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Application of carbonyl acyl reductase in preparing (S)-4-chlorine-3 hydroxyl ethyl butyrate

A technology of ethyl hydroxybutyrate and carbonyl reductase, which is applied in the biological field to achieve the effects of reduced production costs, high yield, and high optical activity

Inactive Publication Date: 2011-02-02
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no report has been found that this carbonyl reductase is used for the asymmetric reduction of COBE to prepare (S)-CHBE

Method used

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  • Application of carbonyl acyl reductase in preparing (S)-4-chlorine-3 hydroxyl ethyl butyrate
  • Application of carbonyl acyl reductase in preparing (S)-4-chlorine-3 hydroxyl ethyl butyrate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Acquisition of carbonyl reductase gene

[0031] Pichia Stipitis CBS 6054 (purchased from Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversiry Centre), medium YPD (g L -1 ): Yeast extract 10g, peptone 20g, glucose 20g, add distilled water to 1L.

[0032] Pichia Stipitis CBS 6054 was inoculated in 5mL LYPD liquid medium and cultured at 30°C to logarithmic growth phase, and the genome was extracted using a genomic DNA extraction kit (Beijing Tianwei Bioengineering Co., Ltd. Yeast Genome Extraction Kit).

[0033] The primers used to construct the expression vectors are provided with enzyme cutting sites, and the primer sequences are as follows:

[0034] The upstream primer (CRII-sense containing Nde I) is:

[0035] 5'-GGAATTC CATATG ACTGTCGAAACCGCCACC-3'

[0036] Downstream primers (CRII-anti containing BamH I) are:

[0037] 5'-CGC GGATCC CTAGACAGAACAGTAACCACCT-3'

[0038] All primers were synthesized by Shanghai Shenergy Gaming Company.

[00...

Embodiment 2

[0041] Example 2: Expression of Genes

[0042] Use Nde I and BamH I to respectively digest pET-22b (pET-22b was purchased from Novagen (Merck China)) and the amplified target gene containing two restriction sites, and recover the double-digested target fragments by gel respectively and the expression vector, the double-digested expression vector pET-22b and the target gene were ligated overnight with T4 ligase, and 10uL of the ligation product pET-22b-PsCRII was added to 100uL of Rosetta (DE3) competent cells, on ice Leave it for 30min, heat shock at 42°C for 90sec. Place on ice for 2 minutes. Add 0.45 mL of pre-warmed medium. 220rpm 37°C for 1h. Add 200 uL of bacterial solution to LB plates containing 100 μg / mL ampicillin and chloramphenicol respectively, and culture overnight at 37°C for 12-16 hours. To build a graph see figure 1 .

Embodiment 3

[0043] Embodiment 3: the mensuration of enzyme activity

[0044] Pick the recombinant bacteria E.coli Rosseta (pET-22b-PsCR) and Escherichia coli Rosseta (DE3) into the LB liquid medium containing antibiotics, and cultivate overnight at 37°C with shaking. Then inoculate into fresh culture medium according to 2% inoculum amount, and cultivate to OD at 37°C 600 At about 0.6, add IPTG to a final concentration of 0.8mmol·L -1 , 25°C, 220rpm, after induction of expression for 10h, centrifuge (4°C, 5000rpm, 15min), resuspend the bacteria sludge with 100mM potassium phosphate buffer (pH7.0), and sonicate the cells (power 300W, ultrasonic 5s, intermittent 5s, total 5min), centrifuge (4°C, 12000rpm, 15min), and measure the enzyme activity in the supernatant.

[0045] The enzyme reaction system includes 100mM potassium phosphate buffer (pH6.0), 5mM NADH, 20mM COBE, 30°C, and the decrease of absorbance value at 340nm is measured. Enzyme activity is defined as the amount of enzyme requ...

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Abstract

The invention discloses an application of a carbonyl acyl reductase in preparing (S)-4-chlorine-3 hydroxyl ethyl butyrate with an amino acid sequence as shown in SEQ ID NO: 2 by utilizing 4-chloracetyl acetidin asymmetrical reduction, which takes the carbonyl acyl reductase with the amino acid sequence as shown in SEQ ID NO: 2 as a catalyst, 4-chloracetyl acetidin as a substrate and NADH (Dihydronicotinamide Adenine Dinucleotide) as a cofactor to prepare the (S)-4-chlorine-3 hydroxyl ethyl butyrate by utilizing the asymmetrical reduction. The invention has the advantages that the carbonyl acyl reductase with the amino acid sequence as shown in SEQ ID NO: 2 is applied to the preparation of the (S)-4-chlorine-3 hydroxyl ethyl butyrate by utilizing the 4-chloracetyl acetidin asymmetrical reduction to obtain good effect; the enzyme activity thereof can reach 15U / mg; the yield of the substrate thereof reaches 91%; the enantiomer excessive value of products is 100%; the yield is high; and the production cost is greatly reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the application of a carbonyl reductase, in particular to a carbonyl reductase (Carbonyl Reductase CR) used in the asymmetric reduction reaction of ethyl 4-chloroacetoacetate to produce (S) - Application of ethyl 4-chloro-3-hydroxybutyrate. Background technique [0002] (S)-4-chloro-3-hydroxybutanoate (Ethyl 4-chloro-3-hydroxybutanoate, (S)-CHBE) is an important chiral intermediate, which can be used in the synthesis of many active drugs, such as statin Drugs - hydroxymethylglutaryl CoA (HMG-CoA) reductase inhibitors and 4-hydroxypyrrolidone (4-hydroxypyrrolidone), etc. [1]. [0003] Ethyl 4-chloroacetoacetate (Ethyl 4-chloroacetoacetate, COBE) is used as the latent chiral substrate of the reduction reaction, which is easy to synthesize and low in price, and it is very easy to obtain (S)-CHBE by asymmetric reduction reaction using it as the substrate. Cost-effective preparation route...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/62
Inventor 应汉杰栗西木叶齐曹厚张月圆王艳熊健陈勇
Owner NANJING TECH UNIV
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