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Method for preparing (R)-2-hydroxy-4-phenyl ethyl butyrate by catalyzing with recombinant carbonyl reductase

A technology of ethyl phenylbutyrate and carbonyl reductase, applied in the field of bioengineering, can solve problems such as expensive reagents, high price, harsh reaction conditions, etc., and achieve the effects of improving conversion effect, solving regeneration problem, and solving inhibition effect

Inactive Publication Date: 2012-08-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Chemical synthesis methods often have complex reaction steps and require expensive reagents, or the reaction conditions are very harsh
The problem with the chemical resolution method is that optically pure phenylethylamine derivatives are used as the resolution agent, which is of little practical value due to its high price. [2]
So far there is no information about the use of this carbonyl reductase for the asymmetric reduction of OPBE ( R )-HPBE Public Reports

Method used

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  • Method for preparing (R)-2-hydroxy-4-phenyl ethyl butyrate by catalyzing with recombinant carbonyl reductase
  • Method for preparing (R)-2-hydroxy-4-phenyl ethyl butyrate by catalyzing with recombinant carbonyl reductase
  • Method for preparing (R)-2-hydroxy-4-phenyl ethyl butyrate by catalyzing with recombinant carbonyl reductase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Acquisition of carbonyl reductase gene

[0032] Bacillus subtilis ( Bacillus subtilis ) CGMCC NO.1.1508 comes from China General Microorganism Culture Collection and Management Center, medium LB (g / L): 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, add water to 1 L. Bacillus subtilis was inoculated in 50 mL of liquid medium and cultured at 37 °C to the logarithmic phase, and the genome was extracted using a genomic DNA extraction kit. The primer sequences were designed as follows:

[0033] The upstream primer IolSf contains N de I

[0034] 5'-GGAATTC CATATG AAAAAAAGCGAAGCTCGG-3'

[0035] The downstream primer IolSr contains X ho I

[0036] 5'-CCG CTCGAG TGCGAACAGCTTATCAAT-3'

[0037] PCR conditions were: denaturation at 95°C for 5 min, followed by 30 cycles with the following parameters: denaturation at 95°C for 1 min, annealing at 62°C for 50 s, and extension at 72°C for 1 min. A final extension at 72°C for 10 min. PCR results such as ...

Embodiment 2

[0038] Example 2 Expression of carbonyl reductase gene

[0039] use N de I and X pET20b and carbonyl reductase genes were digested with ho I respectively, and the recovered products were ligated overnight with T4 ligase. The ligation product was added to Escherichia coli BL21(DE3) competent cells, transformed by heat shock method, spread on LB plates containing 100 μg / mL ampicillin, and incubated at 37°C for 12 h. Positive transformants were selected by ampicillin resistance carried by the vector. Positive recombinants were verified by colony PCR and extracted plasmid double enzyme digestion.

Embodiment 3

[0040] Example 3 Acquisition of Glucose Dehydrogenase Gene

[0041] Strain cultivation and gene extraction were as in Example 1. The primer sequences were designed as follows:

[0042] The upstream primer GDHf contains N de I

[0043] 5'-GGAATTC CATATG TATCCGGATTTAAAAGC-3'

[0044] The downstream primer GDHr contains H ind III

[0045] 5'-CCC AAGCTT TTAACCGCGGCCTGC-3'

[0046] PCR conditions were: denaturation at 95°C for 5 min, followed by 30 cycles with the following parameters: denaturation at 95°C for 1 min, annealing at 55°C for 50 s, and extension at 72°C for 1 min. Finally, extend at 72°C for 10 min. PCR results such as figure 1 shown.

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Abstract

The invention discloses a method for preparing (R)-2-hydroxy-4-phenyl ethyl butyrate by catalyzing recombinant carbonyl reductase, which belongs to the technical field of biological engineering. The method comprises the following steps of: cloning gene segments of carbonyl reductase (IolS) and glucose dehydrogenase (GDH) from bacillus subtilis CGMCC NO.1.1508, expressing an IolS gene and a GDH gene in series by adopting a dual-starter method to construct a recombinant plasmid pET24a-G-T7-I, and introducing the plasmid into escherichia coli BL21(DE3); and under the condition of not adding or adding a small amount of NADP+cofactors, performing biotransformation by taking a cell-free extract of the escherichia coli recombinant plasmid as a catalyst, 2-oxo-4-phenyl ethyl butyrate as a substrate and glucose as a substrate to obtain (R)-2-hydroxy-4-phenyl ethyl butyrate, wherein the enantiomeric excess value of the product is higher than 99.5 percent. In the method, IolS and GDH are co-expressed, so that efficient regeneration of an intra-cellular cofactor NADP(H) is realized, production cost is lowered, and a good industrial application prospect is achieved.

Description

technical field [0001] The present invention relates to a kind of key chiral intermediate ( R )-2-hydroxyl-4-phenylbutyric acid ethyl ester, belongs to the technical field of bioengineering. Background technique [0002] With the improvement of people's living standards, high blood pressure is increasingly endangering people's health. ( R )-2-Hydroxy-4-phenylbutanoic acid ethyl ester (Ethyl ( R )-2-hydroxy-4-phenylbutyrate, ( R )-HPBE) is a key chiral intermediate for the synthesis of numerous angiotensin-converting enzyme (ACE) inhibitors of Pril series, such as lisinopril, enalapril, captopril, etc. [1] . Due to the high efficiency and low side effects of ACEI drugs, they have become one of the best-selling drugs on the market. [0003] With 2-oxo-4-phenylbutyric acid ethyl ester (OPBE) as the latent chiral substrate of the reduction reaction, it is easy to synthesize and cheap, and it is obtained as a substrate by asymmetric reduction reaction ( R )-HPBE is a very ...

Claims

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Application Information

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IPC IPC(8): C12P7/62C12N15/70C12N15/66C12R1/19C12R1/125
Inventor 倪晔宿宇宁李海东薛天芸
Owner JIANGNAN UNIV
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