RU2032744C1 - Strain streptomyces sp - a producer of rifamycin oxidase and a method of rifamycin oxidase preparing - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: method involves screening of strain Streptomyces sp. G-473 VNIIA 2143 - a producer of rifamycin oxidase and development of method of enzyme preparing using strain Streptomyces sp. G-473. Enzyme isolation is carried out from cultural fluid by ultrafiltration concentration up to achievement of activity 120-130 U/ml. EFFECT: detection and isolation of strain indicated above. 3 cl, 2 tbl

Description

 The invention relates to biotechnology, specifically to the production of an enzyme that transforms rifamycin B into rifamycin S through an intermediate stage of formation of rifamycin O.

 Humicola spp strains are known. and Monocillium spp. rifamycin oxidase producers. All of these strains are fungi.

A known method of producing rifamycin oxidase by culturing strains of Monocillium spp. (e.g., strain ATCC 20621) or Humicola spp. (for example, strain ATCC 20620) in a liquid nutrient medium containing nitrogen sources (e.g. soy flour, casein hydrolyzate, peptone, yeast extract), carbon sources (e.g. glucose, lactose), phosphorus and trace elements (corn extract, yeast extract ) for three days with aeration and stirring. Enzyme activity accumulates in the mycelium. The cells are filtered off, the culture fluid is discarded. Cells or cell extract (cells are destroyed, for example, by ultrasound, the supernatant is separated after centrifugation) is stored at 4 ^about C.

 The disadvantage of this method of producing an enzyme using known strains is the low yield of enzyme activity at the stage of biosynthesis and the need for cell destruction for the concentration of enzyme activity, which is not technologically advanced.

 The aim of the present invention is to search for a new strain of the producer of rifamycin oxidase, which allows to increase the yield of enzyme activity at the stage of biosynthesis, and the development of a new method for producing the enzyme, which allows to simplify the procedure for concentration of enzyme activity.

 The goal is achieved by finding a new strain of the producer of rifamycin oxidase, namely, the strain of actinomycetes Streptomyces sp. G-473 (deposited in the collection of VNIIA under the number 2143).

 The use of actinomycetes as producers of rifamycin oxidase is not known.

 Strain Streptomyces sp. G-473 was isolated from a sample of gray forest soil in the Tula region and is characterized by the following cultural, morphological and physiological characteristics.

Aerob, optimum growth 26-28 ^about C.

 Forms an air mycelium of gray, gray-white, yellowish-gray, pinkish-gray or white.

 Spore carriers spiral (S), spores with a smooth shell.

 Mature sporebirds contain 10-50 spores in the chain.

 Aerial mycelium may be poorly developed.

 The substrate mycelium is colorless (does not have a specific color) or brownish yellow, brown, brown.

 There is no soluble pigment or brown, brown.

 Forms melanoid pigments.

 As a carbon source, it metabolizes D-glucose, L-arabinose, D-xylose, D-fructose, sucrose, raffinose, D-mannitol, i-inositol, and poorly metabolizes rhamnose.

Description of cultural-morphological characteristics was conducted on the 14th day of growth at ²⁸ ° C and is given in Table 1.

 The proposed strain G-473 according to the described characteristics is close to the species Streptomyces anandii and Streptomyces diastatochromogenes (determinant Gauze G. F. Preobrazhenskaya T. P. Sveshnikova M. A. Terekhova L. P. Maksimova T. S. “Determinant of actinomycetes.” M. Publishing house "Science", 1983), but differs from them by the presence of a yellowish-gray aerial mycelium on oat agar. It also differs from Streptomyces diastatochromogenes by yellowish-gray aerial mycelium on mineral medium 1. Based on the foregoing, the proposed strain G-473 is assigned to the indefinite species Streptomyces sp.

 The goal is also achieved by a new method for producing the enzyme.

The method consists in cultivating a strain of Streptomyces sp. T-473 in the liquid nutrient medium containing sources of nitrogen, carbon, trace elements, water (e.g., peptone, yeast extract, glucose, etc.) At 28-30 ^{° C,} aeration and stirring, separation by centrifugation or filtration of cells, concentrating and purification of the enzyme from the filtrate by ultrafiltration on membranes.

 The enzyme activity is determined by the reaction rate of transformation of rifamycin B into rifamycin S (see examples). However, the unit of rifamycin oxidase activity corresponds to the formation of one micromole of rifamycin S in 1 h.

 Significant differences of the proposed method for producing the enzyme are the use of a new producer strain (Streptomyces sp. G-473), the accumulation of the enzyme not in the mycelium, but in the culture fluid, concentration and purification of the enzyme from the culture fluid using ultrafiltration.

