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Method for preparing L-tert-leucine by coupling leucine dehydrogenase with glucose dehydrogenase

A technology of leucine dehydrogenase and glucose dehydrogenase, which is applied in organic chemistry, fermentation and other directions, can solve the problems of poor economy, low reaction efficiency, low activity of formate dehydrogenase, etc. The effect of simple process and shortened reaction time

Active Publication Date: 2016-05-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These synthetic pathways all have the following problems: 1. The reaction needs to add coenzyme, and coenzyme is expensive and economical. If coenzyme can not be added in the synthetic engineering, the cost of industrial production process will be greatly reduced; 2. Formic acid dehydrogenation The enzyme activity is low, and a large amount of formate dehydrogenase needs to be added in the asymmetric conversion process to fully exert the catalytic synthesis effect of leucine dehydrogenase, which increases the cost of enzyme input; 3. The reaction efficiency is low, precisely because The less active formate dehydrogenase used in the reaction process affects the conversion efficiency of the coenzyme and increases the reaction time required for the complete conversion of the substrate, making the reaction efficiency lower

Method used

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  • Method for preparing L-tert-leucine by coupling leucine dehydrogenase with glucose dehydrogenase
  • Method for preparing L-tert-leucine by coupling leucine dehydrogenase with glucose dehydrogenase
  • Method for preparing L-tert-leucine by coupling leucine dehydrogenase with glucose dehydrogenase

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Embodiment 1

[0030]Example 1: Construction of Escherichia coli recombinant strains co-expressing leucine dehydrogenase and glucose dehydrogenase

[0031] Use the gene sequence in the NCBI database to design primers, transfer the two enzyme genes of leucine dehydrogenase and glucose dehydrogenase from Bacilluscereus and Bacillussp, respectively, and use restriction enzymes XhoI and NheI to pair the target gene with the expression plasmid pET28a was subjected to double digestion, and the target gene was ligated with the vector to obtain recombinant plasmids pET28a-LDH and pET28a-GDH. Starting from the expression plasmids of the two genes, an SD-AS sequence (GGAGATATACC) was added between the two genes by using the overlap extension PCR technique. Primers containing the SD-AS sequence were designed, the gene GDH-SD-AS-LDH was obtained after two rounds of PCR, the gene was connected to the expression vector pET28a, and transformed into the E. coli host BL21 to obtain the recombinant strain E.c...

Embodiment 2

[0032] Embodiment 2: LB medium induces the expression of genetically engineered strains

[0033] The recombinant bacteria were fermented and cultured using LB medium. The components of LB medium were peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH 7.0, prepared with deionized water, and kanamycin sulfate 50μg / mL was added before use. The culture conditions are as follows: the initial temperature is 37°C, the rotation speed of the shaker is 200r / min, when the cell OD 600 After reaching 0.6-2.0, add IPTG with a final concentration of 0.1 mM, and culture at 18°C ​​for 17h.

Embodiment 3

[0034] Example 3: LB medium added with niacin and other substances induces expression of genetically engineered strains

[0035] Add 1 g / L nicotinic acid (or precursor substances in the coenzyme synthesis pathway such as nicotinamide, L-tryptophan, L-aspartic acid, etc.) to the LB medium. The components of LB medium were peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH 7.0, prepared with deionized water, and kanamycin sulfate 50μg / mL was added before use. The culture conditions are as follows: the initial temperature is 37°C, the rotation speed of the shaker is 200r / min, when the cell OD 600 After reaching 0.6-2.0, add IPTG with a final concentration of 0.1 mM, and culture at 18°C ​​for 17h.

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Abstract

The invention discloses a method for preparing L-tert-leucine by coupling leucine dehydrogenase with glucose dehydrogenase, belonging to the technical field of enzyme engineering and chiral medical intermediate preparation. The method comprises the following steps: adding substrates trimethylpyruvic acid and glucose into a reaction system; adjusting the pH value of the system to 8.0-9.0; and adding a crude enzyme (containing leucine dehydrogenase, glucose dehydrogenase and coenzyme) with broken co-expressed strain and cultured by a culture medium added with substances such as niacin, and then starting reactions while controlling the temperature at 25-30 DEG C and pH value at 8.0-9.0 in the reaction process to generate L-tert-leucine. In the invention, a system in which leucine dehydrogenase is coupled with glucose dehydrogenase is adopted, and the L-tert-leucine is prepared by use of the coenzyme contained in the thalli; and the method has the characteristics of high concentration of single-batch reaction substrates, high reaction efficiency, no coenzyme addition and the like.

Description

technical field [0001] The invention relates to a method for preparing L-tert-leucine by using leucine dehydrogenase coupled with glucose dehydrogenase, and belongs to the technical field of enzyme engineering and preparation of chiral pharmaceutical intermediates. Background technique [0002] Optically pure L-tert-leucine, the tert-butyl group contained not only has steric hindrance effect but also has strong hydrophobicity, so it is widely used as a template for asymmetric catalytic synthesis. At the same time, as a non-natural amino acid, it is often used in pharmaceutically active peptides. Studies have reported that L-tert-leucine is used in anti-tumor drug reagents, anti-AIDS protease inhibitors and other drugs. It is precisely because of the wide application of L-tert-leucine that a large number of synthetic methods have been developed and reported. These mainly include chemical resolution, chemical asymmetric synthesis, enzymatic resolution, and enzymatic reductiv...

Claims

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Application Information

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IPC IPC(8): C12P13/04C07C229/08
CPCC07C229/08C12P13/04
Inventor 穆晓清徐岩聂尧
Owner JIANGNAN UNIV
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