CN105177065B - A kind of method of biotransformation method synthesis α-ketoglutaric acid - Google Patents

Abstract

The invention provides a method for synthesizing α-ketoglutarate by a biotransformation method. The method uses L-glutamic acid as a substrate, and the transformation obtained by culturing Kluyveromyces marxianus ATCC36534 in a transformation medium Add α-ketoglutarate dehydrogenase inhibitor to the culture solution, carry out synthetic transformation culture at 25-30° C. and 200-250 r/min shaking conditions, after the synthetic transformation culture is completed, the synthetic transformation solution is separated and purified to obtain the obtained solution. The α-ketoglutarate described; the yeast cells in the growth state convert L-glutamic acid into α-ketoglutarate, and add α-ketoglutarate dehydrogenase inhibitor to block its further metabolic consumption , so that α-ketoglutarate accumulates in a large amount in the culture medium; when the substrate L-glutamic acid feeding concentration is 50 g/L, the molar conversion rate can reach 83.2%; The high-value α-ketoglutaric acid can be converted into large-scale industrial application, and the production process has the advantages of short cycle, high conversion rate and low environmental pollution.

Description

A kind of method for synthesizing α-ketoglutaric acid by biotransformation

(1) Technical field

The invention belongs to the technical field of biochemical industry, and specifically relates to a method for synthesizing α-ketoglutaric acid by a biotransformation method.

(2) Background technology

α-ketoglutaric acid (α-ketoglutaric acid), also known as α-carbonylglutaric acid, 2-oxoglutaric acid or α-glutonic acid, CAS number is 328-50-7. α-Ketoglutarate is one of the important intermediates in the tricarboxylic acid (TCA) cycle and plays an important role in cellular material metabolism and energy metabolism. As an important intermediate, it is an important precursor for the synthesis of various amino acids and vitamins. Alpha-ketoglutaric acid has important application prospects in the fields of medicine, organic synthesis, nutritional fortifiers, etc. At present, its main application fields are: as a component of sports functional drinks, an intermediate in organic synthesis, and a matching liver function test Reagents and physique enhancers; reduce the body loss of postoperative patients and long-term patients; some studies have shown that α-ketoglutaric acid has anti-cyanide effect, and the combination of sodium nitrite and sodium thiosulfate can improve the anti-cyanide ability, Has anticonvulsant effect.

At present, the production process of α-ketoglutaric acid is mainly chemical synthesis. The chemical synthesis method is prepared by chemical reaction using succinic acid and diethyl oxalate as raw materials, and it is facing elimination due to high cost and serious pollution. The production of α-ketoglutaric acid can also adopt biological fermentation method or biological transformation method. Compared with chemical synthesis method, biological method has the advantages of mild production conditions, simple process steps, and environmental friendliness. In recent years, great progress has been made in the study of biological synthesis of α-ketoglutarate, and the fermentation unit or conversion rate has been greatly improved, but the production cost is still higher than that of chemical synthesis, and there are still certain difficulties in industrial application.

In recent years, there have been many research reports on the biological synthesis of α-ketoglutaric acid in China, and some patents have been applied for. For example, Chen Jian and others from Jiangnan University used recombinant Yarrowia lipolytica to ferment and produce α-ketoglutarate. After 144 hours of fermentation, the yield of α-ketoglutarate reached 47.2g/L (Chinese invention patent ZL201210066444 .6); Chen Ning et al. of Tianjin University of Science and Technology utilized the fermentation of Corynebacterium glutamicum to produce α-ketoglutarate, and the maximum yield of α-ketoglutarate was 47.2g/L ( Chinese invention patent ZL201110392778.8). However, the fermentation method also has its shortcomings, such as a long production cycle, the product and the various components in the fermentation broth are mixed together, resulting in complicated extraction and refining processes and high total cost. The biotransformation method may overcome the shortcomings of the fermentation method, which can increase the product concentration, simplify the extraction process and reduce the cost. For example, Tao Rongsheng et al. used L-glutamate oxidase to biotransform L-glutamate or its salts, and the concentration of α-ketoglutarate was as high as 138.5g/L, and the molar yield was over 80% (China Invention patent application number 201310134674.6). However, in this invention, L-glutamate oxidase needs to be prepared, and commercial catalase needs to be added when catalyzing the reaction, so the cost of the enzyme in production is relatively high. Chen Jian et al. of Jiangnan University also reported a method for the production of α-ketoglutarate by whole-cell transformation of L-glutamic acid. The L-amino acid deaminase gene was transformed by error-prone PCR or site-directed saturation mutagenesis. After expression of Bacillus, L-glutamic acid was transformed to generate α-ketoglutarate, and the molar yield could reach more than 85%, but the substrate concentration was only 15 g/L, and the yield needed to be improved.

