RU2113477C1 - Strain kluyveromyces marxianus varietas bulgaricus t3 - a producer of superoxide dismutase and accompanying enzymes - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: invention proposes the yeast strain Kluyveromyces marxianus var. bulgaricus T that is able to grow on milk-processing industry waste enriched with nicotinic acid source. Superoxide dismutase producing by the culturing strain shows moderately thermophilic property. Strain produces catalase and beta-galactosidase simultaneously. EFFECT: strain indicated above, enhanced effectiveness of superoxide dismutase production. 4 tbl

Description

 The invention relates to biotechnology and relates to microbiological methods for producing enzyme preparations, and more particularly to methods for producing superoxide dismutase and related enzymes.

 Superoxide dismutase is a metalloprotein and is of significant practical interest in connection with widely conducted studies on the use of this enzyme in medicine and the food industry.

 The most famous sources of superoxide dismutase are red blood cells [1] and such animal tissues as the liver, kidneys, lungs, heart, brain, and others [2].

 At the same time, it is also known that superoxide dismutase can also be obtained microbiologically using microorganisms such as Pseudomonas putida [3], Nocardopsis [4], Gluconobacter [5], Saccharomyces cerevisiae [6], recombinant E. Coli strains [7] ].

 It has to be noted, however, that microbiological methods have not yet been found in industrial applications and are used only in laboratory conditions.

 The goal set by the applicant is to find a producer strain of superoxide dismutase suitable for large-scale production of this enzyme under production conditions.

 The applicant also had in mind that enzyme preparations obtained from various natural sources can vary greatly in some properties.

As a result of a long search and selection work, the applicant isolated and identified the strain T _{3 of the} yeast Kluyveromyces marxianus var. bulgaricus with the desired properties.

 The superoxide dismutase producer strain first isolated by the applicant was able to accumulate sufficient amounts of superoxide dismutase to organize its cost-effective production, showed the ability to actively develop and biosynthesize the enzyme complex when grown on dairy waste, showed moderate thermophilicity.

 The most valuable quality of the newly identified producer was the ability to synthesize superoxide dismutase with increased resistance to high temperatures.

The new strain Kluyveromyces marxianus var.bulgaricus T ₃ received by the applicant was deposited with the National Bank of Industrial Microorganisms and Cell Culture of the National Biotechnological Center of Bulgaria under registration number 1984.

 The strain is isolated from cheese and is characterized by the following features.

 Growth on malt extract 15 B: during the growth process forms a precipitate; cells are 0.05-0.1 x 0.2 μm in size; reproduction by budding.

Growth on malt agar 10 B: when grown at 28 ^o C for 3-5 days, smooth colonies with a matte surface, white, soft consistency, concave, with an uneven center and smooth edges are formed.

 Ascospore formation: It forms 1-2-4 spherical ascospores located individually or in tetrads on acetate agar, Mc Clari agar and malt agar.

 Formation of pseudomicelles: does not form pseudomicelles.

 Chlamydospore formation: Chlamydospore does not form on corn and rice agar.

 Tolerance to sodium chloride: resistant to 8-9 sodium chloride.

 Biochemical characteristics: sugar fermentation - ferments glucose, galactose, sucrose (100%) and does not ferment maltose, lactose, raffinose.

 Assimilation of sugars - assimilates glucose, galactose, sucrose, lactose, raffinose, inulin, D-xylose, D-arabinose, ethanol, L - lactic acid.

 Assimilation of potassium nitrate - does not assimilate.

 Growth on vitamin-free environments - does not grow on vitamin-free environments; needs nicotinic acid.

 The formation of pigments - does not form pigments.

 Arbutin Cleavage - Arbutin does not cleave.

 Growth on 50% glucose agar - no growth.

Growth temperature - minimum 5-6 ^o C, optimal 35-42 ^o C, maximum 50 ^o C.

 The strain did not show high demands on cultivation conditions and was able to grow on relatively simple media using dairy waste as a basis, which partially follows from the data in Table 1.

The initial nutrient medium contained 50 ml of whey filtrate and 50 ml of distilled water and was enriched with components of the Reader medium. The initial lactose concentration was 16.4 mg / ml. The cultivation was carried out at 40 ^o C and lasted 20 hours

 The results obtained show that the growth of the culture on halved whey filtrate is limited only by the nitrogen source, and does not need other components of the Reader medium.

 The proposed strain showed a pronounced dependence on the presence of nicotinic acid in the medium and this property is confirmed by the data in table. 2.

The cultivation was carried out on a complete medium of Reader, prepared on diluted twice the filtrate of whey. The initial lactose concentration was 16.4 mg / ml. Cultivation lasted 20 hours at 40 ^o C.

 It is clearly seen that unlimited growth occurs only at a concentration of nicotinic acid of the order of 75 μg / 100 ml. However, it was shown that the need for cells in nicotinic acid can be completely satisfied when introducing into the growth medium up to 20% yeast homogenate filtrate obtained by separating cell debris.

 In parallel with the biosynthesis of superoxide dismutase, the strain biosynthesizes concomitant enzymes such as beta-galactosidase and catalase, which are also of practical interest.

 Superoxide dismutase, obtained using the proposed strain, has original properties that distinguish it from preparations of superoxide dismutase isolated from other natural sources, and this is shown in table. 3.

 It should be noted that the accumulation of concomitant enzymes by the proposed strain — catalase and beta-galactosidase — is quite high and amounts to about 8-10 Δ E / min / mg and 0.750 Δ E / min / mg, respectively.

 The essence of the proposal is further illustrated by the following example.

Example. Strain Kluyveromyces marxianus var. bulgaricus T _{3 was} grown on whey ultrafiltrate - a waste product from a dairy plant in Silistra. Pre-serum was heated at pH 7.3 and ultrafiltered through a membrane with a pore size of 5 kD. Whey ultrafiltrate was enriched with nicotinic acid at the rate of 75 μg of nicotinic acid for every 100 ml of medium.

Cultivation was carried out in a fermenter with a working volume of 4 l at a temperature of 35 degrees, pH 5.0 and pO ₂ 30-40% for 30 hours. The pH value was maintained using a 10% solution of ammonium hydroxide.

 The dynamics of the accumulation of superoxide dismutase and related enzymes are shown in table. 4. The activity of enzymes was determined in the filtrate of destroyed cells.

 According to the applicant, the proposed strain is original and has significant advantages over the known superoxide dismutase precursor strains.

Claims (1)

Strain Kluyveromyces marxianus var. bulgaricus T ₃ - 1984 (Collection of the National Bank of Industrial Microorganisms and Cell Cultures of the National Biotechnology Center, Bulgaria, Sofia) - producer of superoxide dismutase and related enzymes.

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