Alcohol dehydrogenase mutant and application thereof in synthesis of chiral diaryl alcohol compounds

A technology of alcohol dehydrogenase and catalytic center, applied in application, antibody mimics/scaffolds, enzymes, etc., can solve the problems of unknown protein sequence, high stereoselectivity, and no amino acid sequence

Active Publication Date: 2020-05-05
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the efficiency of these whole-cell transformation methods is low, and there is no report on the amino acid sequence of the relevant enzyme
In 2007, Truppo et al. reported the use of commercial carbonyl reductase (ketoreductase, KRED) to asymmetrically reduce prochiral ketones to synthesize (S)-(4-chlorophenyl)pyridine-2-methanol, but the ee value was only 60 %, the sequence of the enzyme protein is unknown, and the source of the species has not been reported (Truppo et al., Org. Lett., 2007, 9(2):335-338)
However, since (S)-(4-chlorophenyl)pyridine-2-methanol is the precursor required for the synthesis of drug intermediates, there is currently no good enzyme that can efficiently catalyze (4-chlorophenyl)pyridine-2-methanone Generates (S)-products with high stereoselectivity

Method used

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  • Alcohol dehydrogenase mutant and application thereof in synthesis of chiral diaryl alcohol compounds
  • Alcohol dehydrogenase mutant and application thereof in synthesis of chiral diaryl alcohol compounds
  • Alcohol dehydrogenase mutant and application thereof in synthesis of chiral diaryl alcohol compounds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0149] Embodiment 1, alcohol dehydrogenase TbSADH and its mutant sequence

[0150] The amino acid sequence of the alcohol dehydrogenase TbSADH in this example is shown in SEQ ID No.2. In this embodiment, the alcohol dehydrogenase TbSADH mutant is specifically any of the following (1)-(39):

[0151] (1) Alcohol dehydrogenase TbSADH mutant P84A: After mutating the 84th amino acid of the amino acid sequence of alcohol dehydrogenase TbSADH from proline (P) to alanine (A), and keeping other amino acid sequences unchanged The protein which becomes.

[0152] (2) Alcohol dehydrogenase TbSADH mutant P84V: After mutating the 84th amino acid of the amino acid sequence of alcohol dehydrogenase TbSADH from proline (P) to valine (V), and keeping other amino acid sequences unchanged The protein which becomes.

[0153] (3) Alcohol dehydrogenase TbSADH mutant P84S: obtained by mutating the 84th amino acid of the amino acid sequence of alcohol dehydrogenase TbSADH from proline (P) to serine ...

Embodiment 2

[0190] Embodiment 2, the preparation of the engineering bacterium of alcohol dehydrogenase TbSADH gene or its mutant

[0191] 1. Preparation of Alcohol Dehydrogenase TbSADH Gene Wild Type Genetic Engineering Strain

[0192] 1. Optimization of Alcohol Dehydrogenase TbSADH Gene Sequence

[0193] The alcohol dehydrogenase TbSADH gene derived from Thermoanaerobacter brockii was codon-optimized with Escherichia coli as the host cell, and the whole gene was synthesized after optimization. The optimized alcohol dehydrogenase TbSADH gene sequence is shown as SEQ ID No. 1.

[0194] 2. Construction of recombinant vector pRSFDuet-1-TbSADH

[0195] Use primers pRSF-NcoI (ggt ata tct cct tat taa agt taa aca aaa tta ttt ctacag g) and pRSF-AvrII (taa cct agg ctg ctg cca ccg ctg agc aat aac) to homolog the DNA fragment shown in SEQ ID No.1 Recombined into the pRSFDuet-1 plasmid. After sequencing, it was shown that the small fragment between NcoI and AvrII of the restriction site of pRSFDu...

Embodiment 3

[0253] Example 3, the expression of alcohol dehydrogenase TbSADH gene or its mutants and the preparation of whole cells, crude enzyme solution and crude enzyme powder

[0254] The alcohol dehydrogenase TbSADH gene wild-type genetic engineering strain prepared in step 1 of Example 2 and the 39 alcohol dehydrogenase TbSADH gene mutant engineering strains prepared in step 2 were induced and expressed to obtain wild-type alcohol dehydrogenase TbSADH and Individual alcohol dehydrogenase TbSADH mutants in Example 1. Specific steps are as follows:

[0255]Pick the recombinant E.coli BL21 (DE3) transformants containing the recombinant plasmid of the alcohol dehydrogenase TbSADH gene or its mutants respectively, and put them in 5 mL LB liquid medium containing 50 μg / mL kanamycin, 37 °C, 220 rpm Shake overnight for 12-16 hours, inoculate into TB liquid medium containing 50 μg / mL kanamycin according to 1% (volume percentage) inoculation amount, and culture at 37°C until OD 600 When the...

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Abstract

The invention discloses an alcohol dehydrogenase mutant and application thereof in synthesis of chiral diaryl alcohol compounds. The alcohol dehydrogenase mutant disclosed by the invention is a protein, which is obtained through mutating amino acid residues located in a catalytic center pocket of alcohol dehydrogenase TbSADH, and particularly is the protein, which is obtained through mutating 39thand/or 42th and/or 84th and/or 85th and/or 86th and/or 104th and/or 110th and/or 294th amino acid residues of an amino acid sequence of the alcohol dehydrogenase TbSADH. According to the alcohol dehydrogenase mutant and the application thereof, the alcohol dehydrogenase TbSADH is subjected to enzyme reforming by applying an orthogenesis technology and method, a series of mutants, which have enzyme activity to diaryl ketones with represented by (4-chlorophenyl)pyrid-2-ketone, are obtained, and the mutants are applied to biocatalytic synthesis of optically-pure chiral diaryl alcohol compounds.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to alcohol dehydrogenase mutants obtained by enzymatic modification of alcohol dehydrogenase derived from Thermoanaerobacter brockii by applying directed evolution technology and methods, and these mutants are used as biocatalysts in Application of asymmetric reduction in the preparation of optically pure chiral bis-aryl alcohol compounds. Background technique [0002] As an important class of building blocks, chiral bisaryl secondary alcohols can be used in the synthesis of a variety of chiral drugs, and the process of synthesizing chiral bisaryl alcohols from prochiral bisaryl ketones through asymmetric reduction has atomic Advantages of high economy. Among them, (S)-(4-chlorophenyl)pyridine-2-methanol is a chiral precursor for the synthesis of antihistamines, which can be used to synthesize antihistamines carbinoxamine (Barouh et al., J. Med.Chem., 1971,14(9):834-836) an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C07K19/00C12N15/53C12N15/62C12P17/12
CPCC12N9/0006C12Y101/01001C12P17/12C07K2319/43C07K2319/21C07K2319/22C07K2319/41
Inventor 孙周通刘贝贝曲戈刘保艳
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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