(3) Contents of the invention

The object of the present invention is to provide a method for synthesizing α-ketoglutarate by using Kluyveromyces marxianus ATCC36534 whole-cell biotransformation of L-glutamic acid, using yeast cells as biocatalysts and L-glutamic acid as Raw material, whole-cell biotransformation to synthesize α-ketoglutarate. In high-glucose and low-nitrogen medium, dehydrogenase inhibitors are added to prevent further metabolism of α-ketoglutarate, thereby promoting the accumulation of α-ketoglutarate, so that cheap L-glutamate can be converted For the higher-priced α-ketoglutaric acid, the problem of high production cost of the current fermentation method, enzymatic method and whole-cell transformation method is solved, and it has high industrial application value.

The whole cell method biotransformation synthesis α-ketoglutaric acid reaction formula of the present invention:

The technical scheme adopted in the present invention is:

The invention provides a method for synthesizing α-ketoglutarate by a biotransformation method. The method uses L-glutamic acid as a substrate, and the transformation obtained by culturing Kluyveromyces marxianus ATCC36534 in a transformation medium Add α-ketoglutarate dehydrogenase inhibitor to the culture medium, carry out synthetic culture at 25-30° C. and 200-250 r/min shaking conditions, after the synthetic culture is completed, the synthetic culture medium is separated and purified to obtain the described α-ketoglutarate; the α-ketoglutarate dehydrogenase inhibitor is one or a mixture of hydrogen peroxide (H ₂ O ₂ ) or methotrexate; the transformation medium The composition is (reagents are all commercially available analytical pure or biological reagents): sucrose 50～100g/L, yeast extract powder 10～20g/L, KH ₂ PO ₄ 3～5g/L, K ₂ HPO ₄ 5～6.5g /L, MgSO ₄ 0.5～1.0g/L, the solvent is water, and the pH value is natural.

Further, the added amount of the substrate in the transformation medium is 20-50 g/L. The substrate L-glutamic acid (CAS: 56-86-0) is a commercially available raw material with a purity of ≥98%.

Further, the added amount of the α-ketoglutarate dehydrogenase inhibitor is 0.0005-50 mmol/L in terms of the volume of the transformed culture medium. When the α-ketoglutarate dehydrogenase inhibitor is H ₂ O ₂ , the H ₂ O ₂ is added in the form of an aqueous H ₂ O 2 solution with a mass concentration of 30%, and the H ₂ O ₂ in the aqueous H ₂ O ₂ solution is added The amount of O ₂ added is 10-50 mmol/L based on the volume of the transformed culture medium. When the α-ketoglutarate dehydrogenase inhibitor is methotrexate, the methotrexate is added in the form of an aqueous solution of methotrexate of 1 mmol/L, and the methotrexate in the aqueous solution of methotrexate is The amount of aminopterin added is 0.5-1 μmol/L based on the volume of the transformed culture medium. When the α-ketoglutarate dehydrogenase inhibitor is a mixture of H ₂ O ₂ and methotrexate, the H ₂ O ₂ is added in the form of an aqueous H ₂ O ₂ solution with a mass concentration of 30%, and H The amount of H ₂ O ₂ added in the ₂ O ₂ aqueous solution is 40 mmol/L in terms of the volume of the transformed culture solution, and the methotrexate is added in the form of a 1 mmol/L aqueous solution of methotrexate, and the aqueous solution of methotrexate is added. The amount of methotrexate added in the medium was 0.8 μmol/L based on the volume of the transformed culture medium. The function of the α-ketoglutarate dehydrogenase inhibitor is to inhibit the activity of α-ketoglutarate dehydrogenase and block the further metabolism of α-ketoglutarate, so that α-ketoglutarate can be obtained. accumulation.

The Kluyveromyces marxianus ATCC36534 of the present invention performs transformation reaction with the substrate in the form of a transformation solution with a dry cell concentration of 6-10 g/L, wherein the transformation solution is to inoculate Kluyveromyces marxianus ATCC36534 slant cells into the transformation culture It is obtained by inoculating the seed liquid with a dry cell concentration of 5-7 g/L into a transformation medium with an inoculation amount of 5-10% by volume, and then transforming and culturing.

