SU584801A3 - Method of preparing ferment oxidizing cholesterin to hydrogen peroxide and cholestenone - Google Patents

Description

one

This invention relates to the field of microbiology.

The method of obtaining the enzyme that oxidizes cholesterol to peroxide, and hydrogen and cholestenone is that the biomass of producer microorganisms is extracted with buffer containing a non-ionic surfactant and the target product is isolated from the cell-free extract, in addition, ProactijioJnvce culture is used as a producer eivthropotis strain NBC 9158. or LTCC 17895, or ATCC 42 77, or culture of Bocardioi formica strain ATCC 14811,

The microorganisms are decomposed, dissolved and removed using a buffer solution containing a non-ionic surfactant.

Preferred non-ionic surfactants are sterile ethylene. and adducts of polyethylene and polypropylene oxides, in particular: their esters. The principal concentration of the surfactant used in decomposition (dissolution) and for extraction in a buffer solution depends on the surfactant used and can be determined by preliminary tests. With

concentrations of 0.01–3%, mostly 0.1–1%, are suitable. The pH of the buffer solution is 5-9, preferably 6-8.

Mostly good results are obtained if the microorganism is washed with a mixture of butanol and water before dissolving and before extraction in the presence of a surfactant.

The resulting extract, containing the enzyme in solution, is purified on an ion exchanger.

Anion exchange resins, in particular low basic varieties, are used as the ion exchanger, and those that carry the diethylaminoethanol group are preferred ion exchangers.

Leaching of the ion exchanger is carried out with 0.01-0.2 M buffer solution with a pH of 5-9. Concentration of the surfactant: advantageously 0.052%.

Preferably the use of 0.03-0.1 M phosphate buffer solution with a content of 0.2-0.7% surfactant non-ionic agent.

From the eluate obtained in this way, the enzyme is precipitated with the help of salts or organic solvents, for example ammok sulfate, methanol and others.

To re-dissolve the precipitated enzyme, a buffer solution is used which contains a small amount of surfactant 1 agent. 5

The enzyme obtained after washing and washing can be used directly for the quantitative determination of cholesterol according to the equation "O colesterin (HH)" (O, cholestenone 4-NaOiE enzyme and the effectiveness of the proposed method is determined by THAT1 forming the fraction of HgO measured by known methods -, definition

Microorganisms are grown on a medium of mineral salts containing peptone; and as soon as the logarithm of the growth phase is reached, water emulsion is added, which is made by wet grinding of an aqueous suspension of cholesterol and subsequent sterilization at high temperature.

In this way, it is possible to achieve 100% use of cholesterol during the 25 one-day leveling of the culture (microorganism) with the release of L-2 g of the dry cell substance. A broth with cuprope is added in such a quantity that 1-20 g of cholesterol 30 y are set per liter during the growth period. Zagrueku is carried out in portions as microorganisms are used.

A small amount of cholesterol is added to the culture broth at the beginning of growth, i.e. before reaching the logarithmic growth phase.

An aqueous solution containing wt.% S 0.1-2 peptone, 0.01-0.2, 0.05i, 0, magnesium sulfate heptahydrate, which contains traces of ferric chloride and calcium chloride, is used as a nutrient medium. pH 6-9. The initial cholesterol loading is 0.01-0.1%.

The specific content of the enzyme is up to 7 V / r of the dry mass of bacteria and 1-3.5 g of the dry mass of bacteria per each liter of an inflammatory medium.

The specific content of microorganisms in the enzyme is improved by increasing the amount of sleep and alaborum on acetate and then on cholesterol as the only carbon source.

So, for example, when growing microorganisms on a medium of mineral salts with the composition indicated above, but when replacing peptone with acetate in a quantity of 0.5% by weight, prior to the addition of the cholesterol emulsion, the specific activity increases to 24 V / r. at the same time, the dry mass content increases to b, 5-g / l. Significant increase in the dry oil content and activity up to $ g

It is fired because a small amount of 0.02-1, preferably 0.05-0.3% by weight of the yeast extract is added as an emulsifier to the aqueous cholesterol suspension.

