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A method for preparing (2s, 3r)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester

A technology of benzamidomethyl and methyl hydroxybutyrate, which is applied in the field of biopharmaceuticals, can solve the problems of reducing catalytic efficiency, affecting AdhR catalytic effect, increasing the complexity of product separation and purification, and achieving the goal of improving conversion rate and purity Effect

Active Publication Date: 2018-06-01
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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

Method used

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  • A method for preparing (2s, 3r)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester
  • A method for preparing (2s, 3r)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester
  • A method for preparing (2s, 3r)-2-benzamidomethyl-3-hydroxybutyric acid methyl ester

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Construction of embodiment 1 plasmid pET30-LbADH

[0045] 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, such as figure 1 .

[0046] The sequence of primer F_LbADH is: 5'CGC GGATCC ATGTCTAACCGTTTGGATG-3';

[0047] The sequence of primer R_LbADH is: 5'CCG CTCGAG CTATTGAGCAGTGTAGCCAC-3'.

[0048] 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 excised 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 pE...

Embodiment 2

[0049] Construction of embodiment 2 plasmid pET30-GdhBM

[0050] The GdhBM gene was cloned with primers F_GdhBM / R_GdhBM to obtain a 786bp GdhBM gene (as shown in SEQID NO.2). Nucleic acid electrophoresis to verify gene size, such as figure 2 .

[0051] The sequence of primer F_GdhBM is: 5'-GGA AGATCTG ATGTATAAAGATTTAGAAGGA-3';

[0052] The sequence of primer R_GdhBM is: 5'CCG CTCGAG TTATCCGCGTCCTGCTTGGAA-3'.

[0053] 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 excised 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 ...

Embodiment 3

[0054] Construction and induced expression of embodiment 3 genetically engineered bacteria

[0055] 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 grows to OD 600 When =0.8, add 0.5mM IPTG (final concentrati...

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Abstract

The invention discloses a method for preparing (2S, 3R)-2-benzamidomethyl-3-hydroxybutyrate methyl ester. The method comprises: preparing engineering bacteria containing carbonyl reductase gene and glucose-containing dehydrogenation Enzyme gene engineering bacteria; respectively prepare resting cell suspensions of two engineering bacteria; after mixing the two resting cell suspensions, and then with the substrate racemic 2-benzamidomethyl-3-carbonylbutyl Acid methyl ester, hydrogen donor and cofactor are mixed, and an asymmetric reduction reaction is carried out to obtain (2S, 3R)-2-benzamidomethyl-3-hydroxybutyrate 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 methyl 2-benzoylaminomethyl-3-carbonylbutyrate to generate (2S,3R)-2-benzoylaminomethyl-3-hydroxybutyrate methyl ester, The conversion rate and purity of the product are improved.

Description

technical field [0001] 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 [0002] (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 bac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/02C12R1/19
Inventor 吴坚平徐佳李洪明黄光东徐丹萍龚伟中徐刚杨立荣
Owner ZHEJIANG UNIV
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