Specifically, the steps of synthesizing α-ketoglutaric acid by the biotransformation method of the present invention are as follows:

(1) Inoculate the cell body of Kluyveromyces marxianus ATCC36534 stored in the refrigerator on a fresh slant medium, and cultivate in a constant temperature incubator at 25-30° C. for 24-36 hours to obtain an activated Kluyveromyces marxianus ATCC36534 strain. The slant culture medium is composed of: glucose 10～20g/L, peptone 5～10g/L, yeast extract powder 3～5g/L, agar 15～20g/L, solvent is water, pH is natural (measured 6.3～6.5). ), sterilized by high pressure steam at 121℃ for 15-20min.

(2) Picking 2 to 3 rings of Kluyveromyces marxianus ATCC36534 slant cells in step (1) after activation and culturing, and inoculating them into the seed medium. The seed after inoculation is cultured for 20-24 hours under the shaking conditions of 25-30 ℃ and 200-250 r/min, and the seed liquid with the dry cell concentration of 5-7 g/L is obtained. The composition of the seed medium is the same as that of the slant medium in step (1) except that agar is not added. Sterilize with high pressure steam at 121℃ for 15-20min.

(3) Pick 3 to 5 rings of Kluyveromyces marxianus ATCC36534 slant cells after step (1) activation and culture with the inoculating loop, or the seed solution prepared in step (2), and then receive 5% to 10% by volume. into the transformation medium, and cultured at 25-30°C and 200-250r/min shaking conditions for 20-24h to obtain dry cells with a concentration of 6-10g/L, adding H ₂ O ₂ with a final concentration of 10-50mmol/L , or methotrexate with a final concentration of 0.5-1.0 μmol/L, or both are added simultaneously; and then L-glutamic acid with a final concentration of 20-50 g/L is added. Synthetic culture in the triangular flask is carried out under the same conditions for 20 to 24 hours to obtain a synthetic culture solution with a concentration of α-ketoglutarate of 17.3 to 42.9 g/L.

The transformation medium is composed of: sucrose 50-100 g/L, yeast extract powder 10-20 g/L, KH ₂ PO ₄ 3-5 g/L, K ₂ HPO ₄ 5-6.5 g/L, MgSO ₄ 0.5-5 g/L 1.0g/L, the solvent is tap water, the pH is natural (measured 6.5), and the filling amount in the triangular flask is 20% to 40% of its volume. The triangular flask is tied with 8 layers and sterilized by high pressure steam at 121°C for 15-20min.

(4) After the synthetic culture solution prepared in step (3) is centrifuged at 3000-4000g for 5-10min to remove the bacterial cells, supernatant a is obtained, activated carbon is added, the activated carbon is filtered off after stirring for 30-60min, and the filtrate is concentrated under reduced pressure To 1/2～1/4 of the original volume, centrifuge again to remove the precipitate to obtain supernatant b, add α-ketoglutaric acid seed crystals, slowly stir and cool to 4°C, stand for 8～12h, and collect by suction filtration The α-ketoglutaric acid crystals were vacuum-dried at 45°C to obtain crystalline α-ketoglutaric acid; the activated carbon was added in an amount of 5-10 g/L in terms of the volume of the supernatant a, and the α-ketoglutaric acid was The added amount is 10-20 g/L in terms of the volume of the supernatant b.

The yeast strain used in the present invention is Kluyveromyces marxianus ATCC36534, which was purchased from American Type Culture Collection (ATCC). Colony characteristics of this strain: on the wort agar plate medium, the colony is milky white, shiny, raised, neatly edged, moist, smooth surface and uniform in texture.

The method for determining the concentration of dry cells of the present invention is as follows: take 10 mL of culture solution, centrifuge at 4000 g and discard the supernatant, resuspend the precipitated cells with 10 mL of distilled water, and centrifuge again to discard the supernatant. After drying the cells at 105°C for 12 hours, weigh the total weight (W _total ), subtract the empty weight of the centrifuge tube (W _empty ) from the total weight, and calculate the cell concentration according to the following formula:

Dry cell concentration (g/L)=(W _total -W _empty )×100.

