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Method for synthesizing (R)-3-amino-1-butanol through double-enzyme cascade catalysis

A technology of amino and butanol, applied in the biological field, can solve problems such as unsuitable for large-scale industrial production, difficult separation and purification, and low safety factor, and achieve good industrial application prospects, high optical purity of products, and convenient operation

Active Publication Date: 2021-05-28
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 1977, Kinas et al. reacted crotonate with (R)-phenethylamine to generate a group of epimers with two chiral centers, which were separated by silica gel column chromatography to obtain a single isomer, and then passed through ester Reduction, debenzylation obtain (R)-3-amino-1-butanol; This method step is less and raw material is easy to get, is a kind of method that has hope to realize suitability for industrialized production, but there is following problem, because the first step reaction The selectivity is poor, and two enantiomers are obtained in almost equal amounts, which makes separation and purification more difficult. Chromatographic column separation is often used, with large eluent consumption, large losses, and low efficiency. At the same time, due to the use of expensive LiAlH 4 As a reducing agent, the cost of raw materials also increases significantly, and this method is not suitable for large-scale industrial production
In 1998, Tatsuya etc. reported a method for synthesizing (R)-3-amino-1-butanol, but the price of raw materials was expensive and the reaction conditions were harsh in this method
[0004] At present, in the synthesis method of (R)-3-amino-1-butanol, the chemical method requires high temperature, high pressure and metal catalyst, harsh reaction conditions, large pollution, low safety factor, and the yield is 60% to 70%.
The biological method has mild reaction conditions, but the conversion rate is low

Method used

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  • Method for synthesizing (R)-3-amino-1-butanol through double-enzyme cascade catalysis
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  • Method for synthesizing (R)-3-amino-1-butanol through double-enzyme cascade catalysis

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Embodiment 1

[0115]Embodiment 1, the preparation of engineering bacteria expressing alcohol dehydrogenase and engineering bacteria expressing amine dehydrogenase

[0116] The genes encoding alcohol dehydrogenase and amine dehydrogenase were respectively sent to Jinkairui Biotechnology Co., Ltd. for synthesis (codon optimization was performed with Escherichia coli as the host as required), and the synthesized genes were connected to various expression vectors to construct become. The expression vectors are various conventional vectors in the art. The vector of the present invention is specifically pET28a, and the small DNA fragment between NcoI and XhoI of the pET28a restriction recognition site is replaced with the coding gene of the related enzyme after the whole gene synthesis to obtain the recombinant expression vector.

[0117] The above recombinant expression vectors are transformed into suitable microbial hosts. The host microorganisms are various conventional host microorganisms i...

Embodiment 2

[0120] Embodiment 2, the preparation of the engineering bacterium that alcohol dehydrogenase and amine dehydrogenase co-express

[0121] The coding genes of alcohol dehydrogenase (or alcohol dehydrogenase mutant) and amine dehydrogenase (or amine dehydrogenase mutant) were respectively subjected to whole gene synthesis (codon optimization was performed with Escherichia coli as the host as required), It is constructed by linking the synthetic gene to various expression vectors. The expression vectors are various conventional vectors in the art. The vector of the present invention is specifically pET28a. Firstly, the small DNA fragment between NcoI and XhoI of pET28a's enzyme-cleaved recognition sites was replaced with the coding gene of alcohol dehydrogenase after the whole gene synthesis to obtain the intermediate vector; Co., Ltd., Cat. No. C112-02) after the whole gene synthesis, the coding gene of amine dehydrogenase was connected to the coding gene of alcohol dehydrogena...

Embodiment 3

[0123] Example 3. Expression or co-expression of alcohol dehydrogenase and amine dehydrogenase and preparation of whole cells and crude enzymes

[0124] 1. Transform the recombinant expression vector constructed in Example 1 or the recombinant expression plasmid constructed in Example 2 into Escherichia coli BL21 (DE3) competent cells to obtain recombinant cells.

[0125] 2. Pick the transformant into 5mL LB liquid medium containing 50μg / mL kanamycin, shake overnight at 37°C and 220rpm for 12h, and then inoculate into 50μg / mL kanamycin containing 50μg / mL kanamycin cultured at 37°C to OD in plain TB liquid medium 600nm When the concentration is 0.7, add IPTG with a final concentration of 0.1mmol / L, induce expression at 20°C and 220rpm for 12h, centrifuge at 4°C and 4000rpm for 10min, collect the precipitated cells (i.e. whole cells), and use the collected cells with phosphate Buffer (50mmol / L, pH 7.4) was resuspended to obtain a bacterial suspension; then the bacterial cells w...

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Abstract

The invention discloses a method for synthesizing (R)-3-amino-1-butanol through double enzyme cascade catalysis. The method comprises the following steps: with 1, 3-butanediol as a substrate, carrying out a catalytic reaction with alcohol dehydrogenase to generate 4-hydroxy-2-butanone; and taking 4-hydroxy-2-butanone as a substrate, and generating chiral (R)-3-amino-1-butanol through a catalytic reaction of amine dehydrogenase or a mutant of the amine dehydrogenase. The invention provides a brand-new green biosynthesis route, and the chiral (R)-3-amino-1-butanol is catalytically synthesized by using cheap 1, 3-butanediol as a raw material through double-enzyme cell co-expression. Meanwhile, the method provided by the invention has a cofactor self-circulation system and has good economic benefits. The method has an important application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for synthesizing (R)-3-amino-1-butanol by cascade catalysis of two enzymes, in particular to a method for synthesizing by cascade catalysis of alcohol dehydrogenase and amine dehydrogenase A method for chiral (R)-3-amino-1-butanol. Background technique [0002] Chiral amino alcohol compounds are structural units of many biologically active molecules, and are important pharmaceutical and fine chemical intermediates, among which (R)-3-amino-1-butanol is a synthetic anti-AIDS integrase inhibitor dulutevir ( Dolutegravir) and can also be derivatized into β-lactams for the synthesis of penem antibiotics. [0003] (R)-3-Amino-1-butanol is mainly synthesized by chemical method, and there are few reports on enzymatic synthesis. In 1995, Gertzmann et al. used chiral (R)-alanine as a raw material. After protection by the amino group, the carbon chain was extended by diaz...

Claims

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Application Information

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IPC IPC(8): C12P13/00C12P7/26C12N9/06C12N9/04
CPCC12N9/0006C12N9/0014C12P7/26C12P13/001C12Y104/99003
Inventor 孙周通王红月曲戈
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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