CN104846025A - Method for preparing (2S, 3R)-2-benzoyl aminomethyl-3-hydroxy methyl butyrate - Google Patents

Abstract

The invention discloses a method for preparing (2S, 3R)-2-benzamidomethyl-3-hydroxybutyrate methyl ester, the method comprising: preparing engineering bacteria containing carbonyl reductase gene and glucose dehydrogenation Enzyme gene engineering bacteria; prepare the resting cell suspensions of two engineering bacteria respectively; mix the two resting cell suspensions, and then racemize 2-benzamidomethyl-3-carbonylbutyl with the substrate Methyl ester, hydrogen donor and cofactor are mixed for asymmetric reduction reaction to produce (2S,3R)-2-benzoylaminomethyl-3-hydroxybutyric acid methyl ester; the base of carbonyl reductase gene The sequence is shown in SEQ ID NO.1, and the base sequence of the glucose dehydrogenase gene is shown in SEQ ID NO.2. The method can catalyze the reaction of the substrate racemic 2-benzoylaminomethyl-3-oxobutyric acid methyl ester to generate (2S, 3R)-2-benzoylaminomethyl-3-hydroxybutyric acid methyl ester, The conversion rate and purity of the product are improved.

Description

A method for preparing (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester

technical field

The invention relates to the technical field of biopharmaceuticals, in particular to a method for preparing (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester.

Background technique

(2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester is a kind of optically active β-hydroxy esters, and is also a kind of chiral alcohol. Its chemical structure is as follows: II) shown. (2S,3R)-2-Benzamidomethyl-3-hydroxybutyrate methyl ester is synthesized from (3R,4R)-3-[(R)-1-tert-butyldimethylsiloxyethyl] - the key starting material of 4-acetoxy-2-azetidinone (abbreviated as 4-AA, chemical structural formula as shown in formula (III)), 4-AA is a kind of important pharmaceutical fine chemicals, It is mainly used for the synthesis of various penem antibiotics, such as imipenem, biapenem, meropenem and faropenem. These drugs have a wide range of uses, and have broad-spectrum and potent antibacterial effects on Gram-negative and positive bacteria, aerobic bacteria, and anaerobic bacteria, so they have received great attention. At present, in terms of the synthesis process of 4-AA, the synthetic route of racemic 2-benzamidomethyl-3-oxobutanoic acid methyl ester as raw material is relatively superior, therefore, how to pass the reaction with high selectivity The precise establishment of the chiral center is the key to the reaction process.

In the existing reports, the chiral catalyst (R)-BINAP-Ru is used as a better effect, but this method needs to use the precious metal ruthenium as a catalyst, and it needs to be carried out under high temperature and high pressure conditions, which requires high requirements for the reactor. , so this route limits the large-scale industrial production of 4-AA. In addition, there are also some reports on utilizing biocatalysis to carry out asymmetric reactions, such as Peter Schneider et al. (US Patent, US 4927507) have reported a method of reducing 2-benzamidomethyl-3-carbonylbutyrate by baker's yeast method, the product configuration obtained is (2S, 3S)-2-benzoylaminomethyl-3-hydroxybutyrate and (2R, 3S)-2-benzoylaminomethyl-3-hydroxybutyrate Ester, thus need to carry out configuration flip through chemical method again, the operation is cumbersome, and the recovery rate is low.

American Codexis Company has screened a carbonyl reductase AdhR, and after a series of improvements in molecular biology, it uses isopropanol as a co-substrate to achieve coenzyme regeneration, allowing AdhR to directly catalyze the production of (2S, 3R) configuration product. However, it is difficult to match the activity of coenzyme consumption and regeneration by using this method, thereby affecting the catalytic effect of AdhR; the reaction is easily limited by the thermodynamic equilibrium of the two substrates and products, and it is difficult to obtain the optimal thermodynamic conditions for the reaction; excessive coenzyme The substrate will also inhibit the enzyme and reduce the catalytic efficiency, and the addition of auxiliary substrates will increase the complexity of product separation and purification and increase the cost.

Contents of the invention

The invention provides a method for preparing (2S, 3R)-2-benzoylaminomethyl-3-hydroxybutyric acid methyl ester, which can catalyze the substrate racemization 2-benzoylaminomethyl- The 3-carbonylbutyric acid methyl ester reacts to generate (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester, which improves the conversion rate and purity of the product.