The supernatant a and the supernatant b of the present invention are both supernatants, and are named for the convenience of distinguishing the supernatants obtained in different steps, and the letters themselves have no meaning.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

The invention provides a method for synthesizing α-ketoglutarate by utilizing Kluyveromyces marxianus ATCC36534 whole-cell biotransformation of L-glutamic acid. Yeast (Kluyveromyces marxianus) ATCC36534 transformation culture medium, adding α-ketoglutarate dehydrogenase inhibitor, the method of synthetic culture, the yeast cells in growth state convert L-glutamic acid into α-ketoglutarate, The addition of α-ketoglutarate dehydrogenase inhibitor blocked its further metabolic consumption, so that α-ketoglutarate accumulated in the medium in large quantities; when the substrate feeding concentration was 50g/L, the culture medium was synthesized The concentration of α-ketoglutaric acid in the product can reach 41.3g/L, and the molar conversion rate is 83.2%. The α-ketoglutaric acid separated by crystallization method has a purity of 96.7% and a yield of 81.4%; Yeast whole cell transformation has the following advantages: low nutritional requirements for yeast cells, short culture time, strong anti-pollution ability; fast growth and high yield , strong enzymatic activity; no harmful substances are produced in the conversion process, and the concentration of by-products is low; the present invention converts low-value L-glutamic acid into high-value α-ketoglutaric acid, which can be applied in large-scale industrialization, and the production process has a cycle It has the advantages of short time, high conversion rate and low environmental pollution.

(4) Specific implementations

The present invention is further described below in conjunction with specific embodiment, but the protection scope of the present invention is not limited to this:

Example 1: Screening of transformed yeast strains

7 yeast strains were purchased in the market, including 2 strains of Saccharomyces cerevisiae, 1 strain of Pichia pastoris, 1 strain of Candida guilliermond, and Sporidiobolus johnsonii ) 1 strain, 1 strain of Kluyveromyces marxianus and 1 strain of Candida tropicalis. Among them, Kluyveromyces marxianus was purchased from the American Culture Collection, and the deposit number was ATCC36534.

First, pick 1 full ring of the above yeast slant strains stored in the refrigerator, inoculate it in a fresh slant medium, and cultivate the slant in a 30°C biochemical incubator for 36 hours to obtain an activated yeast strain slant. Pick 4 full circles of cells from the activated strain slant and inoculate it into a 250 mL conical flask containing 50 mL of transformation medium. 0.80g (final concentration of 20g/L) of L-glutamic acid and 31μL of 30wt% H2O were added to each bottle of culture solution ₂ O ₂ aqueous solution (final concentration is 10 mmol/L), the Erlenmeyer flask was continuously shaken under the same conditions for synthesizing culture for 24 h.

After the synthetic culture, take 40 mL of synthetic culture solution in a 50 mL centrifuge tube, centrifuge at 4000 g for 5 min, pour out the supernatant, filter the supernatant through a 0.22 μm microporous membrane, and analyze the α-ketoglutarate in the filtrate by HPLC concentration.

The above methods were used to compare the ability of seven yeast strains to transform L-glutamic acid into α-ketoglutarate. The results showed that the transformation efficiency of Kluyveromyces marxianus ATCC36534 was the highest, and the α-ketopentane in the transformation solution The diacid concentration was 8.3 g/L, and the molar conversion rate was 41.8%, so this strain was selected as the microbial strain for synthesizing α-ketoglutaric acid by biotransformation.

The slant culture medium is composed of: glucose 10g/L, peptone 5g/L, yeast extract powder 3g/L, agar 20g/L, solvent is water, pH is natural (measured 6.5), and high-pressure steam is sterilized at 121° C. for 15min.

The transformation medium is composed of: sucrose 50g/L, yeast extract powder 10g/L, KH ₂ PO ₄ 3g/L, K ₂ HPO ₄ 5g/L, MgSO ₄ 0.5g/L, the solvent is tap water, the pH is natural (measured 6.5). 50mL transformation medium was placed in a 250mL conical flask, 8 layers of gauze were tied, and sterilized by high pressure steam at 121°C for 15min.

The HPLC method for analyzing the concentration of α-ketoglutaric acid was as follows: Shimadzu LC-20 HPLC instrument, Agilent Zorbax Extend C18 reversed-phase chromatographic column (5 μm, 250 mm×4.6 mm), the detection wavelength was UV 210 nm, and the sample was injected. The volume was 20 μL, the column temperature was 25° C., the mobile phase was ultrapure water containing 0.1% phosphoric acid, and the flow rate was 1.0 mL/min.

Example _{2 :} Selection of H2O2 Concentration

After determining that Kluyveromyces marxianus ATCC36534 was used as the strain for transforming L-glutamic acid into α-ketoglutarate, H ₂ O ₂ was added to the transformation medium as an α-ketoglutarate dehydrogenase inhibitor, In this example, the optimal concentration of H ₂ O ₂ was selected for optimization.

