Alcohol dehydrogenase mutant and application thereof to synthesis of diaryl chiral alcohol

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 effect of high industrial application value

Inactive Publication Date: 2018-08-10
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 to synthesis of diaryl chiral alcohol
  • Alcohol dehydrogenase mutant and application thereof to synthesis of diaryl chiral alcohol
  • Alcohol dehydrogenase mutant and application thereof to synthesis of diaryl chiral alcohol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1: the activity determination method of alcohol dehydrogenase:

[0061] 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.

[0062] Enzyme activity was calculated using the following formula:

[0063] Enzyme activity (U) = EW × V × 10 3 / (6220×l)

[0064] 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.

[0065] Determination method of optical purity ee:

[0066] As: the molar concentration of (S)...

Embodiment 2

[0067] Example 2: Construction of Alcohol Dehydrogenase Mutant Gene and Recombinant Expression Transformant

[0068] The site-directed mutation of the amino acid residue at position 237 was carried out by the whole plasmid PCR method, and the primers were designed as follows (both described in the 5'-3' direction), and the underline represents the mutation site:

[0069] Table 1 Design table of primers for site-directed mutagenesis

[0070]

[0071]

[0072]The PCR reaction system is: PCR reaction system (50 μL) including KOD enzyme (2.5U / mL) 1.0 μL, template (5-50ng) 1.0 μL, dNTP 4.0 μL, 10×reactionbuffer 5.0 μL, upstream and downstream primers 1.0 μL each , ddH 2 O to make up to 50 μL.

[0073] The PCR amplification program is: (1) Denaturation at 94°C for 3min, (2) Denaturation at 94°C for 30sec, (3) Annealing at 54°C for 30sec, (4) Extension at 72°C for 150sec, repeat steps (2) to (4) for 10- After 15 cycles, the final extension was 10 min at 72°C, and the PCR amp...

Embodiment 3

[0075] Example 3: Expression and purification of alcohol dehydrogenase and mutants thereof

[0076] 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 galactofuranose (IPTG) for induction, and the induction temperature is 25°C. After induction for 8 hours, centrifuge at 8000rpm for 10 minutes to obtain highly expressed recombinant alcohol dehydrogenase mutations The collected bacterial cells were suspended in potassium phosphate buffer (100 mM, pH 6.0) and ultrasonically disrupted.

[0077] 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 ...

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Abstract

The invention discloses an alcohol dehydrogenase mutant and application thereof to synthesis of diaryl chiral alcohol and belongs to the technical field of biological engineering. The alcohol dehydrogenase mutant disclosed by the invention has good catalytic activity and stereoselectivity, and a series of chiral diaryl alcohol with R- and S- configurations can be prepared through efficient catalysis. According to the alcohol dehydrogenase mutant, alcohol dehydrogenase is coupled with glucose dehydrogenase or formate dehydrogenase, and can be used for synthesizing various antihistamine drugs, i.e., a chiral diaryl alcohol intermediate. Compared with an existing report, a method for preparing the diaryl chiral alcohol through asymmetric catalysis of the alcohol dehydrogenase has the advantages of simplicity in operation, high substrate concentration, complete reaction and high product purity and has a very good 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 倪晔王岳戴威许国超周婕妤
Owner JIANGNAN UNIV
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