A method for preparing (2S, 3R)-2-benzoylaminomethyl-3-hydroxybutyric acid methyl ester, comprising:

(1) preparing engineering bacteria containing the carbonyl reductase gene and engineering bacteria containing the glucose dehydrogenase gene;

(2) prepare the resting cell suspensions of two kinds of engineering bacteria respectively;

(3) After mixing the two resting cell suspensions, they are mixed with the substrate racemic 2-benzamidomethyl-3-oxobutanoic acid methyl ester, hydrogen donor and cofactor for asymmetric reduction Reaction to obtain (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester;

The base sequence of the carbonyl reductase gene is shown in SEQ ID NO.1, and the base sequence of the glucose dehydrogenase gene is shown in SEQ ID NO.2.

The carbonyl reductase gene (abbreviated as LbADH gene), is cloned from wild bacterium Lactobacillus brevis, and the amino acid sequence of the protein encoded by the carbonyl reductase gene is as shown in SEQ ID NO.7; The glucose dehydrogenase gene (abbreviated as GdhBM Gene), cloned from Bacillus Megaterium, the amino acid sequence of the protein encoded by the glucose dehydrogenase gene is shown in SEQ ID NO.8.

Wherein, the engineering bacterium contains the expression vector pET-30a(+), and the host cell is Escherichia coli BL21(DE3).

The carbonyl reductase gene and the glucose dehydrogenase gene are respectively expressed in Escherichia coli BL21 (DE3) to obtain the carbonyl reductase and glucose dehydrogenase.

Both the carbonyl reductase and the glucose dehydrogenase are in the form of resting cells of genetically engineered bacteria as catalysts in the asymmetric reduction reaction. The preparation method of the resting cell suspension comprises: inoculating the engineering bacterium into the culture medium containing kanamycin, after the shaker is activated, expanding the culture until the OD600 value reaches 0.8-1.2, adding an inducer, and continuing Cultivate, collect cells by centrifugation, and resuspend with buffer to obtain the resting cell suspension.

Preferably, the inducer is IPTG at a concentration of 0.5-0.8mM. Preferably, the culture conditions after adding the inducer are as follows: the culture temperature is 16-25° C., and the culture time is 10-20 h. The buffer is sodium dihydrogen phosphate-disodium hydrogen phosphate, preferably, the buffer has a concentration of 100-150 mM; the buffer also contains 1-10 mM MgCl ₂ .

The reaction formula of asymmetric reduction reaction of the present invention is as follows:

Throughout the reaction, on the one hand, the carbonyl reductase LbADH catalyzes the asymmetric reduction of racemic 2-benzamidomethyl-3-oxobutanoic acid methyl ester to generate stereoisomerically pure (2S,3R)-2- Benzamidomethyl-3-hydroxybutyrate, accompanied by the conversion of the reduced cofactor NADPH to the oxidized cofactor NADP ⁺ , on the other hand, glucose dehydrogenase oxidizes glucose to gluconolactone, At the same time, the reduced cofactor NADPH is regenerated, forming a closed loop of cofactor consumption and regeneration to promote the main reaction.

In the asymmetric reduction reaction, the hydrogen donor is glucose, and the cofactor is NADP ⁺ /NADPH.

During the reaction, the concentration of carbonyl reductase and glucose dehydrogenase has an influence on the yield of the final product. Preferably, in the mixture of the two resting cell suspensions, the mass ratio of the resting cells containing carbonyl reductase to the resting cells containing glucose dehydrogenase is 4:1-1:2. More preferably, the mass ratio of resting cells containing carbonyl reductase to resting cells containing glucose dehydrogenase is 1:1.

Specifically, in the asymmetric reduction reaction, the substrate concentration is 2.5-500 g/L, the concentration of resting cells containing carbonyl reductase is 0.1 g _{dry weight} /L-25 g _{dry weight} /L, and the concentration of glucose dehydrogenase The concentration of resting cells is 0.1g _{dry weight} /L-25g _{dry weight} /L, the concentration of hydrogen donor is 5-750g/L, and the concentration of cofactor is 0-0.5mM.

Preferably, the temperature of the asymmetric reduction reaction is 25-40° C., and the pH of the reaction buffer is 6.0-8.0. More preferably, the reaction temperature is 30-37° C., and the pH of the reaction buffer is 6.5-7.5.