First, pick 1 full ring of Kluyveromyces marxianus ATCC36534 slant strain stored in the refrigerator, inoculate it in fresh slant medium, and cultivate the slant in a 30°C biochemical incubator for 24 hours to obtain an activated Kluyveromyces marxianus ATCC36534 strain bevel. Pick 2 rings of thalli from the slanted surface of the activated strain into a 50mL seed medium (in a 250mL Erlenmeyer flask). Seed liquid with a bacterial concentration of 5.35 g/L. Use a sterile pipette to draw 5 mL of the seed solution of ATCC36534 into a conical flask containing 45 mL of transformation medium (installed in a 250 mL conical flask), and in a constant temperature shaking shaker, 30 ° C, 200 r/min shaking transformation culture for 24 h, to obtain The transformation solution with a dry cell concentration of 7.17 g/L.

In above-mentioned ATCC36534 transformation liquid, add the H ₂ O ₂ (that is, the H ₂ O of mass concentration 30% of the final concentration of 0mmol/L, 10mmol/L, 20mmol/L, 30mmol/L, 40mmol/L, 50mmol/L respectively ₎ 0 μL/vial, 31 μL/vial, 62 μL/vial, 93 μL/vial, 124 μL/vial, and 155 μL/vial) of aqueous solution, and 20 g/L (0.8 g/vial) of L-glutamic acid, each experiment set up 3 replicates , the flask was continuously shaken under the same conditions for 24h of synthetic culture.

After the synthesis culture, according to the method described in Example 1, the α-ketoglutarate concentration in the transformation solution was analyzed by HPLC. The results showed that after Kluyveromyces marxianus ATCC36534 was grown in the transformation medium for 24 hours, if H ₂ O ₂ was not added to the transformation system, the molar conversion rate of α _- ketoglutarate was only 11.6 _% . Significantly promotes the conversion of alpha-ketoglutarate. When the concentration of H ₂ O ₂ was 40 mmol/L, the concentration of α-ketoglutaric acid in the conversion solution was the highest, reaching 15.6 g/L, and the molar conversion rate was 78.5%.

The composition and preparation method of the slant medium, seed medium and transformation medium are the same as in Example 1.

Example 3: Selection of methotrexate concentrations

After determining that Kluyveromyces marxianus ATCC36534 was used as the strain for transforming glutamic acid into α-ketoglutarate, methotrexate was added to the medium as an α-ketoglutarate dehydrogenase inhibitor. The optimal concentration of methotrexate was selected for optimization.

First, pick 1 full ring of Kluyveromyces marxianus ATCC36534 slant strain stored in the refrigerator, inoculate it in fresh slant medium, and cultivate the slant in a 30°C biochemical incubator for 24 hours to obtain an activated Kluyveromyces marxianus ATCC36534 strain bevel. Pick 2 rings of thalli from the slanted surface of the activated strains and put them into a conical flask containing 50 mL of seed medium (installed in a 250 mL conical flask). , a seed solution with a dry cell concentration of 5.43 g/L was obtained. Use a sterile pipette to draw 5 mL of the seed solution of ATCC36534 into a conical flask containing 45 mL of transformation medium (installed in a 250 mL conical flask). , a transformation solution with a dry cell concentration of 7.24 g/L was obtained.

In the above-mentioned ATCC36534 transformation solution, add methotrexate (i.e., 1 mmol/L) of 0 μmol/L, 0.5 μmol/L, 0.6 μmol/L, 0.7 μmol/L, 0.8 μmol/L, 0.9 μmol/L and 1 μmol/L respectively at the final concentrations. /L of methotrexate aqueous solution 0 μL/vial, 20 μL/vial, 24 μL/vial, 28 μL/vial, 32 μL/vial, 36 μL/vial and 40 μL/vial), and 20 g/L (0.8 g/vial) of L- Glutamate, three replicates were set for each experiment, and the flask was continuously shaken for 24h under the same conditions.

After the synthesis culture, according to the method described in Example 1, the α-ketoglutarate concentration of the transformation solution was analyzed by HPLC. The results showed that after the growth of Kluyveromyces marxianus ATCC36534 in transformation culture for 24 hours, the addition of methotrexate could significantly promote the transformation rate of α-ketoglutarate. When the concentration of methotrexate was 0.8 μmol/L, the concentration of α-ketoglutarate in the transformation solution was the highest, reaching 14.8 g/L, and the molar conversion rate was 74.5%.

