SU990813A1 - Strain staphylococcus saprophyticus z-1.10 producing urease - Google Patents

Description

(54) STAPHYLOCOCCUS SAPROPHYTICUS U-1.10 STRAIN PRODUCT UREASE

one

The invention relates to the microbiological industry, in particular to the preparation of a new strain used to obtain the enzyme urease.

The urease producers are various microorganisms: bacteria, fungi, yeast, T-mycoplasmas, as well as protozoa and plants. The most common urease producers are bacteria. A strain of Staphylococcus saprophyticus 1 is known among the cocci synthesizing urease.

The disadvantage of this strain is low urease activity. When the strain of Staphylococcus saprophyticus was reseeded, the level of urease biosynthesis decreased by more than 50%. The closest to that proposed in its cultural-morphological, biochemical, and physiological properties is the strain Staphylococcus saprophyticus L-1 t2} .20

However, the known strain has not enough high urease activity. The activity of this strain per mg acetone

the preparation of cells under cultivation under semi-production conditions is on average 4.5, in flasks - 4.2 reflux tubes.

The aim of the invention is to obtain a new producer strain of urease with high and stable activity under conditions of deep cultivation.

The strain Staphylococcus saprophyticus C-1.10 was obtained by targeted selection of the most active variants of the original strain. In order to select possible more active variants of the original strain. Staphytoqoccus saprophyticus i, -1 produced the sowing of bacterial suspension on agar medium in Petri dishes. In order to differentiate the true variants from mods, 4-5-fold passages were made. Analysis of the natural variability of the original strain showed that the strain is represented by 2 types of colonies. The appearance of the colonies is presented in the photo.

The 1st type of colonies is characterized by morphological properties typical of the original culture of Staphylococcus saprophyticu L, -1j whose diameter is 0.8-2.0 mm. After 48 hours of incubation, the strain in the center of the colony forms a convex greenish hue. The activity of the variants of the 1st type of colonies grown by the deep method is 3.4 ± 0.6 Еg / mg acetone cell preparation and 8.8 u / ml culture liquid. Colonies of the 2nd type are characterized by a smaller diameter, reaching 0.5-1.2 mm, a more intense luster, the resulting protrusion of a greenish tint in the center of the colonies is more vague. with the digestibility of carbohydrates inherent in the original culture of Staphylococcus saprophyticus L-1, the ability to digest lactose was acquired. The optimal level of biosynthesis of urease in cells occurs at a pH of 6.0-6.5, while the optimum values of this value, contributing to the maximum level of accumulation of this enzyme in cells of the original strain 6, 8-7.2. The activity of the variants forming the 2nd type of colonies, fluctuated in broad; In the range of 6.2 to 14 units / mg acetone cell preparation. Taking into account the possibility of stable formation of urease at a high level, further selection of type 2 colonies was carried out. As a result of this work, option 10 was selected, which consistently retains a high level of urease biosynthesis during multiple passages, with an activity of 13.6 units / mg of acetone cell preparation. The strain Staphylococcus saprophyticus L-1.1 is stored in the collection of the Museum of Micro Organisms of Microorganisms of the Institute of Scientific Research Institute of Genetics under the number of CMMP B-2346. The strain has the following characteristics. Morphological. Globular bacteria. Mostly single, often form a bunch, immobile, do not form a spore. The size of the cells of a one-day culture on MPA-0.5-1.5 microns in diameter. Cells are well stained with diluted basic fuchsin and methyl blue, gram-positive. Cultural and physiological properties. H MPA after 24 hours of incubation at 37 ° C form colonies with a diameter of 0.5-1.2 mm. The colonies are grayish-white opaque, smooth and shiny, with regular or sometimes slightly irregular edges. After 48-72 hours, a vague greenish shade develops in the center of the colonies. The MTD initially forms turbidity, which later settles to the bottom of the tube. The medium becomes and os 4 transparent. On the surface, potatoes are growing. The strain is an optional anazro with an optimal growth temperature of ± 37 ° C. rakhmal do not hydrolyze. Milk does not peptoise. Gelatin does not dilute. On a medium with an inertial source of nitrogen does not grow. It reduces to nitrites. Assimilate maltose, glucose, fructose, glycerin, acharose, lactose. Does not assimilate arabinose, silose, sorbitol, rhamnose, mannitol, inositol dulcite. Resistant to lysozyme. The content of + P in DNA ranges from 30.55, 0 mol%. The culture is stored in test tubes in an agar medium under petroleum jelly and in a lyophilized state. Example. For storage and cultivation of the strain Staphylococcus sapraphyticus L-1.10, the following medium is used: Sodium chloride, g5.0 Sodium nitrate, g2.5 Potassium phosphate monosubstituted, g2.0 Enzymatic yeast hydrolyzate, g20.0 Corn extract, ml 20.0 Water, ml Enzyme gvdrolizat yeast and corn extract is suspended in 1.5 volumes of water from the final volume of the medium. 30% caustic soda solution is adjusted to pH 6.0-6.5 and sterilized for 20 minutes. After cooling the solution to room temperature, in order to remove insoluble particles, it was centrifuged at 4000 rpm for 40 minutes. The supernatant is diluted with water to a final medium volume and the remaining components are added. Secondary 1 sterilization of the resulting solution was performed at 135 ° C for 40 minutes. To recover, the strain of Staphylococcus saprophyticus L -1.10 is seeded onto agar medium with Petri dishes and incubated at 37 ° C for 20-24 hours. In laboratory experiments, lively culture is seeded into 750 ml flasks containing 100 ml of culture medium and grown on a rocking chair at 180 rpm at 37 ° C for 20-24 hours. When cultured in fermenters, the seeds used are cells previously grown in flasks on a rocking chair - for 10-12 hours. Growing is performed with aeration 8, 37 ° C in for 20-24 hours, the pH is not maintained during the cultivation process. Control ipammom in all experiments