During the whole reaction process, after reacting until the substrate is completely consumed by HPLC, extract with an equal volume of organic solvent for 2 to 4 times, combine the extract phases, dry over anhydrous sodium sulfate, and distill off the organic solvent under reduced pressure to obtain the target product.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The present invention applies the engineered bacteria containing carbonyl reductase LbADH and the engineered bacteria containing glucose dehydrogenase GdhBM to catalytic substrate racemization 2-benzamidomethyl-3-carbonyl butyrate methyl ester In the reaction to generate (2S, 3R)-2-benzoylaminomethyl-3-hydroxybutyric acid methyl ester, as (2S, 3R)-2-benzoylaminomethyl-3-hydroxybutyric acid methyl ester The production of provides a new preparation route;

(2) The present invention uses carbonyl reductase LbADH and glucose dehydrogenase GdhBM to combine, so that the activity of coenzyme consumption and regeneration in the reaction process is matched, and the best preparation of (2S, 3R)-2-benzamide is obtained The method of methyl-3-hydroxybutyrate methyl ester improves the conversion rate and purity of the product.

Description of drawings

Fig. 1 is the electrophoresis figure of gene Lbadh of the present invention;

M: nucleic acid marker, 1, 2: gene Lbadh sample.

Fig. 2 is the electrophoresis figure of gene GdhBM of the present invention;

M: nucleic acid marker, 1, 2: gene GdhBM sample.

Figure 3 is a map of plasmid pET30-LbADH.

Figure 4 is a map of plasmid pET30-GdhBM.

Fig. 5 is the SDS-PAGE electrophoresis diagram of the protein induced and expressed by the genetically engineered bacterium EcoLbADH;

M: Protein Marker, 1: pET-30a(+) empty plasmid control cell lysis supernatant, 2: genetically engineered bacteria EcoLbADH induced cell lysis supernatant, 3: genetically engineered bacteria EcoLbADH induced cell lysis supernatant.

Fig. 6 is the SDS-PAGE electrophoresis figure of the protein induced and expressed by the genetically engineered bacteria EcoGdhBM;

M: Protein Marker, 1: supernatant of cell lysis induced by genetically engineered bacteria EcoGdhBM, 2: cell lysis precipitate induced by genetically engineered bacteria EcoGdhBM.

Fig. 7 is the HPLC analytical spectrum of 2-benzamidomethyl-3-oxobutanoic acid methyl ester standard.

Fig. 8 is the HPLC analysis spectrum of (2S, 3R)-2-benzamidomethyl-3-hydroxybutyrate standard product.

Fig. 9 is an HPLC analysis standard spectrum of the measured ee value of (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester.

Fig. 10 is the ¹ H NMR spectrum of methyl 2-benzamidomethyl-3-oxobutanoate.

Fig. 11 is the ¹ H NMR spectrum of the reaction product (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester.

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments.

Construction of embodiment 1 plasmid pET30-LbADH

The Lbadh gene was cloned with primers F_LbADH/R_LbADH to obtain a 759bp Lbadh gene (as shown in SEQ ID NO.1). Nucleic acid electrophoresis to verify gene size, as shown in Figure 1.

The sequence of primer F_LbADH is: 5'CGC GGATCC ATGTCTAACCGTTTGGATG-3';

The sequence of primer R_LbADH is: 5'CCG CTCGAG CTATTGAGCAGTGTAGCCAC-3'.

BamHI and XhoI double-digest Lbadh gene, recover the gene band after digestion, BamHI and XhoI double-digest pET-30a(+) plasmid, recover the plasmid band after digestion, and digest the Lbadh gene and enzyme The cut pET-30a(+) plasmid was ligated with ligase and transformed into the cloning host Escherichia coli DH5α. Colony PCR was performed with primers F_LbADH/R_LbADH to verify the transformed recombinant, and then the recombinant plasmid was extracted for sequencing. The recombinant plasmid with correct sequencing results is the recombinant plasmid pET30-LbADH, the plasmid map is shown in Figure 3, and it is stored at -20°C for future use.

Construction of embodiment 2 plasmid pET30-GdhBM

The GdhBM gene was cloned with primers F_GdhBM/R_GdhBM to obtain a GdhBM gene (as shown in SEQ ID NO.2) with a length of 786bp. Nucleic acid electrophoresis to verify gene size, as shown in Figure 2.

The sequence of primer F_GdhBM is: 5'-GGA AGATCTG ATGTATAAAGATTTAGAAGGA-3';

The sequence of primer R_GdhBM is: 5'CCG CTCGAG TTATCCGCGTCCTGCTTGGAA-3'.