The composition and preparation method of the slant medium, seed medium and transformation medium are the same as in Example 1.

Example ₅ : Simultaneous addition of _H2O2 and methotrexate

In this example, H ₂ O ₂ and methotrexate were simultaneously added as α-ketoglutarate dehydrogenase inhibitors in the transformation medium.

First, pick 1 full ring of Kluyveromyces marxianus ATCC36534 slant strain stored in the refrigerator, inoculate it in fresh slant medium, and cultivate the slant in a 30°C biochemical incubator for 24 hours to obtain an activated Kluyveromyces marxianus ATCC36534 strain bevel. Pick 2 rings of thalli from the slanted surface of the activated strain into a 50mL seed medium (in a 250mL Erlenmeyer flask). Seed solution with a bacterial concentration of 5.38 g/L. Use a sterile pipette to draw 5 mL of the seed solution of ATCC36534 into a conical flask (250 mL conical flask) containing 45 mL of transformation medium. The transformation solution with a dry cell concentration of 7.21 g/L.

In the above-mentioned transformation solution of ATCC36534, H ₂ O ₂ with a final concentration of 40 mmol/L (in the form of an aqueous solution of H ₂ O ₂ with a mass concentration of 30%) and 0.8 μmol/L of methotrexate (with 1 mmol/L) were added simultaneously. L of methotrexate aqueous solution), and 20g/L (0.8g/bottle) of L-glutamic acid, the conical flask was continuously shaken under the same conditions to carry out synthetic culture for 24h.

After the synthesis culture, according to the method described in Example 1, the α-ketoglutarate concentration of the transformation solution was analyzed by HPLC. The results showed that after the growth of Kluyveromyces marxianus ATCC36534 in transformation culture for 24h, adding 40mmol/L H ₂ O ₂ and 0.8μmol/L methotrexate at the same time, compared with adding one of them, α-keto The conversion rate of glutaric acid was significantly improved, the concentration of α-ketoglutaric acid in the conversion liquid was 17.3 g/L, and the molar conversion rate reached 87.1%.

The composition and preparation method of the slant medium, seed medium and transformation medium are the same as in Example 1.

Example 6: Preferred Transformation Process

Taking Kluyveromyces marxianus ATCC36534 as the transformation strain, on the basis of Example 5, conditions such as seed medium composition, transformation medium composition, substrate concentration, transformation culture time were optimized, and the preferred biotransformation method was used to synthesize α- The process steps of ketoglutaric acid are as follows:

(1) inoculate the thalline of ATCC36534 stored in the refrigerator on a fresh slant medium, and cultivate in a 30°C constant temperature incubator for 24h to obtain the activated strain of ATCC36534; the slant medium consists of: glucose 10g/L, Peptone 5g/, yeast extract powder 3g/L, agar 20g/L, water solvent, natural pH (measured 6.5), high pressure steam sterilization at 121°C for 15min.

(2) Picking with an inoculation loop Step (1) 2 loops of ATCC36534 slanted cells after activation and culture are inoculated into the seed medium. The seed culture after inoculation is based on culturing for 20 hours at 30 ° C and 200 r/min shaking conditions to obtain a seed liquid with a dry cell concentration of 7.03 g/L; the seed medium is composed of: glucose 20 g/L, peptone 10 g/L, The yeast extract powder is 5g/L, the solvent is water, and the pH is natural (measured 6.3). 50 mL of seed culture medium was put into a 250 mL conical flask, 8 layers of gauze were tied, and high-pressure steam was sterilized at 121 °C for 15 min.

(3) According to the inoculum volume percentage of 5%, the seed liquid prepared in step (2)) is transferred to the transformation medium, and the transformation culture is based on 30 ° C and 200 r/min shaking conditions for 20 hours, and the dry cell concentration is 9.87 g/L transformation solution, add H ₂ O ₂ with a final concentration of 40 mmol/L (in the form of a 30% mass concentration of H ₂ O ₂ aqueous solution) and 0.8 μmol/L of methotrexate (with 1 mmol/L In the form of methotrexate aqueous solution), L-glutamic acid with a final concentration of 50g/L (2.5g/bottle) was added, and the triangular flask was synthesized and cultured for 20h under the same conditions.

The transformation medium is composed of: sucrose 100g/L, yeast extract powder 20g/L, KH ₂ PO ₄ 5g/L, K ₂ HPO ₄ 6.5g/L, MgSO ₄ 1.0g/L, the solvent is tap water, The pH is natural (measured 6.5). 50 mL of transformation medium was put into a 250 mL conical flask, 8 layers of gauze were tied, and high-pressure steam was sterilized at 121 °C for 15 min.

