SU1523569A1 - Strain of citrobacter freundii bacteria for biotransformation of fumarate to l-malic acid - Google Patents

Abstract

The invention relates to the production of organic acids by the microbiological method and can be used in the microbiological, medical and food industries, in perfumery and metallurgy (as a complexing agent in the extraction of certain metals). The aim of the invention is the bacterial strain CITROBACTER FREUNDII VKPMNB-4090, which has a high synthesizing activity, stability of biosynthesis, a higher degree of substrate conversion into L - malic acid, which will increase the yield of the target product. The strain has a high, relatively stable level of fumarate - hydratase, the activity of which depends little on the age and growing conditions of the culture. This makes it possible to obtain more than 99% of the target product from the amount of substrate consumed. Due to the growth of the proposed bacterial strain on the medium with 3% fermentolizat BVK and the subsequent immobilization of native bacterial cells in carrageenan, the yield of the target product increases. 1 tab.

Description

The invention relates to biotechnology, the production of organic acids by the microbiological method and can be applied in the microbiological, medical, food industry, in perfumery and metallurgy (as a complexing agent, in the extraction of certain metals) .;

The purpose of the invention is a strain of bacteria of the genus Citrobacter freundii (strain ZG), which has a high synthesizing activity, stability of biosynthesis, regardless of the culture age, a higher degree of substrate conversion to L-malic acid, which allows to increase the yield of the target product.

Strain Citrobacter freundii ZG obtained from natural substrates (garden soil) as a result of selection on the medium with formate and deposited in the collection of industrial microorganisms VNIIGenetika and has a registration. The current number is B-4090.

The Citrobacter freundii strain VKPN No. 8-4090 is characterized by the following features.

Morphological and physiological signs.

Bacterial cells are Gram-negative motile peritris1 Hc5 CO SP

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chial sticks with rounded ends of 0.4-0.5 X 1-2 them, capsules and spores do not form. Facultative anaerobes. Optimal growth is observed with ZS-SO C. One-day colonies on m-peptone agar (MPA) are round, shiny, transparent, of the same type, with smooth edges, 1-2 mm in diameter, and later grayish-white, shiny. With growth on solid media around the colonies of the day, concentric and circles appear, in the form of shvermerov.

Growth on the moc-peptone broth (MPB) is plentiful, in the form of muti ,. with a grayish tint, the film does not form, in old cultures turbidity, sediment. No formation of indole was observed in 2 hour cultures. Hydrogen sulfide forms.

The strain grows in liquid medium from 21% calcium formate and 0, peptone. In this case, it gives turbidity throughout the test tube, forms a gas and a dense film of chalk on the surface of the medium due to decomposition of formic acid.

With growth on milk, acidification and coagulation occur. Growth on potato agar in the form of a grayish-white bloom. Culture has a characteristic smell. Gelatin does not dilute.

Attitude to carbohydrates. Decomposes with the formation of acid and gas, glucose, fruktoa, galactose, arabinose, mannitol, xylose, starch. Lactose is fermented slowly. Does not grow on sucrose, dextrin, dulcite, inositol. As a carbon source, it uses pyruvate, formate, fumarate, glycerin. KNC does not inhibit growth. Malonate does not use, it can grow on media containing 1% monmonic acid as the sole carbon source.

Relation to nitrogenous substances. It grows well due to ammonium and amino nitrogen, peptone. Nitrates restores to nitrites. It has catalase activity.

The strain grows well in laboratory environments. No need for growth factors. The cultivation of bacteria lasts from 9 to 36 hours, better than 12 to 18 hours, with magnitudes of pH 6.8- 8.0 (better at 7.0-7.2) and temperatures of 20-37 0 (better at 25- 30 ° C).

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Storage conditions of the strain on solid media (MPA) under a layer of sterile vaseline oil or in a lyophilized state.

