RU2575804C1 - STRAIN OF YEAST Candida parapsilosis - PRODUCER OF LIPASE - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of yeast Candida parapsilosis M10, having high lipolytic activity, is deposited in the Russian National Collection of Industrial Microorganisms under the registration number Y-4055 and can be used to produce enzyme preparation of lipase.

EFFECT: invention enables to obtain a strain with improved lipolytic activity.

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Description

The invention relates to microbiology and biotechnology. The strain Candida parapsilosis Μ10, which has high lipolytic activity, was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) FSUE GosNII Genetika (Moscow, 1st Dorozhny proezd 1) under registration number VKPM: Υ-4055 and can be used to obtain the enzyme preparation of lipase. The invention allows to obtain a strain with high lipase activity.

Lipolytic enzymes are hydrolases of esters of saturated and unsaturated aliphatic acids containing at least 12 carbon atoms.

They have a wide range of applications - in the food industry, household chemicals, cosmetics, and medicine. Significant interest in the practical use of enzymes in medicine is due to the important role they play in biological processes, in particular in digestion in humans and animals. Of no less interest is their practical application in the food industry - in cheese making, in baking. Lipases of microorganisms in combination with other enzymes are used for biological wastewater treatment.

The Trichosporon fermentans WU-C12 strain is known (Chen J., Ishii T., Shimura S., Kirimura K. and Usami S. Lipase production by Trichosporon fermentans WU-C12, a newly isolated yeast. Journal of Fermentation and Bioengineering, 1992, vol . 73, p. 412-414) having lipolytic activity. The disadvantage of the strain is the low activity of lipase.

The known recombinant strain of Pichia pastoris (Catoni Ε., Schmidt-Dannert C, Brocca S., Schmid R. Overexpression of lipase A and B of Geotrichum candidum in Pichia pastoris: high-level production and some properties of functional expressed lipase B. Biotechnology Techniques , September 1997, vol. 11, no. 9, p. 689-695) having lipolytic activity. The disadvantage of the strain is the low activity of lipase.

The wild strain Yarrowia lipolytica W29 (ATCC 20460) is known, which served as the parent strain (J. Biotechnol., 2004, vol. 109 pp. 63-81) for a number of recipient strains, including Yarrowia lipolytica Pold (CLIB 139) and Yarrowia lipolytica Polf (ATCC MYA-2613). Despite the fact that they are well characterized biochemically and genetically, the above producers are not able to provide a high level of lipolytic activity.

The closest analogue to the claimed invention is a strain of Yarrowia lipolytica VKPM Y-3600 (RF patent No. 2475532). The patent describes a method for producing a recombinant strain of Y. lipolytica VKPM Y-3600 by transforming a strain of Y. lipolytica Polf (leu +) with the expression plasmid pY-Y1PIR1-LIP2. The disadvantages of the strain include low producer activity, as well as the difficult isolation and purification of the enzyme preparation.

The objective of the claimed invention is to obtain a new yeast strain with high lipolytic activity.

The technical result of the claimed invention is the improvement of lipase biotechnology, the possibility of using this enzyme in the food industry.

The technical result is achieved through the use of a new strain of Candida parapsilosis M10 isolated from a sample of butter. The criteria for the selection of microorganisms were the color change of the indicator in the composition of the selective nutrient medium during the cultivation of yeast colonies, the size of the clarification zones around them, and the amount of accumulated lipase in the filtrate of the culture fluid after the fermentation period.

The unit of lipase activity is taken to be such an amount of an enzyme that at pH 7.0 and a temperature of 37 ° C for 1 hour catalyzes the hydrolysis of a 40% emulsion of olive oil with the formation of 1 μmol oleic acid.

The proposed strain was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) FSUE GosNII Genetics (Moscow, 1st Road passage, 1). VKPM registration number: Y-4055.

The strain of Candida parapsilosis M10 is characterized by the following features:

The belonging of the strain Candida parapsilosis M10 to the indicated species was revealed on the basis of genetic analysis of sequencing of variable regions of 18S rRNA. Homology was 99%.

Cell morphology.

When growing on a liquid Saburo medium (peptone - 10 g / dm ³ , glucose - 40 g / dm ^3, water - the rest), the daily yeast culture is represented by oval, elongated-oval, rounded cells. Cell sizes range from 2.5-4 microns in width and 2.5-9 microns in length. By the second day, most cells are budded and form pseudomycelia. Cells grow well on simple nutrient media.

When growing on a liquid medium, Candida parapsilosis Μ10: turbidity is observed, a precipitate and a ring are formed on the walls of the tube, there is no pigmentation of the medium, no gas evolution, the smell is characteristic of yeast.

Cultural characters of Candida parapsilosis M10.