GdhBM gene was digested with glII and XhoI, and the gene band after digestion was recovered. The pET-30a(+) plasmid was digested with BglII and XhoI, and the plasmid band after digestion was recovered. The digested GdhBM gene and enzyme The cut pET-30a(+) plasmid was ligated with ligase and transformed into the cloning host Escherichia coli DH5α. Colony PCR was performed with primers F_GdhBM/R_GdhBM to verify the transformed recombinants, and then the recombinant plasmids were extracted for sequencing. The recombinant plasmid with correct sequencing results is the recombinant plasmid pET30-GdhBM, the plasmid map is shown in Figure 4, and it is stored at -20°C for future use.

Construction and induced expression of embodiment 3 genetically engineered bacteria

The expression host Escherichia coli BL21(DE3) was transformed with the plasmids pET30-LbADH and pET30-GdhBM constructed in Examples 1 and 2, respectively. Colony PCR was performed with primers F_LbADH/R_LbADH and F_GdhBM/R_GdhBM respectively to verify the transformed recombinants. The verified genetically engineered bacteria are EcoLbADH and EcoGdhBM. Inoculate EcoLbADH and EcoGdhBM into 3-5mL liquid LB test tube culture medium containing kanamycin resistance respectively, activate on a shaking table at 35°C for 12 hours, transfer the culture obtained after activation according to 1% transfer amount Into the liquid LB shake flask medium containing kanamycin resistance, culture in the fermentation medium with constant temperature shaking for 3 hours, the culture condition is 37°C, 200rpm. When the cell concentration reaches OD ₆₀₀ =0.8, add 0.5mM IPTG (final concentration), induce at 16°C for 16h, centrifuge at 10,000g for 5min to collect the cells, wash once with pH7.0 sodium phosphate buffer, discard the supernatant, The resting cells were obtained and stored at -80°C. Protein expression was detected by SDS-pAGE, as shown in Figures 5 and 6.

Construction and induced expression of embodiment 4 genetically engineered bacteria

The expression host Escherichia coli BL21(DE3) was transformed with the plasmids pET30-LbADH and pET30-GdhBM constructed in Examples 1 and 2, respectively. Colony PCR was performed with primers F_LbADH/R_LbADH and F_GdhBM/R_GdhBM respectively to verify the transformed recombinants. The verified genetically engineered bacteria are EcoLbADH and EcoGdhBM. Inoculate EcoLbADH and EcoGdhBM into 3-5mL liquid LB test tube culture medium containing kanamycin resistance respectively, activate on a shaking table at 40°C for 8 hours, transfer the culture obtained after activation according to 1% transfer amount Into the liquid LB shake flask medium containing kanamycin resistance, culture in the fermentation medium with constant temperature shaking for 3 hours, the culture condition is 35°C, 200rpm. When the cell concentration reaches _OD600 = 1.2, add 0.8mM IPTG (final concentration), induce at 25°C for 10h, centrifuge at 10,000g for 5min to collect the cells, wash once with pH7.0 sodium phosphate buffer, discard the supernatant, The resting cells were obtained and stored at -80°C.

Embodiment 5 reaction process monitoring method

The conversion of 2-benzoylaminomethyl-3-oxobutyric acid methyl ester to (2S, 3R)-2-benzoylaminomethyl-3-hydroxybutyric acid methyl ester was determined by HPLC detection method. Sample treatment: Take 50 μL of reaction solution at different time points, add 150-950 μL of acetonitrile, mix well, and centrifuge at 12,000 g for 5 min, take the supernatant and filter it with a 0.45 μm microporous membrane to be injected for detection. The chromatographic conditions are: chromatographic column: Pursuit C18 (150*4.6mm); mobile phase: 10mM ammonium acetate aqueous solution: acetonitrile = 55:45 (v/v); flow rate: 0.5mL/min; injection volume: 5μL; detection wavelength : 254nm. The HPLC spectrum is shown in FIG. 7 . The retention time of 2-benzamidomethyl-3-oxobutyric acid methyl ester is 9.811min, and the H spectrum of its structural identification is as shown in Figure 10; (2S, 3R)-2-benzamidomethyl- The retention time of methyl 3-hydroxybutyrate is 7.438 min, and the H spectrum of its structural identification is shown in Figure 11.

Embodiment 6 product ee value determination method

The ee value of the target product (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester was determined by chiral HPLC. Sample treatment: take 100 μL of the reaction solution at different time points, extract three times with ethyl acetate, combine the extracts, wash once with saturated NaHCO ₃ and saturated NaCl, dry with anhydrous sodium sulfate to remove water, filter, spin evaporate to remove solvent, and dissolve the residue. In 2mL chromatographic grade absolute ethanol, to be detected by HPLC.

