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Application of carbonyl reductase mutants in the synthesis of chiral ortho-halogenated-α-phenylethanol

A mutant and reductase technology, which is applied in the field of biocatalysis and protein engineering, can solve the problems that the catalytic activity cannot meet the requirements of industrial production, and achieve the effects of good industrial application development prospects, high stereoselectivity, and easy preparation

Active Publication Date: 2022-03-04
杭州文德阶生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the catalytic activity of this carbonyl reductase is far from meeting the requirements of industrial production. Therefore, it is necessary to improve the catalytic efficiency of this enzyme through related technologies to fully tap its application value.

Method used

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  • Application of carbonyl reductase mutants in the synthesis of chiral ortho-halogenated-α-phenylethanol
  • Application of carbonyl reductase mutants in the synthesis of chiral ortho-halogenated-α-phenylethanol
  • Application of carbonyl reductase mutants in the synthesis of chiral ortho-halogenated-α-phenylethanol

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preparation example Construction

[0032] The method for preparing a mutant of the above-mentioned carbonyl reductase BaSDR1 is characterized in that the method comprises the following steps: cultivating a recombinant expression transformant of the carbonyl reductase mutant, and inducing the recombinant carbonyl reductase mutant protein; wherein, culturing the recombinant expression The medium used for the transformant is LB medium, including the following: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, pH 7.2; recombinant Escherichia coli is inoculated into the LB medium containing kanamycin Medium culture, when the optical density OD600 of the culture solution reaches 0.5-0.7, under the induction of the final concentration of 0.1-1.0mM isopropyl-β-D-thiogalactopyranoside (IPTG), it can be obtained. The above reaction conditions can be selected according to conventional conditions used in the art, as long as the transformant can grow and produce the carbonyl reductase mutant protein.

[0033] In the...

Embodiment 1

[0034] Embodiment 1: the construction of above-mentioned mutant:

[0035] Using the oligonucleotide fragment containing the mutation point as a primer, as shown in Table 1, the QuickChangeTM method was used to amplify the pET-30a recombinant plasmid containing the carbonyl reductase BaSDR1 gene.

[0036] Table 1 Mutant construction primers

[0037]

[0038] a Mutation sites are underlined.

[0039] PCR reaction system: upstream primer 10 μM, 1.0 μL; downstream primer 10 μM, 1.0 μL; recombinant plasmid template, 10 ng; PrimerSTAR Max DNA Polymerase (2X), 12.5 μL; add ddH 2 O to a total volume of 25 µL.

[0040] PCR program: step (1) 98°C, 1min; step (2) 98°C, 10s; step (3) 55°C, 10s; step (4) 72°C, 6min. Steps (2)-(4) were cycled 15 times and then cooled to 4°C. Get the PCR product.

[0041] After the PCR product was washed, it was digested with the restriction endonuclease DpnI that specifically recognizes the methylation site to degrade the template plasmid. Enzyme ...

Embodiment 2

[0043] Example 2: Induced expression of the above-mentioned carbonyl reductase mutants:

[0044] The engineered bacteria constructed in Example 1 were inoculated into LB liquid medium containing 50 μg / mL kanamycin, cultivated overnight at 37° C., and then inoculated with 1% inoculum size (v / v) into LB liquid medium containing 50 μg / mL kanamycin. In 50mL LB medium, cultured at 37°C and 200rpm until the cell concentration OD600 to about 0.6, added IPTG with a final concentration of 0.1 mM, induced culture at 30°C for 6 hours, and centrifuged at 4°C and 4000rpm for 10min to collect the bacterial cells. Store at -80°C for later use.

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Abstract

The invention discloses the application of carbonyl reductase mutants in the synthesis of chiral ortho-halogenated-α-phenylethanol, a mutant of carbonyl reductase BaSDR1, the mutant is the amino acid sequence SEQ ID No of carbonyl reductase BaSDR1 .2 based on a single or double point mutation at glutamine at position 139 and aspartic acid at position 253. The mutant of the carbonyl reductase BaSDR1 and the enzyme activity of the recombinant engineered bacteria provide a powerful biocatalyst for the synthesis of chiral ortho-halogenated-α-phenethyl alcohol.

Description

【Technical field】 [0001] The invention belongs to the technical field of biocatalysis and protein engineering, in particular to the application of carbonyl reductase mutants in the synthesis of chiral orthohalogenated-alpha-phenylethanol. 【Background technique】 [0002] Chiral ortho-halogenated-α-phenylethanol has become an important tool in the synthesis of chiral drugs because of its special structure of chiral carbon hydroxyl group next to the aromatic ring halogen, as well as the unique biochemical properties of chiral alcohols, aromatic rings and halogens. intermediate. As an important chiral synthesis building block, chiral ortho-halo-α-phenylethanol is also widely used in the synthesis of high value-added chemical products such as fine chemicals, agrochemicals and flavors and fragrances. The important role played by chiral ortho-halogenated-α-phenylethanol compounds in the field of chiral synthesis makes them one of the most important chiral building blocks in the ch...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/70C12N1/21C12P7/22C12R1/19
CPCC12N9/0006C12N15/70C12P7/22C12Y101/01184
Inventor 李爱朋张连兵
Owner 杭州文德阶生物科技有限公司
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