On the surface of dense nutrient media such as Saburo Candida parapsilosis M10 forms colonies with the following symptoms:

- the form is round with a scalloped edge;

- size (diameter) - from 1 to 7 mm;

- surface - large-folded;

- profile - flat, growing into the substrate;

- Shine and transparency - the colony is shiny, opaque;

- color - light dirty beige;

- edge - wavy;

- structure - coarse-grained;

- consistency - dense leathery.

Peculiarities of growth along the line of Candida parapsilosis M10: the line is abundant, continuous with a wavy edge, the surface is coarse-pleated, shiny, light dirty-beige. The dispute does not form.

Physiological and biochemical characteristics.

Obligate aerob. Ferment glucose. Assimilates: glucose, galactose, sucrose, maltose, D-xylose, D-ribose, glycerin, citric and succinic acids. Does not assimilate: cellobiose, lactose, raffinose, inulin, D-arabinose, L-ramnose, lactic acid. Does not assimilate nitrates. It grows in a vitamin-free environment, but biotin is a powerful growth stimulator.

Thins gelatin. Hydrolyzes urea.

The ratio of Candida parapsilosis M10 to the concentration of sodium chloride in the nutrient medium: intensive growth of the culture was observed on media supplemented with NaCl (8%, 10%, 12%).

The ratio of Candida parapsilosis M10 to pH: growth of the culture is observed in the pH range from 3 to 9.

The ratio of Candida parapsilosis M10 to the temperature of cultivation:

- at 20 ° C - weak growth;

- at 25 ° C - abundant growth;

- at 30 ° C - abundant growth;

- at 37 ° C - abundant growth;

- at 50 ° C - weak growth.

Conditions and composition of the medium for long-term storage of the strain: Saburo agarized nutrient medium with the addition of tributyrin - 0.1%, Ca ^{2+ ions} - 0.03-0.08%, t 4-6 ° C. Reseeding on fresh environments - once every three months.

The conditions and composition of the medium for propagation of the strain: Saburo mowed agar medium, incubation temperature 28-30 ° C, incubation time 48 hours

The activity of the enzyme preparation of lipase G20X, obtained on the basis of the cultivation of the proposed strain reaches 30400-30500 units / g

Examples of carrying out the invention.

Example 1

Samples of soil, fruits, oils, meat and dairy products containing an increased amount of lipids served as a substrate for the isolation of yeast-producing lipases. From the moment of selection, the material was stored at a temperature of + 4 ° C for not more than 1 month.

Samples were suspended in physiological saline supplemented with gentamicin and kept in a thermostat at a temperature of 30 ° C for 1 h. The use of antibiotic preparations at the initial stages of screening made it possible to exclude the germination of concomitant bacterial cultures.

The resulting accumulative culture was reseeded in Petri dishes on a dense enriched nutrient medium M9 of the following composition (g / dm ³ ): Na ₂ HPO ₄ - 6; KH ₂ PO ₄ - 3; NaCl - 0.5; NH ₄ Cl - 1; olive oil - 10; agar - 20. Incubation was carried out in a thermostat at a temperature of 30 ° C for 72 hours. At the end of the incubation period, active yeast colonies capable of assimilating olive oil as the sole carbon source were selected. Pure cultures were reseeded on a selective nutrient medium of the following composition (g / dm ³ ): Na ₂ HPO ₄ - 6; KH ₂ PO ₄ - 3; NaCl - 0.5; NH ₄ Cl - 1; tributyrin - 3; agar - 20; in the presence of dye methyl red.

The presence of lipolytic activity was judged by the size of the enlightenment zones around the colonies, and by the color transition of the dense nutrient medium from yellow to orange and then to red.

As a result of the screening, a yeast strain having lipolytic activity was selected.

Example 2

The cultivation of strain Candida parapsilosis Μ10, obtained as a result of screening, on a nutrient medium of the following composition (g / dm ³ ): mustard oil - 26; glucose - 5; yeast extract - 18; soy flour - 10; KH ₂ PO ₄ 0.3; MgSO ₄ · 7H ₂ O — 0.2; CaCO ₃ - 3; tween-80 - 4.2; water - up to 1 dm ³ , for 48 hours at 30 ° C with constant stirring, it is possible to achieve an enzyme activity of 235-240 u / cm ³ in the QL filtrate. The age of the seed is 36-48 hours, the dose is 5% of the volume of the nutrient medium (the number of cells is 5 · 10 ⁷ cells / cm ³ .

Isolation and concentration of the enzyme preparation of the lipase from the centrate of the culture fluid Candida parapsilosis Μ10 carried out by ultrafiltration and sterilizing filtration methods allows to obtain an enzyme preparation with lipase activity of 30400-30500 units / g.

Claims (1)

Candida parapsilosis yeast strain, registration number in the All-Russian collection of industrial microorganisms Y-4055, characterized by the ability to synthesize lipase.