Alcohol dehydrogenase mutant and application thereof to synthesis of chiral biaryl alcohols

An alcohol dehydrogenase and mutant technology, which can be applied in the field of bioengineering, can solve the problems of low stereoselectivity of alcohol dehydrogenase, and achieve the effect of good industrial application prospect and high industrial application value.

Inactive Publication Date: 2018-08-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 to synthesis of chiral biaryl alcohols
  • Alcohol dehydrogenase mutant and application thereof to synthesis of chiral biaryl alcohols
  • Alcohol dehydrogenase mutant and application thereof to synthesis of chiral biaryl alcohols

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: the activity determination method of alcohol dehydrogenase:

[0047] The total reaction system is 200 μL, including: 1.0mM NADPH, 1.0mM substrate CPMK, sodium phosphate buffer (PBS, 100mM, pH 7.0), mix well, keep warm at 30°C for 2min, add an appropriate amount of enzyme solution, and detect light at 340nm Absorption changes.

[0048] Enzyme activity was calculated using the following formula:

[0049] Enzyme activity (U) = EW × V × 10 3 / (6220×l)

[0050] 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.

[0051] Determination method of optical purity ee:

[0052] As: the molar concentration of (S)...

Embodiment 2

[0053] Example 2: Construction of Alcohol Dehydrogenase Mutant Gene and Recombinant Expression Transformant

[0054] The whole plasmid PCR method was used to perform site-directed mutations on the amino acid residues at positions 136, 161, 196, 214, 215, and 237 to construct iterative combination mutants. The primers are designed as follows (both are described in the 5'-3' direction, and the underline represents the mutation site:

[0055] M1 (using the pET28a-KpADH recombinant plasmid as a template)

[0056] E214G-F:AAGAAACTAAAT GGTACT TGT

[0057] E214G-R:AAT TTCACAAGT ACC ATT TAG

[0058] M2 (using the pET28a-KpADH recombinant plasmid as a template)

[0059] E214V-F:AAGAAACTAAAT GTT ACT TGT

[0060] E214V-R: AATTTCACAAGT AAC ATT TAG

[0061] M3 (using the M1 recombinant plasmid as a template)

[0062] S237C-F:AAGACTCACTTC TGT CAATTC

[0063] S237C-R:ATCAATGAATTG ACA GAAG TG

[0064] M4 (using the M3 recombinant plasmid as a template)

[0065] Q136N-F: ACC...

Embodiment 3

[0085] Example 3: Expression and purification of alcohol dehydrogenase and mutants thereof

[0086] 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.

[0087] 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 to synthesis of chiral biaryl alcohols, and belongs to the technical field of bioengineering. The alcohol dehydrogenase mutant has excellent catalytic activity and stereoselectivity and can be used for efficient catalysis for preparation of a series of R-configuration and S-configuration chiral biaryl alcohols. Alcohol dehydrogenase can be applied to synthesis of various chiral biaryl alcohol intermediates of antihistamine drugs by being coupled with glucose dehydrogenase or formate dehydrogenase. Compared withthe existing reports, a method for preparing the chiral biaryl alcohols from alcohol dehydrogenase through asymmetric catalytic reduction has the advantages of simple operation, high substrate concentration, complete reaction and high product purity, and has broad 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): C12N9/04C12N15/53C12N1/21C12P17/12C12R1/19
CPCC12N9/0006C12P17/12C12Y101/01001C12N15/70
Inventor 倪晔周婕妤许国超王岳
Owner JIANGNAN UNIV
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