The preferred nutrient medium consists of an aqueous solution containing 0.1–2 wt.% Acetate (acetate monomer), 0.01–0.2 wt.% KgHPO, 0.051 wt.%, 0.05–0, l weight .% magnesium sulphate as heptahydrate, trace iron chloride and calcium chloride with a pH of 6-9. Upon reaching the logarithmic growth of acetate, the acetate is aamented with Ho-sterol. In order to keep the pH constant during the efastening, ttenpeiX: iBHO or acid is charged in portions. During the phases of growth using acetate, acetic acid is used as a carbon source, and as soon as it is converted to cholesterol, acetic acid is replaced with mineral acid. In this second stage of growth, npHMeansTt hydrochloric acid for p "guided pH value. At the same time reach an activity of up to 30V / r dry s & ssy,

; Example 1.2 l of a nutrient medium consisting of wt.% Casein peptone, 0.05 wt.%, 0.2 wt.% H KjPOv, 0.02 wt.% Magnesium sulfate heptahydrate, traces of ferric chloride and calcium chloride in tap water and using KOH regulate the pH value of 7.5, seeded in a rocking flask with 200 ml of a well-grown pre-culture P g OCSC i 1 by m y e e 5 er-vthropogig LB (SG E15Gi is ventilated with a magnetic stirrer. C the beginning of the logarithmic phase of growth, determined by the resulting increase in turbidity, in the period of further B1: fascations are periodically loaded at prepared by wet grinding, sterilized cholesterol emulsion, in such a way that 10 g of cholesterol is loaded into the flask for 20 h. 5 h after the last loading, the cell mass is collected by centrifugation 4 g of the dry mass of bacteria. ultrasound get 3V / g of enzyme.

Example 2. 15l of the medium described in example 1 together with 0.5l of well-grown pre-culture PfoactinoTTtTces ervttirpogis ATCC 17895 with strong aeration is cultured in a vessel. From the very beginning, 0.05% of cholesterol is loaded. over the course of further cultivation, so that a total of 50 g of cholesterol is loaded into the fermenter for 15 hours of cultivation. A bacterial mass of 40 g is obtained. From the bacterial mass, 7.1 V / g of the enzyme is obtained by sonication (decomposition) with ultrasound.

Example 3. As described in Example 2, bacteria Prooctinomyces .erylhropofis (BC 9158) are cultivated in a working volume of 60 l. Approximately 25 hours later, fresh nutrient medium is introduced into the vessel for growing bacteria, and the solution containing the grown culture is withdrawn from this vessel.

At the same time, a cholesterol suspension was introduced into the fermenter; for 1 h load about 3 g of cholesterol. Thus, with continuous cultivation, from 1.2 to 1.5 g / l of bacterial dry mass with the same specific activity.

Example4. In a fermenter with a capacity of 20 pcs, 15 liters of nutrient medium consisting of 0.5 wt.% Ammonium acetate, 0.05 wt.% Secondary potassium phosphate, 0.2 wt.% Primary ammonium phosphate, 0.02 wt.% Sulfate heptahydrate magnesium and traces of ferric chloride and calcium in tap water, sow 0.5 l of a well-grown pre-culture of bacteria YBC 9158 and aerated with strong aeration.

The culture pH is maintained constant by means of the current load of 20% acetic acid until the biomass concentration is about 1.5 g / l. After this content has been reached, the sterilized suspension is continuously loaded during further growth in such a way that, in general, 30 g of cholesterol is loaded into the fermenter during 20 hours of culture. Cholesterol suspension contains 0.1-0.2% yeast extract. A dilute hydrochloric acid is used to neutralize the culture in place of acetic acid.

After completion of the cultivation, the biomass by centrifuging is separated from the culture solution.