Chiral HPLC analysis conditions are as follows: chromatographic column: Chiralpak ID (4.6 × 250mm); mobile phase: n-hexane: isopropanol: trifluoroacetic acid = 80: 20: 0.1; flow rate: 0.5ml/min; injection volume: 5 μL ; Detection wavelength: 254nm. The HPLC profiles of the four configuration products are shown in Figure 9. The retention time of (2S, 3R) configuration is 38.34min, the retention time of (2R, 3S) configuration is 49.31min, the retention time of (2R, 3R) configuration is 54.14min, the retention time of (2S, 3S) configuration The retention time is 57.97min.

The preparation of embodiment 7 (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester

A certain amount of resting cells of the engineering bacteria EcoLbADH and EcoGdhBM of Example 3 were taken and resuspended with 100 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution at pH 6.0. Add 0.1g _{dry weight} /L of LbADH resting cells and 0.1g _{dry weight} /L of GdhBM resting cells into a 50mL three-necked round bottom flask, add the above buffer to make up the total volume to 20mL, and add substrate Spin 50 mg of 2-benzoylaminomethyl-3-oxobutyric acid methyl ester, 100 mg of auxiliary substrate glucose and NADP ⁺ at a final concentration of 0.2 mM, and then place it in a constant temperature water bath at 37°C for 24 hours, stirring with 1M Sodium hydroxide aqueous solution adjusted the pH value of the reaction system to maintain at 6.0. After the reaction, the reaction solution was centrifuged to remove bacterial cells, the supernatant was filtered with a 0.45 μm microfiltration membrane, and analyzed by HPLC, the product (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid The yield of the methyl ester was 90.3%, and the ee was 86.9%.

The preparation of embodiment 8 (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester

A certain amount of resting cells of the engineering bacteria EcoLbADH and EcoGdhBM of Example 3 were taken and resuspended with 100 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution at pH 7.0. Add 1g _{dry weight} /L of LbADH resting cells and 1g _{dry weight} /L of GdhBM resting cells into a 50mL three-neck round bottom flask, add the above buffer to make up to a total volume of 20mL, and then add the substrate for racemization 2 - 500 mg of benzoylaminomethyl-3-oxobutyric acid methyl ester, 1 g of auxiliary substrate glucose and 0.2 mM NADP ⁺ at a final concentration of 0.2 mM, then placed in a constant temperature water bath at 30 ° C for 24 h, during which time 1 M hydrogen was used for oxidation Sodium aqueous solution was used to adjust the pH value of the reaction system to maintain at 7.0. After the reaction, the reaction solution was centrifuged to remove bacterial cells, the supernatant was filtered with a 0.45 μm microfiltration membrane, and analyzed by HPLC, the product (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid The yield of the methyl ester was 80.5%, and the ee was 89.6%.

The preparation of embodiment 9 (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester

A certain amount of resting cells of the engineering bacteria EcoLbADH and EcoGdhBM of Example 3 were taken and resuspended with 100 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with pH 8.0. Add 5g _{dry weight} /L of LbADH resting cells and 5g _{dry weight} /L of GdhBM resting cells into a 50mL three-neck round bottom flask, add the above buffer to make up to a total volume of 20mL, and then add the substrate for racemization 2 - 2 g of benzoylaminomethyl-3-oxobutyric acid methyl ester, 4 g of auxiliary substrate glucose and NADP ⁺ at a final concentration of 0.2 mM, and then placed in a constant temperature water bath at 40 ° C for 12 h, and oxidized with 1 M hydrogen during the reaction Sodium aqueous solution adjusted the pH value of the reaction system to maintain at 8.0. After the reaction, the reaction solution was centrifuged to remove bacterial cells, the supernatant was filtered with a 0.45 μm microfiltration membrane, and analyzed by HPLC, the product (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid The yield of the methyl ester was 73.5%, and the ee was 91.4%.

The preparation of embodiment 10 (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester

A certain amount of resting cells of the engineering bacteria EcoLbADH and EcoGdhBM of Example 3 were taken and resuspended with 100 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution at pH 7.0. Add 10g _{dry weight} /L of LbADH resting cells and 10g _{dry weight} /L of GdhBM resting cells into a 50mL three-neck round bottom flask, add the above buffer to make up to a total volume of 20mL, and then add the substrate for racemization 2 - 5 g of benzoylaminomethyl-3-oxobutyric acid methyl ester, 10 g of auxiliary substrate glucose and NADP ⁺ at a final concentration of 0.5 mM, and then placed in a constant temperature water bath at 25 ° C for 24 h, during which time 1 M hydrogen was used for oxidation Sodium aqueous solution was used to adjust the pH value of the reaction system to maintain at 7.0. After the reaction, the reaction solution was centrifuged to remove bacterial cells, the supernatant was filtered with a 0.45 μm microfiltration membrane, and analyzed by HPLC, the product (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid The yield of methyl ester was 75.2%, and the ee was 90.2%.

