RU2031947C1 - Strain streptomyces olivocinereus - a producer of glucose isomerase - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: using producer cells for isomerization of glucose to fructose the yield of fructose is up to 10.2% per hour. Productivity of strain S. olivocinereus ВКПМ S-1169 is 444 mcM/min per 1 l nutrient medium. Activity of intracellular glucose isomerase is 74 U/g absolutly dry substance determined at 60 C. EFFECT: increased productivity of strain.

Description

 The invention relates to biotechnology and is a new strain of microorganisms - a producer of the glucose isomerase enzyme, which can be used in the food industry to obtain glucose-fructose syrup and fructose - a sugar substitute, as well as to increase the sweetening ability of glucose-containing molasses in the starch industry.

 There are various strains of microorganisms - producers of glucose isomerase, related mainly to streptomycetes and lactic acid bacteria. Strains exhibit different ability to form glucose isomerase, grow mainly on expensive media containing food raw materials [1].

 As a prototype, one can consider the strain Streptomyces olivocinereus 154 (according to the old classification of Actinomyces olivocinereus) - a producer of glucose isomerase [2]. The strain was isolated from Tajik soils and grows on a nutrient medium containing xylose, glucose, peptone and casein hydrolyzate. The known strain produces glucose isomerase, which converts 45% glucose to fructose in 20 hours. The strain is stored in the culture collection of the Institute of Biochemistry. A.N. Baha RAS.

 The purpose of the invention is to obtain a new strain of Streptomyces olivocinereus, showing a higher than known glucose isomerase activity.

 A new strain of Streptomyces olivocinereus VKPM S-1169 was obtained by sieving the Streptomyces olivocinereus 154 culture and selecting the most active colonies.

 The strain has the following characteristics.

 Morphological signs: spore-bearing spiral, spores have a smooth shell.

 Wednesday Chapek. Aerial mycelium gray-white. Brownish mycelium. Brown pigment is released on Wednesday. There is no aerial mycelium on the Gauze medium. Brown pigment is released on Wednesday.

 On meat and peptone agar, there is no aerial mycelium. A light brown pigment is released on Wednesday. When growing on a medium with glycerin and asparagine, there is no aerial mycelium. A brownish pigment is released on Wednesday.

Physiological signs. Culture grows at 24-38 ^° C Optimum temperature 28-30 ^° C Optimum pH - 6.8-7.1. It uses the only carbon sources: xylose, rhamnose, galactose, sucrose, maltose, raffinose, glucose, inositol, mannitol, L-arabinose. The enzyme forms only in the presence of xylose. Culture thins gelatin, peptones milk. Glucose isomerase from cells of a new strain of Streptomyces olivocinereus was obtained in laboratory and semi-production conditions.

PRI me R. Seed is grown for 5 s on a modified Chapek medium with glucose, xylose and with the addition of cobalt ions of the following composition, g / l: KH ₂ PO ₄ 0.5, MgSO ₄ ˙ 7H ₂ O 0.5, NaCl; 0.5, KNO ₃ 0.5, FeSO ₄ traces, xylose 5.0, glucose 5.0, CoCl ₂ ˙ 6H ₂ O 10 ^-3 M.

From Chapek’s agar medium, the grown mycelium is transferred to a liquid nutrient medium of the following composition, g / l: xylose 5-7; glucose 3-5; peptone 8-10; corn extract 20; MgSO ₄ ˙ 7H ₂ O 0.3-0.6; NaCl 3-6; CoCl ₂ ˙ 6H ₂ O 0.1-0.2 (pH = 6.8-7.0).

Cultivation is carried out at 28-30 ^about C for 3 s. Seed obtained on this medium is introduced into the nutrient medium of the same composition. The maximum accumulation of biomass and glucose isomerase occurs upon cultivation in Erlenmeyer flasks for 24 hours; when cultured in Japanese fermenters (Marubishi company), fermentation ends after 14-17 hours. After separation of the biomass by centrifugation and washing from the residual nutrient medium with phosphate buffer (0.005 M), the mycelium is dried at room temperature. The extraction of the enzyme is carried out with phosphate buffer or distilled water. The extract can be lyophilized while maintaining activity. The glucose isomerase enzyme preparation can be obtained from the extract by precipitation with acetone or ammonium sulfate. Dry cells can also serve as a source of enzyme. The enzyme, located in the cells, catalyzes the process of the glucose isomerization into fructose at 60-80 ^C. The maximum yield of the isomerization of fructose 0,5 M glucose producing cells - 10.2% / h, the average yield - 7.0% / h. Fructose is determined by the method of Percherson.

 The productivity of the strain of Streptomyces olivocinereus - producer of glucose isomerase is 444 μ M / min per 1 liter of culture medium. The activity of intracellular glucose isomerase is 74 Hl IA unit / g a.s.

The determination of the activity of intracellular glucose isomerase is carried out on the basis of the results of a study of the properties of this enzyme. pH - optimum action of glucose isomerase S. olivocinereus is at pH 8.5. Optimum action temperature - at 80 ° ^C. The reaction mixture was (per 1 ml) of 0.5 M glucose solution in 0.2 M K, Na phosphate buffer, pH - 8,5, t - 60 ^o C with adding 3 5 mg MgSO ₄ ˙7H ₂ O and 0.25 mg CoCl ₂ ˙6H ₂ O. The activity is determined not at the optimum temperature, since at 80 ° ^C is more rapid enzyme inactivation than at 60 ° ^C. The value of the activity, determined at 60 ^o C is approximately half the activity determined at 80 ^o C.

 Thus, a new strain with high glucose isomerase activity was obtained.

Claims (1)

 STRAIN STREPTOMYCES OLIVOCINEREUS - PRODUCER OF Glucose Isomerase.  The strain Streptomyces olivocinereus VKPM S-1169 is a producer of glucose isomerase.