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Double carbonyl reductase mutant and its application

A technology of double carbonyl reductase and reductase, which is applied in oxidoreductase, application, genetic engineering, etc., can solve the problem of poor stereoselectivity and achieve high stereoselectivity and high conversion efficiency

Inactive Publication Date: 2011-12-14
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the reaction of catalyzing α-chloroacetophenone to 2-chloro-1-phenylethanol, the stereoselectivity shown is poor, and only the R configuration product with ee of 3.91% can be obtained

Method used

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  • Double carbonyl reductase mutant and its application
  • Double carbonyl reductase mutant and its application
  • Double carbonyl reductase mutant and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Cloning of double carbonyl reductase mutant gene

[0048] 1. PCR amplification of genes containing mutation sites

[0049] Using the principle of overlap extension PCR (OE-PCR), the double carbonyl reductase gene containing the mutation site was obtained through two-step PCR reactions.

[0050] Design a pair of upstream and downstream primers according to the known DNA sequence of double carbonyl reductase, where the underlined parts are the restriction sites of NdeI and BamHI respectively:

[0051] SEQ ID No.: 41 Primer NO.1: 5'-CAACAAGACATATGACCGGCATCACGAAT-3'

[0052] SEQ ID No.: 42 Primer NO.2: 5'-AATGGATCCTCAGTACCGGTAGAAGCCCT-3'

[0053] Design mutation primers containing mutation sites, as shown in the table below:

[0054] Table 1 Mutation primers of double carbonyl reductase

[0055]

[0056]

[0057] In the first step of PCR reaction, PCR amplification was performed using pET22b-DKR (see: NaturePrecedings, http: / / hdl.handle.net / 10101 / npre.2...

Embodiment 2

[0067] Example 2: Mutant Gene Sequencing and Sequence Alignment

[0068] The mutant monoclonal obtained by the method in Example 1 was sent to Shanghai Yingjun Biotechnology Co., Ltd. for sequencing, and the sequencing results were input into the BioXM2.6 software to compare the differences between the gene base sequence and the protein amino acid sequence before and after the mutation.

[0069] The results showed that the amino acid sequences of the 20 mutant proteins contained one or more mutation sites compared with the wild type. The specific mutant protein amino acid sequence and nucleotide sequence changes are shown in Table 2:

[0070] Table 2 Sequence changes of double carbonyl reductase mutants

[0071]

[0072]

[0073] .

Embodiment 3

[0074] Example 3: Expression and purification of double carbonyl reductase mutants in Escherichia coli

[0075] 1. Expression of double carbonyl reductase mutant protein

[0076] The recombinant engineered bacteria obtained by the method described in Example 1 were inoculated into 50 ml of LB liquid medium containing 100 μg / ml ampicillin, and cultured at 37° C. for 12 to 16 hours. Then take 500μl and inoculate it into 50ml of fresh LB liquid medium containing ampicillin, shake and culture at 37℃ until OD 600nm When the value is about 0.8-1.0, add IPTG to a final concentration of 50 μM, and then continue to culture at 15° C. for 12-16 hours to induce expression. The bacterial cells were collected by centrifugation at 13,500 rpm / min, and washed once with 0.1M phosphate buffer solution of pH 7.0. Then the cells were disrupted with a high-pressure cell disruptor, centrifuged at 13,500 rpm / min for 30 min, and the supernatant was collected after centrifugation to obtain the crude ...

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to a mutant of diketoreductase (DKR) that can be used as a catalyst for chiral alcohol synthesis; the invention discloses a mutant of diketoreductase, the diketoreductase The amino acid sequence of the mutant has more than 80% homology with any amino acid sequence shown in SEQ ID NO.1-SEQ ID NO.20. Compared with the wild-type double carbonyl reductase, the double carbonyl reductase mutant of the present invention has higher transformation efficiency and higher stereoselectivity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a mutant of dicarbonyl reductase (diketoreductase, DKR) which can be used as a catalyst for chiral alcohol synthesis. Background technique [0002] Biocatalysis is the most efficient and selective catalytic system known to mankind so far. Enzymes in living organisms can catalyze various biochemical reactions at a speed beyond people's imagination. Enzymes can promote many conversion reactions of natural and synthetic chemical molecules not only in vivo, but also in vitro, and show excellent chemoselectivity, regioselectivity and stereoselectivity. Compared with traditional chemical synthesis, the reaction conditions required for biocatalytic reactions are generally milder: normal temperature, normal pressure, neutral or close to neutral pH. Therefore, biocatalysis is one of the most promising "green" synthetic methods. [0003] An analysis of 134 industrial biotransfor...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/63C12N1/21C12P7/22C12R1/19
Inventor 黄彦陆卓祁建洲陈依军
Owner CHINA PHARM UNIV
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