The preparation of embodiment 11 (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester

A certain amount of resting cells of the engineering bacteria EcoLbADH and EcoGdhBM of Example 3 were taken and resuspended with 100 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution at pH 7.0. Add 20g _{dry weight} /L of LbADH resting cells and 20g _{dry weight} /L of GDHBM resting cells into a 50mL three-neck round bottom flask, add the above buffer to make up to a total volume of 20mL, and then add the substrate for racemization 2 - 10 g of benzoylaminomethyl-3-oxobutyric acid methyl ester, 15 g of auxiliary substrate glucose and NADP ⁺ with a final concentration of 0.5 mM, and then placed in a constant temperature water bath at 30 ° C for 24 h, during which 1 M hydrogen was used for oxidation Sodium aqueous solution was used to adjust the pH value of the reaction system to maintain at 7.0. After the reaction, the reaction solution was centrifuged to remove bacterial cells, the supernatant was filtered with a 0.45 μm microfiltration membrane, and analyzed by HPLC, the product (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid The yield of the methyl ester was 83.2%, and the ee was 87.6%.

The preparation of embodiment 12 (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester

A certain amount of resting cells of the engineering bacteria EcoLbADH and EcoGdhBM of Example 3 were taken and resuspended with 100 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution at pH 7.0. Add 25g _{dry weight} /L of LbADH resting cells and 25g _{dry weight} /L of GdhBM resting cells into a 50mL three-neck round bottom flask, add the above buffer to make up to a total volume of 20mL, and then add the substrate for racemization 2 - 10 g of benzoylaminomethyl-3-oxobutyric acid methyl ester, 15 g of auxiliary substrate glucose and NADP ⁺ with a final concentration of 0.5 mM, and then placed in a constant temperature water bath at 37 ° C for 24 h, during which time it was oxidized with 1 M hydrogen Sodium aqueous solution adjusted the pH value of the reaction system to maintain at 7.0. After the reaction, the reaction solution was centrifuged to remove bacterial cells, the supernatant was filtered with a 0.45 μm microfiltration membrane, and analyzed by HPLC, the product (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid The yield of the methyl ester was 93.6%, and the ee was 84.5%.

The preparation of embodiment 13 (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester

A certain amount of resting cells of the engineering bacteria EcoLbADH and EcoGdhBM of Example 3 were taken and resuspended with 100 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution at pH 7.0. Add 1g _{dry weight} /L of LbADH resting cells and 0.5g _{dry weight} /L of GdhBM resting cells into a 50mL three-necked round bottom flask, add the above buffer to make up to a total volume of 20mL, and then add the substrate for racemization 500 mg of methyl 2-benzamidomethyl-3-oxobutyrate, 1 g of auxiliary substrate glucose and NADP ⁺ at a final concentration of 0.2 mM, and then placed in a constant temperature water bath at 30 ° C for 24 h, with 1 M hydrogen Sodium oxide aqueous solution adjusts the pH value of the reaction system to maintain at 7.0. After the reaction, the reaction solution was centrifuged to remove bacterial cells, the supernatant was filtered with a 0.45 μm microfiltration membrane, and analyzed by HPLC, the product (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid The yield of methyl ester was 73.8%, and the ee was 91.6%.

The preparation of embodiment 14 (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester

A certain amount of resting cells of the engineering bacteria EcoLbADH and EcoGdhBM of Example 3 were taken and resuspended with 100 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution at pH 7.0. Add 1g _{dry weight} /L of LbADH resting cells and 2g _{dry weight} /L of GdhBM resting cells into a 50mL three-neck round bottom flask, add the above buffer to make up to a total volume of 20mL, and then add the substrate for racemization 2 - 500 mg of benzoylaminomethyl-3-oxobutyric acid methyl ester, 1 g of auxiliary substrate glucose and 0.2 mM NADP ⁺ at a final concentration of 0.2 mM, then placed in a constant temperature water bath at 30 ° C for 24 h, during which time 1 M hydrogen was used for oxidation Sodium aqueous solution was used to adjust the pH value of the reaction system to maintain at 7.0. After the reaction, the reaction solution was centrifuged to remove bacterial cells, the supernatant was filtered with a 0.45 μm microfiltration membrane, and analyzed by HPLC, the product (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid The yield of the methyl ester was 83.5%, and the ee was 87.3%.

