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Alcohol dehydrogenase mutant and its application

A technology of alcohol dehydrogenase and mutants, applied in the direction of oxidoreductase, introduction of foreign genetic material by using vectors, recombinant DNA technology, etc., can solve the problems of enzyme activity loss and achieve high conversion efficiency

Inactive Publication Date: 2015-11-18
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] To sum up, the existing research on coenzyme-specific modification has been further developed, but as far as the existing research results are concerned, there are not many studies using rational design, and most of the enzyme activities after modification show varying degrees. Loss, coenzyme specificity changes to a certain extent but cannot be completely changed, etc. There is a large room for development in these aspects

Method used

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  • Alcohol dehydrogenase mutant and its application
  • Alcohol dehydrogenase mutant and its application
  • Alcohol dehydrogenase mutant and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Construction of Alcohol Dehydrogenase Gene.

[0040] 1. Acquisition of alcohol dehydrogenase gene:

[0041] Candida magnoliae (Candida magnoliaeATCC12573), medium YPD (g L -1 ): Yeast extract 10g, peptone 20g, glucose 20g, add distilled water to 1L.

[0042] Candida magnoliae (Candida magnoliae ATCC12573) was inoculated in 5 mL of LYPD liquid medium and cultured at 30°C until the logarithmic growth phase, and the genome was extracted using a genomic DNA extraction kit (Beijing Tianwei Bioengineering Co., Ltd. Yeast Genome Extraction Kit, GD2415YeastgDNAKit).

[0043] The primers used to construct the expression vectors and the primers with restriction sites are as follows:

[0044] The upstream primer is (NdeIsiteisunderlined):

[0045] 5'-GGAATTC CATATG ACGACTACTTCAAATGCGCTCGTCAC-3'

[0046] The downstream primer is (EcoRIsiteisunderlined):

[0047] 5'-CCG GAATTC CTAAGCAATCAAGCCATTGTCGACCAC-3'

[0048] All primers were synthesized by Shanghai Meiji...

Embodiment 2

[0054] Example 2: Construction of Alcohol Dehydrogenase Mutant Gene.

[0055] 1. Site-directed mutation

[0056] The amino acid residues located at Ser13, Ala34, Ser35, and Arg36 positions were subjected to site-directed mutagenesis using Stratagene's rapid conversion site-directed mutagenesis kit. The primers are designed as follows (both are described in the 5'-3' direction, and the underline represents the mutation site):

[0057] Ser35Asp (pET24a-ADH recombinant plasmid as template)

[0058] S35D-1:CAGTGTTACGCTGGCC GAC CGCAGTGTTG

[0059] S35D-2:CAACACTGCG GTC GGCCAGCGTAACACTG

[0060] Ser35Asp / Arg36Ile (mutant Ser35Asp as template)

[0061] S35D / R36I-1:CAGTGTTACGCTGGCCGAC ATC AGTGTTG

[0062] S35D / R36I-2:CAACACT GAT GTCGGCCAGCGTAACACTG

[0063] Ala34Ile / Ser35Asp / Arg36Ile (mutant Ser35Asp / Arg36Ile as template)

[0064] A34I / S35D / R36I-1:CAGTGTTACGCTG ATC GACCGCAGTGTTG

[0065] A34I / S35D / R36I-2:CAACACTGCGGTC GAT CAGCGTAACACTG

[0066] Ser13Ala / Ser35Asp / A...

Embodiment 3

[0080] Example 3: Purification of alcohol dehydrogenase and mutant enzymes thereof.

[0081] 1. Preparation of crude enzyme solution: take the induced LB culture medium at 8000r·min-1, centrifuge for 15min to collect the bacteria, wash twice with sterile water, resuspend the bacteria in pH6.220mmol·L-1 phosphate buffer Cells were disrupted ultrasonically in an ice bath. Centrifuge the ultrasonically crushed sample at 12,000r·min-1 at 4°C for 10min to obtain the supernatant, which is the crude enzyme solution.

[0082] 2. Ammonium sulfate precipitation: put the crude enzyme solution in an ice bath, slowly add saturated ammonium sulfate solution dropwise under magnetic stirring until the final concentration of ammonium sulfate is 30%, and stir overnight at 4°C. Centrifuge and take the supernatant. Also under the condition of ice bath, saturated ammonium sulfate solution was slowly added dropwise to the supernatant to a final concentration of 60%, and stirred overnight at 4°C. ...

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Abstract

The invention discloses an alcohol dehydrogenase mutant which is obtained by the mutation of the amino acid of wild type alcohol dehydrogenase in any one of the following conditions: the 35th-site serine in the sequence of the wild type alcohol dehydrogenase mutates into aspartic acid; the 35th-site serine in the sequence of the wild type alcohol dehydrogenase mutates into aspartic acid, and the 36th-site arginine mutates into isoleucine; the 34th-site alanine, valine or cysteine in the sequence of the wild type alcohol dehydrogenase mutates into isoleucine, the 35th-site serine mutates into aspartic acid, and the 36th-site arginine mutates into isoleucine; and the 13th-site serine in the sequence of the wild type alcohol dehydrogenase mutates into alanine, the 34th-site alanine, valine or cysteine mutates into isoleucine, the 35th-site serine mutates into aspartic acid, and the 36th-site arginine mutates into isoleucine, wherein the sequence of the wild type alcohol dehydrogenase is shown by the SEQ ID No:2, 4, 6 or 8.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an alcohol dehydrogenase (Alcohol Dehydrogenase, ADH) mutant which can be used as a chiral alcohol synthesis catalyst and its application. Background technique [0002] Chiral Alcohols are widely used in the preparation of chiral drugs, agricultural chemicals and various types of chiral materials. Take (S)-4-chloro-3-hydroxybutyric acid ethyl ester [(S)-CHBE] as an example, (S)-CHBE can be used in the synthesis of many active drugs, it is enantioselective synthesis of SlageninsB and C and Statins - key chiral intermediates of hydroxymethylglutaryl CoA (HMG-CoA) reductase inhibitors, and (S)-CHBE can also be converted to 1,4-dihydropyridine β-blockers agent [1]. [0003] Carbonyl reductase biocatalyzed asymmetric reduction of COBE to prepare (S)-CHBE has attracted widespread attention due to its high efficiency, high stereoselectivity, mild reaction conditions, and good ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/70C12N1/21C12P7/62
CPCC12N9/0006C12P7/62
Inventor 严明许琳邱晓鸾
Owner NANJING TECH UNIV
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