SU1521774A1 - Method of obtaining lactatoxidase - Google Patents

Abstract

This invention relates to the microbiological industry and relates to methods for producing lactate oxidase. The purpose of the invention is to simplify the method and expand its functionality and increase the yield of the enzyme. The strain ACETOBACTER SUBOXYDANS VAR is used as the producing microorganism. MUCIPARUM FRATEUR N C1B 5595 (1950-1280), capable of synthesizing lactate oxidase, which oxidizes lactate to pyruvate and H ₂ O ₃ , the seed of which is grown on curd whey, which is a cheap natural source of lactic acid. The method allows to obtain lactate oxidase with an activity of 7.75 units / ml, a specific activity of 1.2 units / mg of protein at a yield of 790.5 units / l.

Description

The invention relates to the microbiological industry and relates to a method for producing the enzyme lactate oxidase, which converts lactic acid (lactate) to pyruvic acid (pyruvate) and which can be used in analytical systems used in medicine, food and microbiological industries.

The purpose of the invention is to simplify the method, expand its functionality and increase the yield of the enzyme,

 The method is carried out in the following manner.

Acetobacter suboxygans var. Strain is used as a producer. muciparim Frateur No. CIB 5595 (1950 1280). The seed of the producer is grown on a nutrient medium containing lactic acid, the source of which is curd cheese. During the fermentation of milk, the lactose contained in it is converted into lactic acid, which, after the separation of milk protein clumps, i.e. cottage cheese, remains in the serum and is 0.92-1.24%. Concentrations of MO-t of lactic acid in curd whey less than 0.96% and more than 1.24% are unlikely, based on the technological process of preparation of 1 bar. Curd whey is a cheap waste from the curd making process.

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The activity of the resulting lactate corticone is determined by spectrophotometric method, m, based on the scheme of a given method for the conversion of lactic acid: lactic acid + Oo--

 lactatoxy --- pyruvic acid +. Daz

. The method involves the interaction of lactate oxidase with a substrate — L-lactic acid in pI 6.5 gstral phosphate buffer, and detection of peroxidase formed, which is visualized by the presence of an ionic mixture of phenylenediamine in the packer. Unit ac- The lactic acid oxidase tivi is expressed by the amount of the enzyme that converts 1 μmol of lactic acid into pyruvic acid and H / jC at pH 6.5, 25 ° C in 1 minute.

The method is explained in the following examples.

Example 1 (control). The method includes the following steps: maintaining the culture in test tubes, growing seed in flasks and cultivating the microorganism in flasks in the presence of lactic acid, separating the biomass, destroying the cells, centrifuging their homogenate to obtain a soluble enzyme.

Maintaining Acetobacc culture “r suboxydane var. muciparum frateur 1950-1289.

The culture is maintained in test tubes on beveled agar and in flasks with liquid nutrient medium variable. The composition of the agar medium next,

g / l: yeast extract 10, glucose 10; peptone 5; chalk 10, agar 15. Carbon sources — glucose, sorbitol, glycerin, periodically Change. Liquid media to maintain the culture of the pre

to

20

25

17744

 Wort, 500 ml phthalate buffer, pH 5.5. The seed is grown for 1 day at 25 ° C on a rocking chair at 150 vibrations / min.

The seed in the amount of 5% is transferred into flasks with a fermentation medium, the composition of which corresponds to that indicated in the prototype, g / l: 1.5; yeast extract 1.0; 5.0; 4.0; MgSO 7H20 0.2; MnSO H, 0, 0.08; 37.5 ml / l of 40% D, L-lactic acid, pH equilibrated to 5.2. Cultivation of the producer was carried out for 24 hours at 25 ° C on a rocking chair at 150 vibrations / min.

Separation of biomass, its homogenization and centrifugation of the cell homogenate.