serves as a strain of Staphylococcus saprophyticus I -1.

During the growth process, urease activity in cells dried with acetone is determined. From the weight of the acetone cell preparation, the level of urease activity in ml of blood fluid is calculated. Urease activity is determined by a colorimetric method using Nessler's reagent. Per unit of activity take

In each xerfusion fermentation, bacterial cells were isolated by centrifuging at 14000 rpm for 30-40 minutes. Collected and weighted cells are destroyed mechanically using glass beads with a diameter of 0.1-0.4 mm. The resulting cell homogenate is centrifuged at 14,000 rpm for 40 minutes, and

As can be seen, the strain Staphylocpccus saprophyticus C-1.10 in the culture fluid accumulates two times more urease. the outcome of the strain Staphylococcus saprophyticus

LI -1. The specific activity in the cell extract of the new strain is approximately 4 times higher than the specific activity in the cell extract of the original strain.

90813 .6

This amount of enzyme by the action of which releases 1 µmol of ammonia per 1 minute at AIA.

J It was found that the maximum accumulation of ureash in the cells of a given strain. grown in flasks and in the fermenter takes place from 12 to 18 hours. The test results of the proposed strain are presented in Table 10. one.

Table

supernatant liquids measure urease activity and protein concentration. Protein is determined by the Lowry method. Using ethyl alcohol from the cell extract as an organic precipitant, the urease enzyme preparation is isolated.

The data obtained are presented in Table. 2

Table 2

Claims (2)

The use of the Staphylococcus Pirophyticus L-1.10 strain makes it possible to increase the urease activity of the dry enzyme preparation by a factor of 3.2 and the specific activity of the isolated urease by a factor of 5, as compared to using the original Iamma Staphylococcus saprophyticus, -1. Isolation of urease and cultivation of the strain in the fermenters does not change the material and labor costs for the production of this enzyme compared to using the original Staphylo strain (ccus saprophyticus. Therefore, the use of a new strain reduces the specific (urease activity) cost of urease produced. Staphylococcus saprophyticus L-1.10 is 0.05 rubles / 1000 units, compared with 0.10 rubles / 1000 units of enzyme preparation isolated from the original strain. The XI pentrum is 600000000 units. The expected annual savings from the production of urease 38 in the All-Russian Research Institute of Epidemiology from the Institute of Staphylococcus saprophyticus U-1.10 cells is 30000 rubles. Invention. B-2346) - urease producer. Sources of information taken into account during the examination 1.Van WyK and Steyn. P. Journal of benerat Microbiology, 91.2, 225-232, 1975. 2. USSR author's certificate No. P575986, cl. C 12 N 15/00, 1979 (prototype).