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Alcohol dehydrogenase mutant, and application thereof in synthesis of bisaryl chiral alcohols

An alcohol dehydrogenase and mutant technology, which is applied in the field of bioengineering, can solve the problem of low stereoselectivity of alcohol dehydrogenase, and achieve the effects of good industrial application prospect, excellent stereoselectivity and high industrial application value.

Inactive Publication Date: 2018-09-07
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0015] Aiming at the problem of low stereoselectivity of alcohol dehydrogenase in the prior art, the present invention provides a series of mutant proteins of alcohol dehydrogenase, nucleic acid sequences encoding the mutant proteins, and recombinant expression vectors containing the nucleic acid sequences and the recombinant expression transformant, and the alcohol dehydrogenase mutant protein or the recombinant transformant expressing the alcohol dehydrogenase mutant protein is used as a catalyst in asymmetric reduction and preparation of optical chiral bis-aryl alcohol

Method used

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  • Alcohol dehydrogenase mutant, and application thereof in synthesis of bisaryl chiral alcohols
  • Alcohol dehydrogenase mutant, and application thereof in synthesis of bisaryl chiral alcohols
  • Alcohol dehydrogenase mutant, and application thereof in synthesis of bisaryl chiral alcohols

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Embodiment 1: the assay method of the activity of alcohol dehydrogenase and product optical purity:

[0043] The total reaction system is 200μL, including: 1.0mM NADPH, 1.0mM substrate CPMK, sodium phosphate buffer (PBS, 100mM, pH7.0), mix well, keep warm at 30°C for 2min, add an appropriate amount of enzyme solution, and detect at 340nm Changes in light absorption values.

[0044] Enzyme activity was calculated using the following formula:

[0045] Enzyme activity (U) = EW × V × 10 3 / (6220×l)

[0046] In the formula, EW is the change of absorbance at 340nm within 1 minute; V is the volume of the reaction solution, the unit is mL; 6220 is the molar extinction coefficient of NADPH, the unit is L / (mol cm); 1 is the optical path distance, the unit is for cm. One activity unit (U) corresponds to the amount of enzyme required to catalyze the oxidation of 1 μmol NADPH per minute under the above conditions.

[0047] Determination method of optical purity ee:

[0048] ...

Embodiment 2

[0049] Example 2: Construction of Alcohol Dehydrogenase Mutant Gene and Recombinant Expression Transformant

[0050] The site-directed mutation of amino acid residues at positions 161 and 196 was carried out by whole-plasmid PCR method to construct iterative combinatorial mutants. The primers are designed as follows (both are described in the 5'-3' direction, and the underline represents the mutation site:

[0051] M1 (using the pET28a-KpADH recombinant plasmid as a template)

[0052] S196V-F: ACTATCCACCCA GTT TTCGTT

[0053] S196V-R:TCCGAAAACGAA AAC TGGGTG

[0054] M2 (using the pET28a-KpADH recombinant plasmid as a template)

[0055] S196W-F: ACTATCCACCCA TGG TTCGTT

[0056] S196W-R:TCCGAAAACGAA CCA TGGGTG

[0057] M3 (using the pET28a-KpADH recombinant plasmid as a template)

[0058] S196P-F: ACTATCCACCCA CCT TTCGTT

[0059] S196P-R:TCCGAAAACGAA AGG TGGGTG

[0060] M4 (using the pET28a-KpADH recombinant plasmid as a template)

[0061] S196G-F: ACTATCCAC...

Embodiment 3

[0069] Example 3: Expression and purification of alcohol dehydrogenase and mutants thereof

[0070] The recombinant Escherichia coli carrying the stereoselective improvement mutant was inoculated into the LB medium containing kanamycin sulfate (50 μg / mL) by 2% of the transfer amount, 37 ° C, 200 rpm shaker culture, the absorbance of the culture solution OD 600 When it reaches 0.8, add 0.2mM isopropyl-β-D-six-generation galactofuranoside (IPTG) for induction, the induction temperature is 25°C, after induction for 8h, centrifuge at 8000rpm for 10min to obtain highly expressed recombinant alcohol dehydrogenase mutation The collected bacterial cells were suspended in potassium phosphate buffer (100 mM, pH 6.0) and ultrasonically disrupted.

[0071]The column used for purification is HisTrap FF crude, a nickel affinity column, which is accomplished by affinity chromatography using the histidine tag on the recombinant protein. First use solution A to equilibrate the nickel column,...

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Abstract

The invention discloses an alcohol dehydrogenase mutant, and an application thereof in the synthesis of bisaryl chiral alcohols, and belongs to the technical field of bioengineering. The alcohol dehydrogenase mutant has excellent catalytic activity and stereoselectivity, and achieve efficiently catalyzed preparation of a series of chiral bisaryl alcohols with R- and S- configurations. Alcohol dehydrogenase is coupled with glucose dehydrogenase or formate dehydrogenase in order to synthesize multiple antihistamine chiral bisaryl alcohol intermediates. Compared with existing reports, the methodfor preparing the bisaryl chiral alcohols by asymmetric catalytic reduction of the alcohol dehydrogenase has the advantages of simplicity in operation, high substrate concentration, completeness in reaction, high product purity and great industrial application prospect.

Description

technical field [0001] The invention relates to an alcohol dehydrogenase mutant and its application in the synthesis of biaryl chiral alcohols, belonging to the technical field of bioengineering. Background technique [0002] Chiral bisaryl alcohol compounds are key chiral intermediates for the synthesis of many drugs and fine chemicals, among which chiral (4-chlorophenyl)-(pyridin-2-yl)-methanol (CPMA) is a synthetic antihistamine The key chiral intermediate of the drug betahistine. Using latent chiral (4-chlorophenyl)-(pyridin-2-yl)-methanone (CPMK) as raw material, the synthesis of chiral CPMA by chemical asymmetric reduction is mainly realized by the following five technologies: [0003] 1. Under the condition of substrate concentration of 1.0mM, trans-RuCl 2 [(R)-xylbinap][(R)-daipen] is used as a catalyst, reacted at room temperature for 24 hours under the pressure of 40-60psi nitrogen, and reduced to obtain (S)-(4-chlorophenyl)-(pyridine-2- base)-methanol ((S)-CPMA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/70C12N1/21C12P17/12C12R1/19
CPCC12N9/0006C12P17/12C12P41/002C12Y101/01001
Inventor 倪晔周婕妤许国超王岳
Owner JIANGNAN UNIV
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