Comparative example 1

Get the quiescent cells of a certain amount of engineering bacteria EcoLkADH (this engineering bacteria contains the carbonyl reductase gene cloned from Lactobacillus kefiri DSM 20587, and the specific construction method is the same as in Examples 1 and 3), and use 100mM sodium dihydrogen phosphate of pH7.0 - Resuspend in disodium phosphate buffer. Add 1g _{dry weight} /L LkADH resting cells to a 50mL three-necked round-bottom flask, add 500mg of racemic 2-benzamidomethyl-3-oxobutanoic acid methyl ester as the substrate, and 500mg of the auxiliary substrate isopropyl Alcohol 1mL, add the above buffer solution to make up to a total volume of 20mL, and finally add NADP ⁺ with a final concentration of 0.2mM, then place in a constant temperature water bath at 30°C and stir for 24h. After the reaction, the reaction solution was centrifuged to remove bacterial cells, the supernatant was filtered with a 0.45 μm microfiltration membrane, and analyzed by HPLC, the product (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid The yield of methyl ester was 31.6%, and the ee was 95.7%.

Comparative example 2

Get a certain amount of engineering bacteria EcoCR (this engineering bacteria contains the carbonyl reductase gene cloned from Candida magnoliae CGMCC 2.1919, and the specific construction method is the same as in Examples 1 and 3) and the quiescent cells of EcoGdhBM, and use 100mM phosphate diphosphate of pH7.0 Resuspend in sodium hydrogen phosphate-disodium hydrogen phosphate buffer. Add 1g _{dry weight/} L CR resting cells and 1g _{dry weight} /L GdhBM resting cells to a 50mL three-neck round bottom flask, add the above buffer to make up to a total volume of 20mL, and add substrate racemization 2 - 500 mg of benzoylaminomethyl-3-oxobutyric acid methyl ester, 1 g of auxiliary substrate glucose and 0.2 mM NADP ⁺ at a final concentration of 0.2 mM, then placed in a constant temperature water bath at 30 ° C for 24 h, during which time 1 M hydrogen was used for oxidation Sodium aqueous solution was used to adjust the pH value of the reaction system to maintain at 7.0. After the reaction, the reaction solution was centrifuged to remove bacterial cells, the supernatant was filtered with a 0.45 μm microfiltration membrane, and analyzed by HPLC, the product (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid The yield of methyl ester was 0.13%, ee was 0.

Comparative example 3

Get a certain amount of engineering bacteria EcoDkR (this engineering bacteria contains the carbonyl reductase gene cloned from Acinetobacter baylyi EU273886.1, and the specific construction method is the same as that of Examples 1 and 3) and the quiescent cells of EGdhBM, and use 100mM phosphoric acid of pH7.0 Sodium dihydrogen-disodium hydrogen phosphate buffer for resuspension. Add 1g _{dry weight} /L of DkR resting cells and 1g _{dry weight} /L of GdhBM resting cells in a 50mL three-neck round bottom flask, add the above buffer to make up to a total volume of 20mL, and then add the substrate for racemization 2 - 500 mg of benzoylaminomethyl-3-oxobutyric acid methyl ester, 1 g of auxiliary substrate glucose and 0.2 mM NADP ⁺ at a final concentration of 0.2 mM, then placed in a constant temperature water bath at 30 ° C for 24 h, during which time 1 M hydrogen was used for oxidation Sodium aqueous solution was used to adjust the pH value of the reaction system to maintain at 7.0. After the reaction, the reaction solution was centrifuged to remove bacterial cells, the supernatant was filtered with a 0.45 μm microfiltration membrane, and analyzed by HPLC, the product (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid The yield of methyl ester was 0.31%, ee was 0.

Comparative example 4

Get a certain amount of engineering bacteria EcoXRADH (this engineering bacteria contains the carbonyl reductase gene cloned from Rubrobacter xylanophilus DSM9941, and the specific construction method is the same as in Examples 1 and 3) and the quiescent cells of GdhBM, use 100mM dihydrogen phosphate of pH7.0 Sodium-sodium hydrogen phosphate buffer to resuspend. Add 1g _{dry weight} /L of XRADH resting cells and 1g _{dry weight} /L of GdhBM resting cells in a 50mL three-neck round bottom flask, add the above buffer to make up to a total volume of 20mL, and then add the substrate for racemization 2 - 500 mg of benzoylaminomethyl-3-oxobutyric acid methyl ester, 1 g of auxiliary substrate glucose and 0.2 mM NADP ⁺ at a final concentration of 0.2 mM, then placed in a constant temperature water bath at 30 ° C for 24 h, during which time 1 M hydrogen was used for oxidation Sodium aqueous solution was used to adjust the pH value of the reaction system to maintain at 7.0. After the reaction, the reaction solution was centrifuged to remove bacterial cells, the supernatant was filtered with a 0.45 μm microfiltration membrane, and analyzed by HPLC, the product (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid The yield of methyl ester is 0, ee is 0.