Example 1. One-day cells of bacteria C.freundiiBKHM No. were washed from the surface of MPA with 1-2 ml of sterile liquid tap water and transferred with a sterile pipette into a sterile inoculum liquid of the same composition (BCH), the initial pH value which is 7.0-7.2. The culture is grown for 10-12 hours at D3-ZO C in 750 ml rocking chair flasks (medium volume 50 ml). Then, seed in the amount of 2-3 is introduced into the medium of the same composition and grown for h in the same way, in similar rocking flasks with a medium volume of 125 ml. Cells are separated from the medium by centrifugation at 8000 rpm for 10 minutes.

Biomass is suspended in 0.01 M phosphate buffer (p. H 7.3) at the rate of 6-16 mg of protein of bacteria cells in 1 ml of suspension. The determination of the fumarate hydratase activity of bacterial cells is carried out in a known manner by the amount of L-block acid formed from potassium fumarate. The lactic acid synthesis reaction is carried out for 1 hour with the rocking chair at 175 rpm.

The specific activity of fumarase of 9 clock cells of C.freundii bacteria is 3 g equal to 2.6 1U MM malic acid per mg of protein per second, which corresponds to 97.9 of the fumarate consumed; in 24-hour cell culture of 3.1 malic acid (99.2 from consumed fumarate); have a 48-hour culture. 3, block acid (98.9 from consumed fumarate) and 72-hour cells 2, which corresponds to 98.3 conversion of fumarate to malate.

The results of the comparison of the fumarate activity of the strain with the known strain Paracolobacter aerogenoides f 14 When the whole cells of bacteria are incubated with fumaratomikal for 1 h, are presented in the table.

PRI mme R 2. The cultivation of the biomass of the cells of the bacteria C.freundii VKPM No. B-4090 is carried out as described in Measure 1 for 12-14 h.

The reaction mixture for the synthesis of malic acid contains in 1 ml

1.65 mg: protein of cells, either 3.3 mg of protein, or, 95 mg of protein of bacterial cells. The determination of fumarate-hydratase activity is carried out in a known manner. The consumption of fumarate from its initial amount in the reaction mixture is 52.8, 76.8 or 79.21, respectively, with the conversion of the consumed fumarate into malic acid, equal to 87.2, or 95.3, or 99.2.

The specific activity of fumarase cells of C.freundii VKPM No. in dependence1523369

in a sterile synthetic medium with a pH value of 7.2, prepared in distilled water, of the following composition,%: NayEP04 1,

5 0.15; (mg, S04 0.2; MgSO ,.x7H20 0.02 CaCl2 6H O 0.01; FeS04 0.00005;, Zn + 2 traces; glucose or glycerin 1.0. Culture

Q is grown for 2 hours at 28–30 ° C in 750-ml swing-flasks with a volume of 0 ml of medium. Then, seed amount R of 3-5 is added to

sterile. liquid medium of the same proportion of the amount of protein, containing 5 similar flasks per 1 ml of the reaction mixture, corresponding to a medium volume of 125 ml and grown is either 5.1-10 or 3.7 -10, or 2.6-10-mI per 1 mg of protein per 1 second.

at the same temperature for 30- 48h.

Cells are separated from medium by centrifuge.

PRI me R 3. One-day cells 20 girovania at 8000 rpm for

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C.freundii VKPM No. B-j090 is washed from the surface of the agar (1.5 agar) medium from the fermentolizate of the biomass of microorganisms grown on hydrocarbons (3% of the fermentolizate BVK in 25-way water) and using a sterile pipette, they are transferred to a liquid inoculum medium of the same composition (initial pH value 7.2). The culture is grown for 10–12 hours at 28–30 ° C in 750 ml capacity containers with a volume of 50 ml of medium. Then, seed in the amount of 2-3% is introduced into the medium of the same composition and the culture is grown for 12–16 h in the same way in similar rocking flasks with a medium volume of 125 ml. Cells are separated from the medium by centrifugation at 8000 rpm for 10 minutes. ,

The biomass is suspended in 0.01 M phosphate buffer pH 7.5 (6-20 mg of the protein of bacteria cells in 1 ml of suspension) and the activity of fumarate-hydratase is determined by a known method.