 Use of a new rifamycin oxidase producer strain, Streptomyces sp. G-473, and a new method for producing the enzyme can increase the productivity of the biosynthesis process and simplify the process of enzyme isolation (by eliminating the stages of cell destruction and centrifugation), which makes it possible to use the method industrially.

 PRI me R 1. The suspension of spores of the culture of Streptomyces sp. G-473, grown on an agar medium, is introduced into an 750 ml Erlenmeyer flask containing 100 ml of seed medium of the following composition (wt.): Glucose 1.0; casein hydrolyzate 1.0; yeast extract 1.0; copper sulfate 0.005; water the rest.

The inoculum was grown for 48 hours at 28 ^{° C} on a shaker with 220 / min and in an amount of 10% is used to inoculate a fermentation medium of the same composition (10 flasks of 750 ml capacity containing 100 ml of medium). Cultivate for 4 days at 28 ^about C on a rocking chair with 220 rpm. Get 1 liter of culture fluid with an activity of 12 units / ml.

 From 1 l of culture fluid, mycelium is separated by centrifugation at 3000 rpm or by filtration. By ultrafiltration, the supernatant is purified and concentrated to 100 ml. A liquid enzyme concentrate with an activity of 120 u / ml is obtained.

 PRI me R 2. The suspension of the spores of the culture of Streptomyces sp. G-473, grown on an agar medium, is introduced into an 750 ml Erlenmeyer flask containing 100 ml of seed medium of the following composition (wt.): Corn extract 2.0; soy flour 1.0; copper sulfate 0.005; yeast extract 0.5; water the rest.

The inoculum was grown for 48 hours at 28-30 ^{° C} on a shaker with 220 / min and in an amount of 10% is used to inoculate a fermentation medium of the same composition. Further, analogously to example 1 (cultivated at 30 ^about C).

 Get 1 liter of culture fluid with an activity of 10 units / ml and 100 ml of enzyme concentrate with an activity of 100 units / ml.

 The obtained enzyme concentrate is used in the transformation of rifamycin B into rifamycin S and the enzyme activity is determined by the amount of rifamycin S formed. The transformation reaction is carried out in accordance with the conditions of examples 2 and 8 of the known method (US patent 4431735, NKI 435/119 publ. 14.02.84).

EXAMPLE EXAMPLE 3 To 500 ml of 0.16% solution of rifamycin B (10 mM phosphate buffer pH 7.8) was added 1 ml of the enzyme concentrate obtained according to Example 1. The reaction was carried out at ³⁷ ° C, stirring Vania and aeration for 8 hours. At the end of the reaction, the precipitate of rifamycin S is separated by centrifugation, dried over anhydrous sodium sulfate. Obtain 0.64 g of rifamycin S. The enzyme activity is determined by the amount of rifamycin formed. The activity of the enzyme is 120 units per 1 ml of enzyme concentrate, or 12 units per 1 ml of the original culture fluid.

 PRI me R 4. The transformation reaction is carried out according to example 3, using 1 ml of the enzyme concentrate obtained in example 2. Obtain 0.53 g of rifamycin S. The enzyme activity is 100 units per 1 ml of concentrate, or 10 units per 1 ml source culture fluid.

Example 5. 100 ml of the enzyme concentrate obtained according to example 1, is added to 3 l of a native solution of rifamycin B (rifamycin B content of 8000 μg / ml) obtained from the culture fluid of the producer Nocardia mediterranei P-84 by separating the mycelium of the centri - by fugging. The reaction is carried out at a pH of 7.8-8.2 for 8 hours with stirring and aeration. The reaction mixture was cooled to 4 ^° C, the precipitate (rifamycin S) was separated by filtration through a glass filter and washed with 500 ml of acetone. Acetone is distilled off. Obtain 30.2 g of the crude preparation of rifamycin S. Purity 74% yield 92%. The enzyme activity is 40 units per 1 ml of concentrate or 4 units per 1 ml of the original culture fluid.

 Some indicators of the compared methods are additionally given in table 2.

 Thus, the culture fluid and the enzyme concentrate obtained using a new producer strain and a method for producing rifamycin oxidase have increased (about 1.6 times) rifamycin oxidase activity both in the reaction with a pure substrate and in the reaction using a rifamycin producer as a culture fluid B, containing in addition to rifamycin B also enzyme inhibitors.

Claims (2)

 1. Strain Streptomyces sp. VNIIA 2143-producer of rifamycin oxidase. 2. A method of producing rifamycin oxidase, comprising cultivating the producer on a nutrient medium containing sources of carbon, nitrogen, trace elements and water at 28-30 ^° C, mixing and aeration, separating the mycelium and isolating the target product, characterized in that the producer uses Streptomyces sp strain . VNIIA 2143, and the isolation of the enzyme is carried out by ultrafiltration until an activity of 120-130 u / ml is achieved.

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