SU1124027A1 - Culture medium for culturing producer of l-alpha-amino-epsilon-carbolactam hydrolase - Google Patents

Abstract

NUTRITIONAL ENVIRONMENT FOR Bbff ABILITIES OF THE PRODUCER L-L-AMINO-KAPROLAKTAMHYDROPAZY, containing lamiHo-g-caprolactam and inorganic salts - monosubstituted potassium phosphate, magnesium sulphate, manganese chloride, and the company will be at the beginning of the program. it additionally contains glycerin and corn extract in the following ratio of components, g / l of water: N L-ct-amino-E-caprolactam4, 5-5.5 Glycerin17-23 Corn extract, 5.5-6.5 Potassium phosphate 0 , 8-1,2 Sulphate magnesium

Description

The invention relates to the microbiological industry, in particular to the production of nutrient media for growing the producers of the intracellular enzyme L-ct-amino-b-caprolactam hydrolase. The proposed nutrient medium can be used for the first half of the biomass of the producer of the aminolactamase used for the production of L-lysine by the enzyme-enzymatic method. Nutrient media for growing producers of aminolactamase containing Pb-o6-amino-caprolactam (abbreviated as DL-aminolactam), yeast extract, peptone, as well as glucose and malt extract Cl3 and C2j are known. The inclusion of these expensive substances into the composition of these media — yeast extract, peptone, and malt extract — prevents their industrial use. Ll cultivation of amino lactamase nutrients is the most widely used nutrient medium containing, g / l of water: glucose 10, L-aminolactam 50, yeast extract 0.5 and inorganic salts ammonium nitrate 3, potassium phosphate monosubstituted 1, magnesium sulfate 0.5 and manganese chloride 0,2 C3J. However, the enzymatic activity of the biomass obtained on this medium is relatively low, the medium contains expensive components (yeast extract and L-amino caprolactam in high concentrations), as well as a component having nutritional value (glucose). The purpose of the invention is to increase the enzymatic activity of biomass and reduce the cost of the environment. The goal is achieved by the fact that the nutrient medium for growing the producer of L-o-amino-caprolactam rollase, sobranch L-O6-amino-caprolactam and inorganic salts - mono-potassium phosphate, magnesium sulphate, manganese chloride additionally contains glycerol and corn extract, trace agent, and tracer. the ratio of components, g / l of water: L-cC-amino- -caprolactam4-6. Glycerin17-23 Corn extract 5.5-6.5 Potassium phosphate monosubstituted 0.8-1.2 Magnesium sulphate (7-water) 0.4-0.6 Manganese chloride (4-water) 0.15-0.25 Essence The invention consists in the fact that glycerin and corn extract in the proposed medium, being a food source, are simultaneously necessary for another purpose of economically and efficiently obtaining L-° C-amino-caprolactam hydrolase in yeast cells, i.e. their use leads to the elimination of undesirable effects of catabolite repression, a significant decrease in the amount of biosynthesis inducer and an increase in the rate of formation of the enzyme. Glycerol, being equivalent to glucose as a carbon source, at the same time eliminates the effect on the biosynthesis of L-c-amino-caprolactam hydrolase of the undesirable effect of catabolite repression and thereby significantly reduces the concentration of L-aminocaprolactam in the medium without decreasing the enzymatic activity of the biomass. Thus, glycerin, showing properties as a food source, in the case of cultivation of the inductive enzyme producer b-X-amino-caprolactam hydrolase, significantly changes the composition of the components of the medium with the creation of a positive result. The nutritional value of kzgkuruzy extract can be considered equivalent to yeast extract. However, they differ significantly in their effect on the specific rate of formation of L-o-amino-b-caprolactam hydrolase in cells. When growing Sgurtococcus sp. 59 on the medium with glycerol, aminocaprolactam, phosphates and trace elements with a yeast or corn extract content of 0.4%, the specific growth rate in the exponential phase is 0.40 and 0.35 h, respectively, and the specific formation rate of the target enzyme is 0.08 h in the case of yeast extract and 0.33 h in the case of corn extract. A significant (about 4 times) increase in the specific biosynthesis rate at almost the same ipocTa rate is a distinctive feature of the corn extract when the L-O6-amino-caprolactam hydrolase producer is overpowered, leading to an extra-large result — an increase in the enzymatic activity of the producer

more than 2 times. This effect of the corn extract is explained by the lack of repressive biosynthesis of compounds in it, for example, L-lysine, which probably acts by the mechanism of repression by the product of metabolism.

To obtain a nutrient medium, the components (except for phosphoric acid, feces are dissolved in water, 10% NaOH solution is added to 7.0 and sterilized for 30 minutes while. Potassium phosphate is prepared and sterilized separately. Cryptocccus species should be produced with the proposed nutrient medium 5 being an amino lactamase producer.

Example 1. The cultivation of the producer of aminolactamase in flasks. .

For growing the producer of Cryptococcus cpecies 59 aminolactamase, a nutrient medium of the following composition is prepared, g / l of water:

L-aminolactam5

Glycerin20

Corn extract 6 KN2P04-1

MgSO -THgO0,5

MnClgAH O .0.2

The pH of the nutrient medium is adjusted to 7.0 with 10% NaOH solution and sterilized for 30 minutes at 120 s. The solution is sterilized separately and added to the nutrient medium before sowing. Nutrient medium is poured into 750 ml rocking flasks, the volume of medium in the flask is 100 ml.

