SU1151217A3 - Method of obtaining cholesterolesterase - Google Patents

Abstract

A method for producing cholesterol ester, which involves culturing the producing microorganism Pseudomonas on a nutrient medium containing mineral salts, phosphate buffer and inducer, followed by release of the enzyme from the culture fluid and / or cells, characterized in that, in order to increase the activity of the target product, as a producing microorganism strains of Pseudomonas spec. DSM 1281 or Pseudoraonas spec, DSM 1280, a as an inducer lecithin or soy lecithin in an amount of 0.5-2% by weight of the medium. (L

Description

FIELD OF THE INVENTION The invention relates to the microbiological industry and concerns a method for producing cholesterol esterase.

A method of producing cholesterol esterase is known, involving the cultivation of a strain producing Nocardia cho estero icum NRRL 5767 or NRRL 5768 on a nutrient medium containing the necessary mineral salts and the inducer is 1-10 g / l and yeast extract followed by release of the enzyme. The maximum enzyme activity is 49.2.

The closest to the proposed technical essence and the achieved positive effect is the method of obtaining cholesterol esterase 2J, involving the cultivation of producing strains of Pseudomonas fluorescens KY 3955, 4032., Etc. on a nutrient medium containing mineral salts, phosphate buffer and inducer - cholesterol ester, followed by release of the enzyme from the culture fluid and / or cells. According to the method, a nutrient medium containing, g / l: cholesterol ester-5, corn extract 2, NaNO, 2, KjHPQ, 1, KC2 0.5 and MgSO2, is used. The enzyme activity is 17.

Known methods inherent in a common drawback - low enzyme activity.

The purpose of the invention is to increase the activity of the target product.

The goal is achieved in that according to the method of obtaining cholesterol esterase, which controls the cultivation of the producing microorganism Pseudomonas on a nutrient medium containing mineral salts, phosphate buffer, and an inducer, followed by release of the enzyme from the culture fluid and / or cells, the Pseudomonas spec strains are used as the producing microorganism . DSM 1281 or P seudomonas spec. DSM 1280, a as an inductor - lecithin or soy lecithin in the amount of 0.5-2% by weight of the medium.

 The microorganisms used according to the invention are very similar and have the following characteristics: gram-negative; necessarily aerobic; yellowish columns on peptone macar agar; growth at 25, 30 and 37 C; no growth at 10j over cell size from 0.8 to 1 μm, from 2 to 4 μm; mobility, lophotrich begeipelt; tendency to the formation of chains, the absence of spores or prolonged stages: cytochrome - oxidase +; catalase +; indole-; nitrite-; gelatin monosubstrate - destruction: adonite + citrate +; galactose +; glucose +; isonite +; lactose +; mannitol +; mannose +; salicin +; sorbitol +.

The cultivation process is carried out under aerobic conditions at 15-45 ° C, preferably between 25 and, using sowing cultures grown on a rocking chair or in the air - Pumbers. The maximum yield is reached after 1-2 days of cultivation.

Example 1. A strain of Pseudomona spec, DSM 1280, transferred from a cold-cooled ampoule to a fast agar tube, was pre-cultured for two days in the main culture medium at 30 ° C under aerobic conditions (flask) and then in an amount up to 10 % were sown on Wednesday, which had the following composition per 1 l of water:

Disubstituted sodium phosphate 7 g Odisubstituted phosphate potassium 3 g Ammonium chloride 1 g Sodium chloride 0.05 g 1% ferric chloride solution 0.1 ml 0.2% copper chloride solution. 0.1 ml 1% solution of zinc sulphate 0.1 ml 10% solution of calcium chloride 1.0 ml 12% solution of magnesium sulphate 5.0 ml Soy lecithin 1.5% PH: 7.0

Cultivation was carried out with a rocking flask under aerobic conditions. After 2 days, an activity of approximately 1500 v / l is achieved (solution and biomass: substrate: cholesteryl oleate). Approximately the same yield was obtained when, ceteris paribus, instead of Pseudomonas spec. DSM 1280 Pseudomonas .spec. DSM 1281. Example 2. An insoluble cell mass was isolated from the culture solution prepared in accordance with Example 1 by centrifugation and used to determine cholesterol ester. The determination was carried out in accordance with the following reaction scheme: 1. Cholester-Cholesterol-Cholesterol + RHINOVA + Exterase acid N. O fatty ester 2. Cholester-Cholester-Cholestenonone rin + 1/2 0 Noxidase + 0 (catalysis) For measurement whether the following solutions: 1. Phosphate buffer 0.5 M, pH 7.5, 0.4% tesite. 2. Cholesterol oleate with 4 in tezitdioxane (v / v 1/1). 3.Hj, 0 approximately 0.6 m (5 ml perhydrol / 100 ml). 4. Catalase (0.01 mg protein / ml). 5. Cholesterol oxidase (min. 50 v / ml). 6. Cultural solution (approximately 5000 v / l, 1: 5 diluted, 0.01 ml test). To measure, 2.95 ml of solution 1 is mixed with 0.02 ml of solution 3. After 5 minutes, 0.01 ml of solution 6 and 0.02 ml of solution 5 are added and after 1 minute the reaction is initiated by addition. 0.1 ml of solution 2. The calculation is carried out according to the formula 3,12-51000. , T5 5 0 OTLE / MIN v / l culture solution. Thus, the proposed method allows to significantly increase the activity of cholesterol esterase.

Claims (1)

METHOD FOR PRODUCING CHOLESTERINESTERASINE, which involves culturing a producing microorganism Pseudomonas on a nutrient medium containing mineral salts, a phosphate buffer and an inducer, followed by isolation of the enzyme from the culture fluid and / or cells, characterized in that, in order to increase the activity of the target product, as a producing microorganism use strains of Pseudomonas spec. DSM 1281 or Pseudomonas spec. DSM 1280, and as an inductor lecithin or soya lecithin in the amount of 0.5-2% by weight of the medium. ω 1151217 2