After the synthesis culture, according to the method described in Example 1, the α-ketoglutarate concentration of the transformation solution was analyzed by HPLC. The results showed that: α-ketoglutaric acid was synthesized according to the above preferred process biotransformation method. When the concentration of substrate feeding was 50 g/L, the concentration of α-ketoglutaric acid in the conversion solution could reach 42.9 g/L, and the molar conversion rate was 42.9 g/L. was 86.4%.

Example 7: Amplification and separation and purification

Taking Kluyveromyces marxianus ATCC36534 as the transformed strain, on the basis of Example 6, the shake flask scale was put into 250 mL, and the steps were as follows:

(1) inoculate the thalline of ATCC36534 stored in the refrigerator on a fresh slant medium, and cultivate in a 30°C constant temperature incubator for 24h to obtain the activated strain of ATCC36534; the slant medium consists of: glucose 10g/L, Peptone 5g/, yeast extract powder 3g/L, agar 20g/L, water solvent, natural pH (measured 6.5), high pressure steam sterilization at 121°C for 15min.

(2) Picking with an inoculation loop Step (1) 2 loops of ATCC36534 slanted cells after activation and culture are inoculated into the seed medium. The seed culture after inoculation is based on culturing for 20 hours at 30 ° C and 250 r/min shaking conditions, and the dry cell concentration is 6.96 g/L seed liquid; the seed medium is composed of: glucose 20 g/L, peptone 10 g/, yeast The leaching powder is 5g/L, the solvent is water, and the pH is natural (measured 6.3). 100 mL of seed culture medium was put into a 500 mL conical flask, 8 layers of gauze were tied, and high-pressure steam was sterilized at 121 °C for 20 min.

(3) Transfer the seed liquid prepared in step (2) to the transformation medium according to the inoculum volume percentage of 5%, and the transformation culture is based on 30 ° C and 250 r/min shaking conditions for 20 hours, and the dry cell concentration is 10.23 g /L transformation solution, add H ₂ O ₂ with a final concentration of 40 mmol/L (in the form of a 30% mass concentration of H ₂ O ₂ aqueous solution) and 0.8 μmol/L of methotrexate (in 1 mmol/L of H 2 O 2 ). In the form of methotrexate aqueous solution), L-glutamic acid with a final concentration of 50g/L (12.5g/bottle) was added, and the triangular flask was synthesized and cultured for 20h under the same conditions to obtain a synthetic culture medium.

The transformation medium is composed of: sucrose 100g/L, yeast extract powder 20g/L, KH ₂ PO ₄ 5g/L, K ₂ HPO ₄ 6.5g/L, MgSO ₄ 1.0g/L, the solvent is tap water, The pH is natural (measured 6.5). 250mL of transformation medium was put into a 1L conical flask, 8 layers of gauze were tied, and high pressure steam was sterilized at 121°C for 20min.

(4) 5 bottles of synthetic culture solution prepared in step (3) were combined, and after centrifugation at 3000 g for 10 min to remove bacterial cells, 1.2 L of the remaining supernatant was obtained, 12 g of activated carbon was added, and the activated carbon was filtered off after stirring for 60 min. The filtrate was concentrated under reduced pressure to 300 mL, centrifuged again to remove the precipitate, and the remaining supernatant obtained (volume in 300 mL) was added with 6 g of crystalline α-ketoglutaric acid, slowly stirred and cooled to 4 ° C, and after standing overnight, suction filtration The α-ketoglutaric acid crystals were collected and dried under vacuum at 45°C to obtain crystalline α-ketoglutaric acid. According to the above process biotransformation method to synthesize α-ketoglutaric acid, when the concentration of substrate feeding is 50 g/L, the concentration of α-ketoglutaric acid in the conversion solution can reach 41.3 g/L, and the molar conversion rate is 83.2%. The α-ketoglutaric acid isolated by crystallization has a purity of 96.7% and a yield of 81.4%.