The culture of Cryptococcus species 59 is renewed by growing in Petri dishes on an agar medium of the same composition for 48 hours at 30 hours. To prepare the seed, the culture is introduced into a shake flask with the medium and incubated for 14 h at a shaker at a frequency of 200 rpm and temperature. The inoculum obtained is seeded into rocker flasks with medium using 2.4 mg of culture per 100% of medium. They are grown on a pumper at a frequency of 200 rb / min, temperature at 18 h. Then the biomass is separated by centrifugation (min) and 4500 g, washed twice with water and lyophilized.

To determine the aminolactamase activity of microbial cells in woven on magnetic bag and

The cell thermostatted at 50 ° C is poured with 3 ml of a 0.1 M solution of L-aminolactam (pH 8.0) at 50 ° C and an accurately weighed amount (3-5 mg, depending on the activity) of the producer’s lyophilized cells is introduced. The mixture is stirred for several minutes, after which a control sample with a volume of 0.01 ml is taken to determine the background concentration of the hydrolysis product of aminolactam, lysine. After taking the control sample, the mixture is stirred for exactly 30 minutes. The reaction is stopped by adding 0.5 ml of 2.5 n. HC1. Then, 3 samples of 0.01 ml each were taken from the reaction mixture, applied to chromatographic paper, and the lysine content was determined spectrophotometrically, measuring the optical density of the colored product of the reaction of lysine with furfural by the method of M.Patthy, A.Patthy.

Aminolactamase activity calculated by the formula

LV-U 1000,

And product / min

  per 1 g of cells, where D is the difference in optical density of the test and control samples;

V is the volume of the reaction mixture in the cell (after octane reaction), ml; E is the mole extrusion coefficient; .

At - reaction time, min g - dry weight of cells containing, in the reaction cell, g.

. In order to determine the activity of the test cell lot, three reactions of enzymatic hydrolysis of aminolactam are set in parallel and the arithmetic average is calculated.

Example 2. The cultivation of the producer of amino lactamase in the fermenter.

Use laboratory fermenter type Biolafntt capacity of 20 liters. The preparation of the nutrient medium, the renewal of the culture and the preparation of the inoculum are carried out in the same way as in Example 1.

Seeds are sown in the fermenter using 0.5 g of culture per 10 liters of medium. 350 rpm, temperature 30 ° C, aeration 8-10 l / min, are grown under stirring for 18 hours. The biomass is cooled to + 4 ° C, centrifuged at 4500 g, washed twice with water and lyophilized. In tab. Table 1 shows the results of growing amirolactam producer. Method of growing Number of experiments In flasks 10 In a fermenter 5 Main technical and economic indicators of nutrient media confidence interval with a confidence probability of 0.95) in flasks and in a fermenter. Table 1 Enzymatic Biomass yield, activity jg / l CMMOL / MING g 7121642.5 + 0.4 730t585.0 ± 0.5 The growth of aminolactamases is presented in Table. 2. T and l and c a 2 stingenna environment un (in flasks) Offered in flasks in a fermenter 1064152 152 320712 730

As follows from the above data, the proposed nutrient medium is 7 times cheaper compared to the prototype, the specific enzymatic activity of 40% biomass increases 2.2 times, moreover, components with nutritional value are not used, which

Especially important in industrial applications.

With a good amount of fermentation of aminolacty ase in 500 m, the economic effect will be 456 thousand rubles. for sc (lt reduce the cost of the nutrient medium.

Claims (1)

NUTRITIONAL MEDIUM FOR GROWING Ι-Λ-ΑΜΗΗΟ-δ-ΚΑΠΡΟ LACTAMHYDROLASE PRODUCER, containing L- Ho-f-caprolactam and inorganic salts - monosubstituted potassium phosphate, magnesium sulfate, manganese chloride, characterized in that, in order to increase the enzymatic activity of biomass and reduce the cost of the medium, it additionally contains glycerin and corn extract in the following ratio of components, g / l of water : s L-ct-amino-E-caprolactam Glycerin Corn extract. Monosubstituted potassium phosphate Magnesium sulphate (7-water) Manganese chloride (4-water) 0.15-0.25 4.5-5.5 17-23 5.5-6.5 0.8-1.2 0.4-0.6 SU „„ 1124027 Magnesium Sulfate (7-Aq) Manganese Chloride (4-water) The essence of the invention is that glycerin and corn 0.4-0.6 0.15-0.25 is the extract in the proposed environment, being a food source, is simultaneously necessary for another purpose of economically and efficiently producing b-c (-amino-β-caprolactamhydrolase in yeast cells, i.e. their use. inducer of biosynthesis and an increase in the rate of formation of the enzyme. Glycerin, being equivalent to glucose as a carbon source, simultaneously excludes the effect on the biosynthesis of Ld-amino-E-caprolactamhydrolases rather than The effect of catabolite repression and thereby allows one to significantly reduce the concentration of L-aminocaprolactam in the medium without decreasing the enzymatic activity of biomass. caprolactamhydrolase significantly changes the composition of the components of the medium with the creation of a positive result. .Corn extract in its nutritional value can be considered equivalent to a yeast extract. However, they differ significantly in their effect on the specific rate of formation of Ld-amino-E-caprolactamhydrolase in cells. When growing Cryptococcus sp. 59 on a medium with glycerin, aminocaprolactam, phosphates and microelements with a yeast or corn extract content of 0.4%, the specific growth rate in the exponential phase is 0.40 and 0.35 h ' ^1, respectively, and the specific rate of formation of the target enzyme is 0, 08 hours in the case of yeast extract and 0.33 hours' ¹ in the case of corn extract. A significant (about 4 times) increase in the specific rate of biosynthesis at almost the same growth rate is a hallmark of the corn extract when growing the producer of L-cfc-amino-E-capro • lactamhydrolase, leading to an ultimately total result - increasing the enzymatic activity of the producer producer of aminosirok widely 1 1124027