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Carbonyl reductase and method for preparing ethyl (R)-4-chloro-3-hydroxybutyrate by using carbonyl reductase

A technology of carbonyl reductase and reductase, which is applied in the field of bioengineering, can solve the problems of expensive metal catalysts, harsh reactor requirements, and harsh reaction conditions, so as to improve biotransformation efficiency, increase reaction substrate concentration, and stereoselectivity Good results

Pending Publication Date: 2022-05-24
NANJING CHEMPION BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The chemical synthesis method has harsh reaction conditions and needs to be reacted under high temperature and high pressure conditions, which requires high energy consumption and strict requirements on the reactor, and the metal catalysts used at the same time are expensive and costly

Method used

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  • Carbonyl reductase and method for preparing ethyl (R)-4-chloro-3-hydroxybutyrate by using carbonyl reductase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1. Construction and culture of genetically engineered bacteria E.coli BL21(DE3) / pET28a-CR1

[0022] The gene sequence of carbonyl reductase CR1 as shown in SEQ ID NO.2 was cloned into pET28a plasmid, and the restriction sites were NdeI and EcoRI to obtain recombinant plasmid pET28a-CR1, and then recombinant plasmid pET28a-CR1 was imported into Escherichia coli E. coliBL21(DE3) was constructed to obtain a genetically engineered strain E. coli BL21(DE3) / pET28a-CR1.

[0023] The engineered bacteria E.coli BL21(DE3) / pET28a-CR1 constructed above were inoculated into LB medium containing kanamycin (50 μg / mL), and cultured at 37°C at 200 rpm. When the optical density of the bacterial solution (OD600) When it reaches 0.6-0.8, add 0.1mM isopropyl-β-D-thiogalactoside (IPTG) for induction, the induction temperature is 16°C, and the induction time is 20 hours. Genetically engineered bacterial wet cells with carbonyl reductase CR1.

[0024] 2. Construction and culture of genetical...

Embodiment 2

[0033] In this example, the genetically engineered bacteria E.coli BL21(DE3) / pET28a-CR1 and the genetically engineered bacteria E.coli BL21(DE3) / pET28a-GDH obtained in Example 1 were used to synthesize (R)-CHBE, Specifically include the following steps:

[0034] 40g COBE was dissolved in 20mL n-octanol to obtain an oil phase; 2.0g GR1 engineering bacteria wet cells (E.coliBL21(DE3) / pET28a-CR1), 2.0g GDH engineering bacteria wet cells (E.coli BL21(DE3) ) / pET28a-GDH), 3.0 g glucose and 0.03 g NADPH were added to 90 mL of 0.1 mol / L KH, pH=7.5 2 PO 4 -K 2 HPO 4 In the buffer solution, the aqueous phase is obtained; the oil phase and the aqueous phase are mixed, the pH value is adjusted to about 7.0, and the reaction is stirred at room temperature for 3 hours. After the reaction is completed, it is extracted three times with an equal volume of ethyl acetate. The organic solvent was distilled off under reduced pressure to obtain 37.9 g of (R)-CHBE (ee value>99.1%, yield 95%, con...

Embodiment 3

[0036]In this example, the genetically engineered bacteria E.coli BL21(DE3) / pET28a-CR1 and the genetically engineered bacteria E.coli BL21(DE3) / pET28a-GDH obtained in Example 1 were used to synthesize (R)-CHBE, Specifically include the following steps:

[0037] Dissolve 400g COBE in 50mL n-octanol to obtain an oil phase; 5.0g GR1 engineering bacteria wet cells (E.coliBL21(DE3) / pET28a-CR1), 5.0g GDH engineering bacteria wet cells (E.coli BL21(DE3) ) / pET28a-GDH), 7.5g glucose and 0.3g NADPH were added to 950mL KH of 0.1mol / L, pH=7.5 2 PO 4 -K 2 HPO 4 In the buffer solution, the water phase was obtained; the oil phase and the water phase were mixed, the pH value was adjusted to about 7.0, and the reaction was stirred at room temperature for 2 h. After the reaction was completed, it was extracted three times with an equal volume of ethyl acetate. The organic solvent was distilled off under reduced pressure to obtain 388.2 g of (R)-CHBE (ee value>99.5%, yield 97%, conversion 10...

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Abstract

The invention discloses a carbonyl reductase and a method for preparing ethyl (R)-4-chloro-3-hydroxybutyrate by using the carbonyl reductase. The amino acid sequence of the carbonyl reductase is as shown in SEQ ID NO. 1. The preparation method of the (R)-4-chloro-3-hydroxybutyric acid ethyl ester comprises the following steps: dissolving 4-chloroacetoacetic acid ethyl ester in n-caprylic alcohol to obtain an oil phase; adding the engineering bacterium wet cells for expressing carbonyl reductase, the engineering bacterium wet cells for expressing glucose dehydrogenase, glucose and NADPH (Nicotinamide Adenine Dinucleotide Phosphate) into a phosphate buffer solution to obtain a water phase; mixing the oil phase and the water phase, adjusting the pH value, and stirring at room temperature for reaction; and after the reaction is finished, extracting with ethyl acetate, and carrying out reduced pressure distillation on extract liquor to remove the organic solvent, thereby obtaining the target compound (R)-4-chloro-3-hydroxybutyric acid ethyl ester. According to the method, the reaction time is short, the ee of the product is larger than 99.5%, the yield is 97%, the conversion rate is 100%, and the method is obviously superior to the existing technical level.

Description

technical field [0001] The invention relates to the technical field of biological engineering, in particular to a carbonyl reductase and a method for preparing (R)-4-chloro-3-hydroxybutyric acid ethyl ester by using the enzyme. Background technique [0002] 4-Chloro-3-hydroxybutyric acid ethyl ester (CHBE) is an important intermediate in organic synthesis. It can be introduced into a variety of pharmaceutical intermediates through chlorine replacement reaction, reduction reaction, etc. For example: (R)-CHBE can be used to synthesize the weight loss nutritional supplement L-carnitine. At present, there are two main methods to prepare CHBE using 4-chloroacetoacetate (COBE) as raw material: chemical method and biocatalysis method. The chemical synthesis method has harsh reaction conditions, requires high temperature and high pressure reaction, high energy consumption and harsh requirements for the reactor, and the metal catalyst used is expensive and expensive. Biocatalytic s...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/04C12N1/21C12N15/70C12P7/62C12P41/00C12R1/19
CPCC12N9/0006C12N15/70C12P7/62C12P41/002C12Y101/01184C12Y101/01047Y02P20/584
Inventor 周佳海陈剑许雪霞余长泉杨林松
Owner NANJING CHEMPION BIOTECHNOLOGY CO LTD
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