Claims (4)

1. a kind of method of biotransformation method synthesis α-ketoglutaric acid, it is characterised in that the method is with kluyveromyces marxianus (Kluyveromyces marxianus) ATCC36534 is catalyst, using Pidolidone as substrate, ties up ferment in Marx's Crewe In the conversion culture solution that female inverted culture medium culture of ATCC36534 obtains, ketoglurate dehydrogenase inhibitor is added, 25 ~30 DEG C, conversion culture is carried out under 200~250r/min oscillating condition, after conversion culture, conversion culture solution is pure through separating Change and obtains the α-ketoglutaric acid；Additive amount of the substrate Pidolidone in conversion culture solution is 50g/L；The α- Ketoglutaric dehydrogenase inhibitor is the mixing of one or both of hydrogen peroxide or methotrexate (MTX), and the hydrogen peroxide is with matter The form for measuring the aqueous hydrogen peroxide solution of concentration 30% is added, and the additional amount of hydrogen peroxide is in aqueous hydrogen peroxide solution to convert training Nutrient solution volume is calculated as 40mmol/L；The methotrexate (MTX) is added in the form of 1mmol/L methotrexate (MTX) aqueous solution, the first ammonia butterfly Methotrexate (MTX) additional amount is calculated as 0.8 μm of ol/L to convert nutrient solution volume in purine aqueous solution；The composition of the conversion culture medium are as follows: Sucrose 100g/L, yeast extract powder 20g/L, KH₂PO₄5g/L, K₂HPO₄6.5g/L, MgSO₄1.0g/L, solvent are water, and pH is certainly So.

2. the method for biotransformation method synthesis α-ketoglutaric acid as described in claim 1, it is characterised in that Marx's Crewe Tie up yeast ATCC36534 in the form of the conversion culture solution of 6~10g/L of dry mycelium concentration with substrate carry out conversion reaction, described turn Changing culture solution is kluyveromyces marxianus ATCC36534 inclined-plane thalline to be seeded to conversion culture medium or by dry mycelium concentration 5 ~7g/L seed liquor is seeded to what the inverted culture of conversion culture medium obtained with the inoculum concentration of percentage by volume 5-10%.

3. the method for biotransformation method synthesis α-ketoglutaric acid as described in claim 1, it is characterised in that the method is by as follows Step carries out:

(1) thallus of kluyveromyces marxianus ATCC36534 is inoculated in fresh slant medium, is trained in 25~30 DEG C of constant temperature It supports and cultivates 24~36h, the kluyveromyces marxianus ATCC36534 strain after being activated in case；The slant medium Composition are as follows: 10~20g/L of glucose, 5~10g/L of peptone, 3~5g/L of yeast extract powder, 15~20g/L of agar, solvent are Water, pH are natural；

(2) with 2~3 ring of kluyveromyces marxianus ATCC36534 inclined-plane thalline after oese picking step (1) activation culture, Be seeded to seed culture medium, the seed culture after inoculation be based on 25~30 DEG C, culture 20 under 200~250r/min oscillating condition~ For 24 hours, the seed liquor that dry mycelium concentration is 5~7g/L is obtained；The seed culture medium composition are as follows: 10~20g/L of glucose, egg White 5~10g/L of peptone, 3~5g/L of yeast extract powder, solvent are water, and pH is natural；

(3) with 3~5 ring of kluyveromyces marxianus ATCC36534 inclined-plane thalline after oese picking step (1) activation culture, Or step (2) preparation seed liquor by percentage by volume 5%~10% amount access conversion culture medium, in 25~30 DEG C, 200~ It is cultivated under 250r/min oscillating condition, obtains the conversion culture solution of 6~10g/L of dry mycelium concentration, α-ketoglutaric acid dehydrogenation is added Enzyme inhibitor adds Pidolidone, carried out under 25~30 DEG C, 200~250r/min oscillating condition conversion culture 20~ For 24 hours, after conversion culture, conversion culture solution is through isolating and purifying to obtain the α-ketoglutaric acid.

4. the method for biotransformation method synthesis α-ketoglutaric acid as claimed in claim 1 or 3, it is characterised in that the conversion culture The method that liquid isolates and purifies are as follows: after conversion, conversion culture solution is centrifuged off thallus through 3000~4000g, 5~10min Afterwards, supernatant a is obtained, active carbon is added, filters out active carbon after stirring 30~60min, filtrate decompression is concentrated into the 1/2 of original volume ~1/4, it is centrifuged off sediment again, obtains supernatant b, α-ketoglutaric acid crystal seed is added, be slowly stirred and be cooled to 4 DEG C, it is quiet 8~12h is set, collected by suction α-ketoglutaric acid crystal is dried in vacuo in 45 DEG C, obtains lenticular α-ketoglutaric acid；The activity Charcoal additional amount is calculated as 5~10g/L with supernatant a volume, and the α-ketoglutaric acid Seed charge is calculated as 10 with supernatant b volume ~20g/L.