A kind of recombinant carbonyl reductase mutant, gene, engineering bacteria and application thereof

A mutant, reductase technology, applied in genetic engineering, oxidoreductase, applications, etc., can solve the problems of insufficient diastereomeric induction, failure to meet production requirements, low optical purity of products, etc., and shorten the reaction time. , the effect of reducing the reaction cost and simplifying the chemical catalysis steps

Active Publication Date: 2020-11-13
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The catalytic functions of the three are similar, but the structure is quite different
The chemical synthesis of (3R,5S)-CDHH often starts from a simple compound, such as (S)-epichlorohydrin, and undergoes a series of chemical reactions to synthesize the target intermediate product. The introduction of the C3 chiral center requires the use of easy Flammable and explosive NaBH 4 As a reducing agent, at the same time, the synthesis reaction needs to be carried out at a low temperature of minus 65°C, which causes great environmental pollution and high energy consumption.
In addition, the chemical synthesis of (3R,5S)-CDHH diastereomer induction is insufficient, the optical purity of the product is low, the production requirements cannot be met, and the final yield is not high

Method used

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  • A kind of recombinant carbonyl reductase mutant, gene, engineering bacteria and application thereof
  • A kind of recombinant carbonyl reductase mutant, gene, engineering bacteria and application thereof
  • A kind of recombinant carbonyl reductase mutant, gene, engineering bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Obtaining of recombinant carbonyl reductase mutants

[0037] Using the recombinant bacteria (E.coli BL21(DE3) / pET28b-SCR) containing the expression vector pET28b-SCR as the starting strain, through random mutation and site-directed saturation mutation techniques, the ability of carbonyl reductase to substrate (S)-CHOH was further improved. Catalytic activity and substrate tolerance.

[0038] (1) Error-prone PCR and PCR with large primers

[0039] Error-prone PCR upstream primer 1: 5'-TATGTCTTCGCCTACTCCCAAC-3'

[0040] Error-prone PCR downstream primer 2: 5'-TCTACCATGGCAAGAACGTCC-3'

[0041] Error-prone PCR by changing the Mn in the PCR system 2+ , Mg 2+ The wrong bases are randomly incorporated into the amplified gene at a certain frequency, so as to obtain a randomly mutated DNA population. The present invention uses the plasmid DNA where the SCR gene (the nucleotide sequence is shown in SEQ ID NO.1 and the amino acid sequence is shown in SEQ ID NO.2) a...

Embodiment 2

[0115] Example 2: Preparation of recombinant carbonyl reductase mutant wet thallus

[0116] The recombinant Escherichia coli (recombinant Escherichia coli BL21(DE3) / pET28b-mut-Phe145Met / Thr152Ser, recombinant Escherichia coli BL21(DE3) / pET28b-mut-Phe145Tyr / Thr152Ser) was inoculated into LB liquid medium containing kanamycin resistance at a final concentration of 50 μg / mL, cultured at 37°C and 180 rpm for 8 hours, and then inoculated with a 1% inoculum amount (v / v) into fresh LB medium containing a final concentration of 50 μg / mL In mL kanamycin-resistant LB liquid medium, culture at 37°C and 180rpm until the cell OD 600 After reaching 0.6-0.8, add IPTG with a final concentration of 0.1mM, induce culture at 28°C for 12h, then centrifuge at 8000rpm for 10min at 4°C, discard the supernatant, collect the precipitate, and obtain the recombinant carbonyl reductase mutant gene. Recombinant Escherichia coli wet cells. The wet thalline can be used directly as a biocatalyst or for pr...

Embodiment 3

[0117] Embodiment 3: Separation and purification of carbonyl reductase mutant

[0118] The wet cells obtained in Example 3 were washed three times with physiological saline, and the wet cells were resuspended according to 1 g of wet cells plus 20 mL of 20 mM, pH 7.0 potassium phosphate buffer, and ultrasonically crushed under ice bath conditions (60 W, continuous 1s, intermittent 1s, continuous disruption for 30min) to obtain cell disruption liquid. The cell lysate obtained after ultrasonic disruption was centrifuged at 8000 rpm and 4° C. for 25 min, and the obtained supernatant was the required crude enzyme solution, which was designated as crude enzyme solution I.

[0119] Fractional salting out of ammonium sulfate: determination of the optimum precipitation concentration, the most commonly used salt in salting out is (NH 4 ) 2 SO 4 、Na 2 SO 4 , MgSO 4 . (NH 4 ) 2 SO 4 Because of its high solubility, small temperature coefficient, good separation effect, generally ...

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Abstract

The invention discloses a recombinant carbonyl reductase mutant, genes, a carrier, engineering bacteria, and applications thereof. The recombinant carbonyl reductase mutant is obtained through combination mutation of the 145th and the 152th sites of an amino acid sequence represented by SEQ ID NO.2, the 145th site phenylalanine is changed into methionine, and the 152th site threonine is changed into serine; the 145th site phenylalanine is changed into tyrosine, and the 152th site threonine is changed into serine. The invention also provided a recombinant carbonyl reductase mutant capable of reducing (S)-6-chloro-5-hydroxy-3-tert-butyl oxohexanoate, the catalytic activity and the substrate survivability of the recombinant carbonyl reductase mutant are much better than those of a wild type enzyme in the above conversion, and reaction process is shortened obviously.

Description

[0001] (1) Technical field [0002] The invention belongs to the field of biopharmaceuticals and biotransformation, and specifically relates to a recombinant carbonyl reductase mutant, gene, carrier, engineering bacteria and application thereof, and recombinant Escherichia coli biocatalyzing (S)-6-chloro- Preparation of (3R,5S)-6-chloro-3 , tert-butyl 5-dihydroxyhexanoate (tert-butyl(3R,5S)-6-chloro-3,5-dihydroxyhexanoate, (3R,5S)-CDHH) method. [0003] (2) Background technology [0004] Stereoselective carbonyl reductase (Carbonyl reducatase, E.C.1.1.1.x) belongs to the oxidoreductase system, which is a class of enzymes that can catalyze the bidirectional reversible redox reaction between alcohols and aldehydes / ketones, and requires the coenzyme NAD(H) (nicotinamide adenine dinucleotide) or NADP(H) (nicotinamide adenine dinucleotide phosphate) as a hydrogen transporter capable of reducing a large number of endogenous or exogenous carbonyl compounds. NADH and NADPH participat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12P7/62
CPCC12N9/0006C12P7/62C12Y101/01184
Inventor 柳志强郑裕国尹欢欢张晓建王亚军
Owner ZHEJIANG UNIV OF TECH
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