The specific activity of fumarase of 12-hour cells of C.freundii bacteria grown on the medium in 3% of BBKjpaBHa 2.4 malic acid fermentolizate per 1 mg of protein of bacterial cells in 1 s, which corresponds to 99% conversion of the fumarate consumed.

EXAMPLE 4 One-day cells of C.freundii VKPM N-4090 bacteria were washed from the surface of an agar (1.5 agar) MPA medium. 1-2 ml of sterile tap water and transferred with a sterile pipette.

10 minutes, then the biomass is suspended in 0.01 M phosphate buffer at pH 7.5 at the rate of 10–20 mg protein of bacterial cells and the activity of fumarate-hydrotase is determined by a known method.

The specific activity of fumarase cells of C.freundii VKPM B-4090 bacteria grown on synthetic medium with glucose or glycerol is equal to 2.910 and 2.1 malic acid per 1 mg of bacteria cells, respectively, per 1 s.

PRI me R 5. Growing the biomass of the bacteria C.freundii VKPN GV-4090- is carried out either on the BCH, as described in example 1, or on a medium with 3% of fermentolizat BVK, as described in example 3.,

The initial specific fumarate hydratase on the activity of C.freundii bacteria cells grown on the medium with BCH equals 8, on the medium with the fermentative BVK 5.7-10 mG s.

A portion of the volume of the bacterial cell suspension is stored for 7 days at (-4) - (6) ° C in phosphate buffer. After this time, the specific activity of the fumarase of bacteria cells grown on the BCH is 82.7%, and on the medium with the fermenolizate of BVK b1.4 from the initial specific activity of the bacterial cells.

Another pbloain volume of the cell suspension of bacteria C.freundii is stored for 7 days, at (-4) - (6) C in the fumarate solution. After this time, the specific enzymatic activity of cells grown on the BCH is 108.6, and on the medium with fer40

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in a sterile synthetic medium with a pH value of 7.2, prepared in distilled water, of the following composition,%: NayEP04 1,

 0.15; (mg, S04 0.2; MgSO ,.x7H20 0.02 CaCl2 6H O 0.01; FeS04 0.00005;, Zn + 2 traces; glucose or glycerin 1.0. Culture

They are grown for 2 hours at 28–30 ° C in 750-ml swirl flasks with a medium volume of 0 ml. Then, seed amount R of 3-5 is added to

sterile. liquid medium of the same composition in the same rocking flasks with a medium volume of 125 ml and grown

at the same temperature for 30- 48h.

Cells are separated from medium by centrifuge.

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10 minutes, then the biomass is suspended in 0.01 M phosphate buffer at pH 7.5 at the rate of 10–20 mg protein of bacterial cells and the activity of fumarate-hydrotase is determined by a known method.

The specific activity of fumarase cells of C.freundii VKPM B-4090 bacteria grown on synthetic medium with glucose or glycerol is equal to 2.910 and 2.1 malic acid per 1 mg of bacteria cells, respectively, per 1 s.

PRI me R 5. Growing the biomass of the bacteria C.freundii VKPN GV-4090- is carried out either on the BCH, as described in example 1, or on a medium with 3% of fermentolizat BVK, as described in example 3.,

The initial specific fumarate hydratase activity of the cells of C.freundii bacteria grown on the medium with the BCH equals 8 on the medium with the fermentative BVK 5.7-10 mG s.

A portion of the volume of the bacterial cell suspension is stored for 7 days at (-4) - (6) ° C in phosphate buffer. After this time, the specific activity of the fumarase of bacteria cells grown on the BCH is 82.7%, and on the medium with the fermenolizate of BVK b1.4 from the initial specific activity of the bacterial cells.

Another pbloain volume of the cell suspension of bacteria C.freundii is stored for 7 days, at (-4) - (6) C in the fumarate solution. After the indicated time, the specific enzymatic activity of cells grown on the BCH is 108.6, and on the medium with fer0

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mentholizatom BVK 98.0% of the initial specific activity of bacterial cells.

