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Alcohol dehydrogenase mutant, gene thereof, and application thereof in preparation of chiral diaryl alcohol

An alcohol dehydrogenase and mutant technology, which can be used in applications, genetic engineering, plant genetic improvement, etc., can solve the problems of low catalytic activity and stereoselectivity, and achieve good industrial application prospects, high catalytic activity and enantioselectivity. sexual effect

Inactive Publication Date: 2016-09-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0015] Therefore, the technical problem to be solved by the present invention is: aiming at the problem that the catalytic activity and stereoselectivity of the currently obtained alcohol dehydrogenase are low, to provide a mutant protein that improves alcohol dehydrogenase, and to encode the mutant protein The nucleic acid sequence of the protein, the recombinant expression vector and recombinant expression transformant containing the nucleic acid sequence, and the alcohol dehydrogenase mutant protein or the recombinant transformant expressing the alcohol dehydrogenase mutant protein are used as catalysts in carbonyl asymmetric reduction, Application in Preparation of Optical Chiral Biaryl Alcohols

Method used

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  • Alcohol dehydrogenase mutant, gene thereof, and application thereof in preparation of chiral diaryl alcohol
  • Alcohol dehydrogenase mutant, gene thereof, and application thereof in preparation of chiral diaryl alcohol
  • Alcohol dehydrogenase mutant, gene thereof, and application thereof in preparation of chiral diaryl alcohol

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Example 1 Construction and screening of random mutation library of alcohol dehydrogenase KpADH

[0050] First, design primers for random mutation, as shown in Table 1 and SEQ ID No. 2 and SEQ ID No. 3 in the sequence table.

[0051] Table 1 Alcohol dehydrogenase KpADH random mutation primers

[0052]

[0053]

[0054] PCR reaction system (25μL): template 10-20ng, 10×rTaq buffer, 2.5mM dNTP mix, 100μMMnSO 4 , 500μM MgCl 2 , 1.25U rTaq polymerase (TaKaRa, Japan), add sterile distilled water to 25μL.

[0055] The error-prone PCR amplification procedure 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 90s, repeat steps (2) to (4) After 30 cycles, the final extension was at 72°C for 10 minutes, and the PCR amplified product was stored at 4°C.

[0056] The amplified product and pET28a(+) vector were digested with restriction enzymes Nde I and BamH I, respectively, to form complementary sticky ends. 4 ...

Embodiment 2

[0060] Example 2 Expression and purification of alcohol dehydrogenase mutants

[0061] Transform the mutants with increased activity into fresh LB culture at 2% transfer volume and cultivate to OD 600 When it reaches 0.6-0.8, add 0.2mM IPTG, 30℃induction culture for 6 hours, 4℃, 8000r / min centrifugation for 10min to collect the bacteria. Suspend the collected bacteria in potassium phosphate buffer (100mM, pH 6.0), and ultrasonically disrupt

[0062] The column used for purification is the affinity column HisTrap FF crude (nickel column), which uses the histidine tag on the recombinant protein for affinity binding. First use solution A to equilibrate the nickel column, load the crude enzyme solution, continue to use solution A to elute the breakthrough peak, and use solution B (20mmol·L -1 Sodium phosphate, 500mmol·L -1 NaCl, 1000mmol·L -1 Imidazole, pH 7.4) was subjected to gradient elution, and the recombinant protein bound to the nickel column was eluted to obtain a recombinant a...

Embodiment 3

[0063] Example 3 Kinetics and selectivity analysis of alcohol dehydrogenase mutants

[0064] Determine the activity of KpADH at different substrate concentrations and coenzyme concentrations, and make a double reciprocal curve based on the reciprocal of the activity and substrate concentration to calculate the kinetic parameters.

[0065] Table 2 shows the k of wild-type alcohol dehydrogenase KpADH to CPMK cat / K m And ee are respectively 16.8L·s -1 ·Mmol -1 And 82.0%, mutant enzyme KpADH M131F , KpADH S196Y And KpADH S237A K for CPMK cat / K m And ee are respectively 17.8L·s -1 ·Mmol -1 And 82.1%, 19.3L·s -1 ·Mmol -1 And 74.7%, 79.2L·s -1 ·Mmol -1 And 96.1%. Mutant enzyme KpADH S237A Shows the highest catalytic activity and stereoselectivity, the k of CPMK cat / K m It is 4.71 times that of the wild type.

[0066] Table 2 Kinetic parameters and stereoselectivity of alcohol dehydrogenase mutants

[0067]

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Abstract

The invention discloses an alcohol dehydrogenase mutant, an encoding gene thereof, and an application thereof in the preparation of chiral diaryl alcohol. The mutant is obtained through substituting serine in the 237th position of alcohol dehydrogenase from Kluyveromyces sp.CCTCC M2011385 and with the amino acid sequence represented by SEQ ID No.1 with alanine. The alcohol dehydrogenase mutant has greatly higher reduction activity and stereoselectivity than wild enzymes. The mutant is especially suitable for asymmetric reduction of diaryl ketone to prepare chiral diaryl alcohol, and can be used to synthesize various antihistamine medicines. The alcohol dehydrogenase mutant has good industrial application prospect.

Description

Technical field [0001] The present invention belongs to the technical field of bioengineering, and specifically relates to an alcohol dehydrogenase mutant and its encoding gene and the mutant using the alcohol dehydrogenase to asymmetrically reduce latent diaryl ketones to prepare chiral diaryl alcohols In the application. Background technique [0002] Chiral bisaryl secondary alcohols are an important class of chiral compounds. The molecular structure of many drugs has the building block structure of bisaryl secondary alcohols, among which chiral (4-chlorophenyl)-(pyridine-2) -Yl)-methanol can be used to synthesize the anti-allergic drug bepotastine. The process of synthesizing chiral bisaryl alcohol from latent bisaryl ketone via asymmetric reduction reaction has the advantage of high atom economy. [0003] The chemical asymmetric reduction method is mainly realized by the following three technologies: [0004] 1. Using (4-chlorophenyl)-(pyridin-2-yl)-methanone (CPMK) as raw mat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12P17/12
CPCC12N9/0004C12P17/12
Inventor 许国超倪晔周婕妤韩瑞枝董晋军
Owner JIANGNAN UNIV
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