linear chain of C10 <>18. Degradation is carried out by a system of oxidoreductases, which convert hydrocarbons to alcohols, aldehydes and carboxylic acids. In most microorganisms, the initiation step of degradation is oxidation of the terminal methyl group. Carboxylic acids are further degraded by beta-oxidation, whereby the carbon chains are degraded to 2 to 3 carbon residues.

The presence of lipase activity is also necessary for the degradation of ester bonds of lipids. Lipases catalyze the hydrolysis of ester bonds of triacylglycerols or other lipids to fatty acids and glycerol or other alcohol present. Fatty acids and possibly fatty alcohols are degraded by the oxidoreductase system. Yeasts of the genus Candida produce the above-mentioned enzyme systems, but in normal cultivation only to a small extent and with properties unsuitable for applications such as detergents, washing agents, wastewater treatment, etc.

These disadvantages are eliminated by the microbial multienzyme agent that degrades ester bonds of lipids, carbon chains of fatty alcohols and acids, hydrocarbons and their derivatives of organic and synthetic origin according to the invention, the essence of which lies in the fact that it contains lipases with minimal activity

- 2 2 units per mg of protein and oxidoreductase with an alkane hydroxylase activity of at least 0.5 nkat per mg of protein and alcohol dehydrogenase with an activity of at least 0.5 units per mg of protein.

The multienzyme preparation is prepared by cultivating the microorganism Candida lipolytica, preferably the mutant strain Candida lipolytica DBM 358, on a medium containing inducers of the desired enzyme activities, i.e. alkanes, hardened fat or other compounds with a longer carbon chain, longer yeast autolysate and minerals at pH 7 to 9, temperature 28 to 37 °C.

Oxidoreductases prepared from Candida lipolytica contain about 50% NADH-independent alkane hydroxylase activity. The method of isolation of the enzyme complex does not dissociate even NADH from dependent hydroxylases. Raw materials for the preparation of the microbial enzyme complex are commonly available, not expensive, so the preparation is economically advantageous and technically undemanding. The multienzyme system works in a wide range of pH and temperatures. It does not metabolize components of common detergents.

Example 1

In the cell homogenate of cells of the mutant strain Candida lipoïtica DBM 358, obtained by cultivation on medium with 1.5 g /UH ₄ / ₂ PO ₄ , 3.2 g /NH ₄ / ₂ SO ₄ , 0.2 g MgSO ₄ . 7H ₂ 0, 1 g K ₂ S0 ₄ per 1,000 ml and 2 to 3% carbon source, pH 7, lipase, alkane hydroxylase and alcohol dehydrogenase activities were measured. After 72 hours of cultivation at 28 °C, the cells were disintegrated in a disintegrator so that the centrifuged cells were mixed with the same weight proportion of ballotin and twice the volume of 0.05 M phosphate buffer pH 7.2. Enzyme activities were measured in the homogenate. The results are given in the table. ·

carbon source alkane hydroxylase nkat/mg protein. alcohol dehydr. unit/mg protein lipase units/mg . hardened fat 0.67 '1.79 21.5 acid· oleic 0.13 3.34 to bear n-heptane 0.07 1.66 « n-octanol 0.16 4.46 - « alkanes 1.54 3.37 16.9 alkanes + glucose 0.43 0.75

- 3 Example 2

To 100 ml of phosphate buffer solution with variable pH was added 0.1 wt. % of the crude multienzyme preparation prepared as described in Example 1. The resulting solution was used to degrease cotton linens soiled with standard dirt by immersing them in the solution at 50 °C for 15 min. The results of degreasing linens with phosphate solution alone and phosphate solution with enzyme depending on pH are shown in the following table:

pH activities degreasing % alcohol dehydrogenase lipase alkane hydroxylase without eizyme with enzymes 6 0.21 27.0 2.1 22.4 24.0 7 0.97 29.6 1.5 25.9 27.0 8 0.75 26.9 1.7 31.8 41.3 9 0',32 22.5 2.2 28.0 38.6 10 unsettled _____ ........2Σ»8.............,,,,,.......,,,,·..

Claims (2)

SUBJECT OF THE INVENTION 1. A microbial multi-enzyme composition which degrades the ester bonds of triacylglycerols, the carbon chains of fatty alcohols, fatty acids, hydrocarbons and their derivatives of organic and synthetic origin, characterized in that it comprises lipases with a minimum activity of 2 units per mg of protein and oxidoreductase with alkanhydroxylase activity . 0,5 nkat per mg of protein and alcohol dehydrogenases with activity min. 0.5 units per mg protein. 2. A process for the preparation of a microbial multienzyme composition according to claim 1, wherein the microorganisms of the genus Candida lipolytica, preferably a mutant strain of Candida lipolytica DBM 358, are cultured on an alkane-containing medium, solidified quill, yeast autolysate, sulfate, phosphate, magnesium and ammonium. ions at a temperature of 28-37 ° C and a pH of 7-9, extracting the enzyme complex from the biomass obtained, which is stabilized by freeze-sublimation or precipitation methods.