Comparative example 5

Get a certain amount of engineering bacteria EcoYDL326 (this engineering bacteria contains the carbonyl reductase gene cloned from Saccharomyces cerevisiae, and the specific construction method is the same as in Examples 1 and 3) and the quiescent cells of engineering bacteria EcoGdhBM, and use 100mM phosphate diphosphate of pH7.0 Resuspend in sodium hydrogen phosphate-disodium hydrogen phosphate buffer. Add 1g _{dry weight} /L of YDL326 resting cells and 1g _{dry weight} /L of GdhBM resting cells into a 50mL three-neck round bottom flask, add the above buffer to make up to a total volume of 20mL, and then add the substrate for racemization 2 - 500 mg of benzoylaminomethyl-3-oxobutyric acid methyl ester, 1 g of auxiliary substrate glucose and NADP ⁺ with a final concentration of 0.2 mM, and then placed in a constant temperature water bath at 30 ° C for 24 h, and oxidized with 1 M hydrogen during the reaction Sodium aqueous solution was used to adjust the pH value of the reaction system to maintain at 7.0. After the reaction, the reaction solution was centrifuged to remove bacterial cells, the supernatant was filtered with a 0.45 μm microfiltration membrane, and analyzed by HPLC, the product (2S, 3R)-2-benzamidomethyl-3-hydroxybutyric acid The yield of methyl ester was 1.72%, ee was 0.

Claims (8)

1. prepare a method for (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters, it is characterized in that, comprising:

(1) engineering bacteria of preparation containing carbonyl reductase gene and the engineering bacteria containing glucose dehydrogenase gene;

(2) the resting cell suspension of two kinds of engineering bacterias is prepared respectively;

(3) by after two kinds of resting cell suspension mixing, mix with substrate racemize 2-benzoyl aminomethyl-3-carbonyl methyl-butyrate, hydrogen donor and cofactor again, carry out asymmetric reduction reaction, obtained (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters;

The base sequence of described carbonyl reductase gene is as shown in SEQ ID NO.1, and the base sequence of described glucose dehydrogenase gene is as shown in SEQ ID NO.2.

2. the method for claim 1, is characterized in that, described engineering bacteria contains expression vector pET-30a (+), and host cell is e. coli bl21 (DE3).

3. the method for claim 1, is characterized in that, described hydrogen donor is glucose, and described cofactor is NADP ⁺/ NADPH.

4. the method for claim 1, is characterized in that, in the mixed solution of two kinds of resting cell suspensions, the resting cell containing carbonyl reductase is 4: 1 ~ 1: 2 with the mass ratio of the resting cell containing Hexose phosphate dehydrogenase.

5. the method for claim 1, is characterized in that, in asymmetric reduction reaction, described concentration of substrate is 2.5 ~ 500g/L, and the concentration containing the resting cell of carbonyl reductase is 0.1g _{dry weight}/ L ~ 25g _{dry weight}/ L, the concentration containing the resting cell of Hexose phosphate dehydrogenase is 0.1g _{dry weight}/ L ~ 25g _{dry weight}/ L, the concentration of hydrogen donor is 5 ~ 750g/L, and the concentration of cofactor is 0 ~ 0.5mM.

6. the method for claim 1, is characterized in that, the temperature of described asymmetric reduction reaction is 25 ~ 40 DEG C, and reaction buffer pH is 6.0 ~ 8.0.

7. the method for claim 1, it is characterized in that, the preparation method of resting cell suspension, comprising: be inoculated into by described engineering bacteria and receive in the substratum of mycin containing card, after shaking table activation, when enlarged culturing to OD600 value reaches 0.8 ~ 1.2, add inductor, continue to cultivate, centrifugal collecting cell, resuspended with damping fluid, the resting cell suspension described in acquisition.

8. the method for claim 1, is characterized in that, described inductor is IPTG, and the concentration of inductor is 0.5 ~ 0.8mM.