The biomass is separated by centrifugation at 3000 rpm for 30 minutes. The biomass yield is 12 g / l nutrient medium, i.e. 2.4 g dry weight / l on the basis that the dry matter of the cell is about 20%,

The cells are washed 3 times with citrate-phosphate buffer, pH 6.5, suspended in the same buffer in a 1: 3 ratio and sonicated at 22 kHz for 6 minutes. The cell homogenate is centrifuged at 100,000 g for 3.5 hours at 4 ° C. The supernatant is a raw enzyme, the lactate oxidase activity of which reaches 6100 units / l (6.1 units / ml). The protein concentration is 6.0 mg / ml, i.e. the activity of 1 ml of enzyme in terms of the amount of protein in the same volume of 1 u / ml. The yield of the raw enzyme in terms of 1 liter of the nutrient medium (the mass of biomass is multiplied by the volume of the buffer in which it is suspended, and the activity is 1 ml

thirty

35

40

are nutrient media, with -. raw enzyme) 219.6 units

50

exchangeable for growing inoculum, the composition of which is listed as No. 1. On canted agar, the culture is maintained for 1 month, and in liquid media for 2-3 days,

Growing seed and cultivating the producer in flasks.

Bacteria with oblique agar re-. sow in flasks with a nutrient medium, the composition of which corresponds to that indicated in the Prototype: 10 g / l of 85% D, L-molar acid; 5.0 g / l yeast extract; 2.0 g / l, j2.0 g / l MgSO4. 7HiO ,. 500 ml beer

Example 2. A producer culture with the use of serum as a true acid for a grown material.

Maintaining culture suboxydans var. Muciparu 1950–1280 is carried out in case of seeding the material on a nutrient medium, while on the basis of a curd syrup, 5.0 g / l of yeast pH of the nutrient medium, which is 0.96% lactic acid, is nutrient-rich.

raw enzyme) 219.6 units

Example 2. Cultivation of a producer using curd whey as a source of lactic acid for growing seed material.

Maintaining Acetobaccer suboxydans var. muciparum frerateur. 1950-1280 is carried out as in example 1, the seed is extruded in flasks on a nutrient medium prepared on the basis of cheese whey containing 0.96% lactic acid, with an additive of 5.0 g / l of yeast extract, pH of the nutrient medium 5, 2 Cottage cheese

the serum is alkalinized to pH 5.2, autoclaved at 0.5 atm for 30 minutes and stored at 4 ° C. Filter before use twice through a paper filter.

Further, the cultivation of the seed material and the cultivation of the producer are carried out as in Example 1. The biomass yield is 34 g / l of nutrient medium (dry weight is 6.8 g / l).

The preparation of the raw enzyme is carried out as in Example 1. The activity of the obtained sample of lactate oxidase is

7750 u / l, i.e. 7.8 units / mp The protein concentration is 7.6 MI / MSI, i.e. the activity of 1 MP enzyme in terms of the ratio with the amount of protein in the same volume of 1.2 units / mg. The output of the enzyme in terms of 1 l nutrient medium 790,3 units

Example 3. Maintaining Acecobacter oxydans var culture. mucipa-rum Trateur 1950–1280 is carried out in prima py 1. The seed material is grown in flasks on a nutrient medium prepared on the basis of curd whey containing 0.96% lactic acid, with the addition of 7 MP / l of 40% lactic acid ( total concentration 1.24%) and 5.0 g / l yeast extract, pH of the nutrient medium 5.2.

I,.

5% (5 ml) of inoculum was transferred to the fermentation medium, and the process was then carried out as in Example 1.

The biomass yield is 32 g / p nutrient medium (based on a dry weight of 6.4 g / l). The activity of the obtained sample of lactate oxidase is 7700 units / l, i.e. 7.7 u / mp The output of the enzyme in terms of 1 l nutrient medium 739.2 units

The advantage of the method is to simplify the production of lactate oxidase, converting lactic acid to pyruvic acid and using an available microorganism and simple nutrient medium. ;

Claims (1)

Formula isobuty A method of producing lactate oxidase, which involves growing the seed material of a producer from the genus Acetobacter on a nutrient medium containing a source of lactic acid and yeast extract, introducing the inoculum of the seed into fermentation. cultivation of the enzyme, separation of biomass, its homogenization and centrifugation, characterized in that, in order to simplify the method, expand its functional capabilities and control the yield of the enzyme, Acecobaccer suboxy strain is used as a producer dans vSr muciparura Frateur NCI B 5595, and cottage cheese whey is used as the basis of the nutrient medium and source of lactic acid for growing seed.