Method for preparing chiral (S)-acetoin by virtue of whole-cell biological catalysis

A technology of biocatalysis and biocatalyst, which is applied in the direction of biochemical equipment and methods, methods based on microorganisms, microorganisms, etc., can solve the problems of cost reduction, achieve the effect of improving regeneration ability, simple operation process, and high-tech economic value

Inactive Publication Date: 2016-09-28
GUANGXI ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The technical problem to be solved by the present invention is to optimize the composition of the co-expression system of reductase and dehydrogenase on the basis of the prior art, improve coenzyme regeneration ability, and significantly improve (S)- The output of acetoin, and keep the characteristics of simple and efficient process, so that the cost is greatly reduced

Method used

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  • Method for preparing chiral (S)-acetoin by virtue of whole-cell biological catalysis
  • Method for preparing chiral (S)-acetoin by virtue of whole-cell biological catalysis
  • Method for preparing chiral (S)-acetoin by virtue of whole-cell biological catalysis

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Embodiment 1

[0035] This implementation confirms that the new method of co-expression of formate dehydrogenase and diacetyl reductase to produce (S)-acetoin of the present invention is superior to co-expression of glucose dehydrogenase in terms of production efficiency, yield and optical purity through comparative experiments system.

[0036] 1. Preparation of recombinant Escherichia coli

[0037] The formate dehydrogenase gene fdh from Saccharomyces cerevisiae and the glucose dehydrogenase gene gdh from B. subtilis168 were artificially synthesized after codon optimization, and cloned into the expression vector pETDuet-1 to obtain a recombinant plasmid pETDuet-fdh and pETDuet-gdh. The diacetyl reductase gene bdh from Enterobacter cloacae was artificially synthesized after codon optimization, and cloned into the plasmids pETDuet-1, pETDuet-gdh and pETDuet-fdh respectively to obtain the recombinant plasmids pETDuet-bdh and pETDuet -bdh-gdh and pETDuet-bdh-fdh, which can be transformed into...

Embodiment 2

[0051] 1. Preparation of recombinant Escherichia coli

[0052] The diacetyl reductase gene adr derived from Rhodococcus erythropolis was artificially synthesized after codon optimization, and cloned into the recombinant plasmid pETDuet-fdh obtained in Example 1 to obtain the recombinant plasmid pETDuet-adr-fdh. E. coli E. coli BL21 (DE3) competent cells were transformed to obtain recombinant E. coli E. coli BL21 (DE3) / pETDuet-adr-fdh. The nucleotide sequence of the artificially synthesized codon-optimized Rhodococcus erythropolis diacetyl reductase gene adr is shown in SEQ ID NO.4.

[0053] 2. The preparation of recombinant Escherichia coli resting cells comprises the following steps:

[0054] (1) Plate culture: Streak the recombinant E. coli E. coli BL21(DE3) / pETDuet-adr-fdh on the LB plate medium containing 1.8% agar by mass volume ratio and 100 μg / mL ampicillin, at 37°C Overnight culture;

[0055] (2) Seed culture: under sterile ultra-clean bench conditions, pick a singl...

Embodiment 8

[0065] 1. Preparation of recombinant Escherichia coli

[0066] The formate dehydrogenase gene fdh from Saccharomyces cerevisiae was artificially synthesized after codon optimization, and cloned into the expression vector pETDuet-1 to obtain the recombinant plasmid pETDuet-fdh. The diacetyl reductase gene bdh derived from Enterobacter cloacae was artificially synthesized after codon optimization, and cloned into the plasmid pETDuet-fdh to obtain the recombinant plasmid pETDuet-bdh-fdh, which was then transformed into Escherichia coli E .coliBL21(DE3) competent cells can be obtained from recombinant Escherichia coli E.coli BL21(DE3) / pETDuet-bdh-fdh. The nucleotide sequences of the artificially synthesized codon-optimized formate dehydrogenase gene fdh and the diacetyl reductase gene bdh are shown in SEQ ID NO.1 and SEQ ID NO.3.

[0067] 2. The preparation of recombinant Escherichia coli resting cells comprises the following steps:

[0068] (1) Plate culture: Streak the recombi...

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Abstract

The invention discloses a method for preparing chiral (S)-acetoin by virtue of whole-cell biological catalysis. The method comprises the following steps: firstly carrying out coexpression on butanedione reductase genes and formate dehydrogenase genes in escherichia coli, so as to obtain recombinant escherichia coli; and carrying out asymmetric reduction reaction in a water phase by taking a resting cell of the recombinant escherichia coli as a whole-cell biocatalyst, butanedione as a substrate and formic acid or formate as a coenzyme NADH regenerated hydrogen donor, so as to generate chiral (S)-acetoin. A whole coenzyme regeneration system of the cell is utilized, and only a proper amount of formic acid or formate needs to be added as the hydrogen donor, so that the transfer ability of a thallus is improved; expensive coenzyme NADH does not need to be added, and the yield of chiral (S)-acetoin can reach up to 46.1g/L; the process is simple and efficient, the production cost is low, and the problems of low efficiency and high cost of traditional conversion are solved; and the method has an extremely high economic value and is applicable to large-scale business application.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and in particular relates to a method for preparing chiral (S)-acetoin by a whole-cell biocatalyst. Background technique [0002] Acetoin has obvious characteristics of cheese aroma and fat aroma, and is naturally present in various foods such as corn, grapes, apples, strawberries, butter, cheese, wine, cocoa, etc., and the national standard GB2760-86 stipulates that it is Edible food spices are allowed. Acetoin is mainly used in the production of cream, dairy products, coffee, yogurt and other spices. In addition, acetoin is an important chemical synthesis intermediate. The U.S. Department of Energy lists acetoin as one of the 30 priority platform compounds for development and utilization, which can be widely used in food, pharmaceutical, chemical and other fields. There are two optical isomers of (S)-acetoin and (R)-acetoin in acetoin, and the optical isomers of (S)-acetoin and (R)-acetoin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/26C12N1/21C12N15/70C12R1/19
CPCC12N9/0006C12N9/0008C12N15/70C12N2800/101C12P7/26C12Y102/01002
Inventor 谢能中李检秀黄日波黄艳燕杜奇石严少敏陈先锐刘祺霞
Owner GUANGXI ACAD OF SCI
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