Froze With a two-step continuous growing method, two fermenters are combined with working volumes of 15 liters for the first and 70 liters for the second stage, so that when the flow rate is 4 liters, the nutrient medium described in Example 1 gives a flow ratio of 0.25-0.30 for the first stage and 0.055-0.065 for the second. By the beginning of the cultivation, the second stage is seeded with a well-grown Prvac-α pre-culture. tinomyces eirithropolis, ATGC 17895 In the amount of 1.5 liters and the second stage are inoculated with the same preliminary culture in the amount of 0.5 liters with a strong one; aeration of both steps, the first step being neutralized with acetic acid, and the second step with (hydrochloric acid).

At the same time in the second stage include the inflow system (4 l nutrient medium / enchantment). The pH value is kept 7-7.5. So for 1 h get 4 l of broth with the culture at 7 g / l with an activity of 30 / g dry weight. The culture solution is collected in a cooled tank and the bacterial mass is isolated from it in a continuously operating centrifuge. Approximately 10 g is required per hour for adjusting the pH in the first stage: 100% acetic acid.

fipHMepS. 40 ml of the suspension with the bacteria Hocapdia erythropofis (about 10 g dry weight) are mixed into 40 ml of 1 g β-butanol and stirred for several minutes at room temperature. The mixture was centrifuged, and butanol and excess aqueous phase were removed. The precipitate is again mixed into 40 ml of water and, after making a homogeneous suspension of the cells, is again mixed with 40 ml of p-butanol, mixed for a few minutes and. centrifugal-t yut. The precipitate is suspended in 40 ml of 0.01; a phosphate buffer solution (pH 7.0), to which 0.3% non-ionic surfactant (alkylaryl polyethylene glycol) is added and stirred for 30 mic at room temperature. Then centrifuged and residual is removed. Extract contains about 120 units. enzyme with an activity of 3 u / m protein.

This extract is mixed into the solid; house ammonium sulfate to a concentration of 1.3 M in ammonium sulfate at a temperature of 0 ° C, then the suspension is centrifuged, the precipitate is collected on the basis of an obvious lipophilic fraction on the surface of the solution. The sintered sediment is filtered, dissolved with 0.01 M phosphate buffer solution, pH 7 and dialyzed at a temperature of 0 ° C with respect to the same buffer solution. The dialyzed solution is passed through equilibrated with 0.01 M phosphate buffer, solution of a column of commodity anion exchange resin with diethylminoethanol groups based on dextran. In this case, the enzyme is adsorbed. After washing the column with the same buffer solution npoj, washing with solutions with 0.5%, 2% and 5% of the specified above-surfactant in water is successively performed. In this case, large quantities of contaminants are washed out.

Claims (2)

Then, successively, rinsing is carried out with 0.1 M phosphate buffer solution, pH 7; 0.05 M phosphate buffer solution, pH - 7, 0.2m phosphate buffer solution, pH 7, and 0.5 M phosphate buffer solution pH 7. Then the washing is again performed using a 0.05 M phosphate buffer solution (pH 7 ) and an enzyme with a 0.05 M phosphate solution, to which 0.05% surfactant is added, is extruded. The eluate contains about 80% of the enzyme activity adsorbed on the column in the enzyme with a specific activity of 25-30 V / mg protein. Example 7. The method of example 6 was repeated, but no water was washed with butanol. Thus, an extract was obtained for extracting ammonium sulfate, which contains about 120 units. enzyme with a specific activity of 1 u / ml protein. Further enrichment in a known manner results in a product with an activity of 8-10 V / mg protein. Example The method of Example 6 is repeated, but the anion exchanger is washed out with an aqueous solution which contains 5% of the same surfactant in the dissolved state. The enzyme is absorbed from vonit (anion exchange resin). Claim 1. Method for producing the enzyme, acid kidego cholesterol to hydrogen peroxide and cholestenone, characterized in that the biomass of microorganism producers is extracted with a buffer containing a nonionic surfactant and the desired product is isolated from the cell-free extract. 2. The method according to claim 1, characterized in that as a producer, Cent uses a culture of Proociinomyoes ' r thropotis strain KBC 9158, or ATCC 17895, or ATCC 4277, or Nosalka culture formica ATCC 14811