RU2328529C2 - Medium for arthrobacter globiformis vniishm-479 -coproporphyrin iii producing agent - Google Patents

Abstract

FIELD: bioengineering.

SUBSTANCE: medium contains, in mass percent: 9.0 of glucose, 3.0 of corn-steep extract, 0.1 of the twice-substituted ammonium phosphate, 0.05-0.2 of cystine, 50.0-85.0 of the water extract from soybean prepared on basis of 7% suspension of the fat-free soybean flour as a nutritious substrate, up to 10.0 of water.

EFFECT: provides increased efficiency of coproporphyrin III biosynthesis.

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Description

The invention relates to the microbiological industry and relates to a nutrient medium for the production of coproporphyrin III.

Known culture medium for the cultivation of Arthrobacter globiformis strain B-658, which allows you to get from 60 to 240 mg of coproporphyrin III in 1 liter of culture fluid (A.S. No. 1482946). The closest in technical essence is the environment for the biosynthesis of coproporphyrin III according to the Patent of the Russian Federation 2078138, 1993 using the strain Arthrobacter globiformis VNIISKHM-479. In the specified patent, the cultivation is carried out on a medium of the following composition, wt.%:

Glucose 8.0

Corn Extract 3.0

A mixture of hydrolyzed amino acids 0.2-0.5

Ammonium Phosphate Disulfide 0.05-0.2

Water - up to 100,

at a pH of 6.8-7.2 and a temperature of 32-36 ° C on a rocking chair 180 rpm in Erlenmeyer flasks with a capacity of 750 ml for 8-10 days.

However, this nutrient medium provides an insufficiently high level of coproporphyrin formation.

In Arthrobacter globiformis, it is well known that amino acids are involved in the formation of the precursors of coproporphyrin III (200-300 mg / L). As sources of nitrogen, individual amino acids were used, a mixture of hydrolyzate amino acids (SAG) produced by the domestic industry (prototype), also fat-free soy flour, soy flour isolate, soy flour enzymatic hydrolyzate and an aqueous extract from soy flour were used.

Fat-free soy flour and water extract from soy flour as one of the most affordable and cheap sources of raw materials provided a high level of porphyrin formation. A high level of coproporphyrin III (320 μg / ml) was accumulated on a medium with defatted soy flour, but the suspension contained in the culture fluid made it impossible to purify it using the ion exchange method to isolate coproporphyrin III and to obtain a highly purified water-soluble salt of coproporphyrin III. In this regard, an aqueous extract from soy flour was used. It was also established that the greatest effect on the biosynthesis of coproporphyrin III is exerted by the amino acid cystine, the content of which in the aqueous soy extract is insufficient. Therefore, cystine was additionally introduced into the medium.

The culture medium we proposed for the cultivation of Arthrobacter globiformis VNIISKHM-479 had the following composition: glucose, ammonium disubstituted phosphate; in the composition of the medium with the commonly used corn extract was introduced an aqueous soybean extract prepared on the basis of a 7% suspension of defatted soy flour and cystine in the following ratio of components in the medium, wt.%:

Glucose 8.0

Corn Extract 3.0

Ammonium disodium phosphate 0.1

Cystine 0.05-0.2

Water soy extract cooked on

based on a 7% suspension of low fat soy flour 50.0-85.0

Water - up to 100.

An aqueous extract from soy flour was prepared as follows: a 7% suspension of defatted soy flour in water (pH 7.5) was heated at 100 ° C for 30 min, cooled, centrifuged at 2000 rpm for 20 min. The supernatant was used to prepare the culture medium.

Such a composition of the fermentation medium provided, at the end of the fermentation, a concentration of coproporphyrin III up to 450 mg / L.

The feasibility of the proposed method for the biosynthesis of coproporphyrin III is confirmed by the following examples.

Example 1

The cultivation of the strain Arthrobacter globiformis VNIISKHM-479 was carried out in Erlenmeyer flasks with a capacity of 750 ml in a nutrient medium (100 ml) of the following composition, wt.%:

Glucose 8.0

Corn Extract 3.0

Ammonium disodium phosphate 0.1

Cystine 0.05

Water soy extract cooked on

based on a 7% suspension of low fat soy flour 50.0

Water - up to 100.

Cultivation was carried out at 34 ° C for 10 days. The content of coproporphyrin III is 340 mg / l.

Example 2

The cultivation of the strain Arthrobacter globiformis VNIISKHM-479 was carried out in Erlenmeyer flasks with a capacity of 750 ml in a nutrient medium (100 ml) of the following composition, wt.%:

Glucose 8.0

Corn Extract 3.0

Ammonium disodium phosphate 0.1

Cystine 0.2

An aqueous soybean extract prepared on the basis of a 7% suspension of fat-free soybean flour 85.0

Water - up to 100.

Cultivation was carried out at 34 ° C for 10 days. The content of coproporphyrin III is 370 mg / l.

Example 3

The cultivation of the strain Arthrobacter globiformis VNIISKHM-479 was carried out in Erlenmeyer flasks with a capacity of 750 ml in a nutrient medium (100 ml) of the following composition, wt.%:

Glucose 8.0

Corn Extract 3.0

Ammonium disodium phosphate 0.1

Cystine 0.1

Water soy extract cooked on

based on a 7% suspension of low fat soy flour 75.0

Water - up to 100.

Cultivation was carried out at 34 ° C for 10 days. The content of coproporphyrin III 450 mg / l.

Claims (1)

Culture medium for cultivating the strain Arthrobacter globiformis VNIISXM-479 is a producer of coproporphyrin III containing glucose, disubstituted ammonium phosphate, a nutrient base is corn extract and water, characterized in that it additionally contains cystine and an aqueous soy extract prepared on the basis of a 7% suspension soy flour as a nutritional basis in the following quantitative ratio of components, wt.%: glucose 8.0 corn extract 3.0 ammonium disodium phosphate 0.1 cystine 0.05-0.2 water soy extract cooked on based on a 7% suspension of low fat soy flour 50.0-85.0 water up to 100.0