Example 6. The cultivation of the biomass of C.freundii VKPM M ° B-4090 bacteria is carried out on the BCH, as described in Example 1.

The resulting cell suspension is divided in half and in one portion determines the specific activity of fumarate hydratase, which is taken to be 100%.

A biocatalyst is prepared from another portion of the cell suspension by immobilizing the native cells of the C.freundii bacterium in carrageenan. To do this, 6 ml of a physiological solution (0.8S% NaCl in distilled water) are placed in a 500 ml glass beaker and 0.35 g of carrageenan is added. The suspension of carrageenan in physiological solution is left for 30-40 minutes at room temperature (18-22 ° C) for swelling. After this time, suspension I is heated in a water bath until the carrageenan is completely dissolved (PP-EO C). The resulting solution is cooled to 5 and 1 ml of the bacterial cell suspension is added, the mixture is mixed thoroughly and the mixture is cooled for 10 minutes in an ice bath or in the freezer of a refrigerator. The block of immobilized cells is transferred into a glass lOO ml glass beaker containing 300 ml of 0.3 M KCl and kept for 1 h at room temperature to increase the rigidity of the gel. After the indicated times, the obtained gel block with immobilized bacteria cells is wiped through a sieve with a cell diameter of 1 ml. The biocatalyst do-irradiated granules are washed 2-3 times with 0.05 M Phosphate buffer pH 7.5. The determination of fumarate hydratase activity is carried out by a known method for the consumption of fumarate and the formation of L-block acid.

The biocatapist obtained in this way contained 0.8202 mg of bacterial cell protein in 1 ml of the reaction mixture and exhibited an activity equal to 180 from the initial one.

When immobilized to carrageenan, the amount of formation of the side pro;

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The L-aspartic acid product is from 0-0.3 to 2.1-2.

Example 7. The cultivation of the biomass of the bacteria C.freundii VKPM No. is carried out on a medium with fermentolizat BVK, as described in example 3.

The resulting cell suspension is divided in half and in one portion of the suspension the initial specific fumarase activity is taken, which is taken as 100.

A biocatalyst is prepared from the other half of the bacterial cell suspension by immobilizing the native bacterial cells in-carrageenan, as described in Example 6,

The biocatalyst obtained in this way contains 0.1028 mg of protein in 1 ml of the reaction mixture and exhibits a specific activity equal to 290% of the initial activity of native cells.

Forms of barking 3 gains Strain bacteria Citrobacter freun-i dii VKPM f B-4090 for biotransformation of fumarate into L-block acid.

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Cell incubation time, h Formation of malic acid, g / l Conversion of fumarate to malic acid,% Speed of transformation of fumarate into malic acid, g / l-h - Specific activity, g of malic acid .- dry biomass MIN

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60.9

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Claims (1)

<claim-text> <table border = "1"> <tbody> <tr> <td> Parameters </ td> <td> C., £ geip &lt; XY VKPM № B4090 </ td> <td> RagasoHoSeg Aego * §EnocSec No. 743 </ td> </ tr> <tr> <td> Cell incubation time, h </ td> <td> 1 </ td> <td> 5 </ td> </ tr> <tr> <td> Formation of malic acid, g / l </ td> <td> 60.9 </ td> <td> 26.0 </ td> </ tr> <tr> <td> Conversion of fumarate to malic acid,% </ td> <td> 78 </ td> <td> 39 </ td> </ tr> <tr> <td> The rate of transformation of fumarate into malic acid, g / l - h * <sup> 1 </ sup> </ td> <td> 60.9 </ td> <td> 5.2 </ td> </ tr> <tr> <td> Specific activity, g malic acid- · G "’ dry biomass ‘Min '<sup> 1, </ sup> </ td> <td> 5.87 </ td> <td> 0.21 </ td> </ tr> </ tbody> </ table>