Alcohol dehydrogenase mutant and application thereof

An alcohol dehydrogenase and mutant technology, which is applied in the field of enzyme engineering to achieve the effects of improving catalytic activity, improving activity expression rate, and improving fermentation enzyme activity

Active Publication Date: 2020-04-24
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, by introducing a strong promoter gene of a molecular chaperone into the genome of Escherichia coli genetically engineered bacteria, a new kind of Escherichia coli genetically

Method used

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  • Alcohol dehydrogenase mutant and application thereof
  • Alcohol dehydrogenase mutant and application thereof
  • Alcohol dehydrogenase mutant and application thereof

Examples

Experimental program
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[0031] Example 1 Microbial cultivation and enzyme activity determination

[0032] 1.1 Cultivation of microorganisms

[0033] The composition of LB liquid medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water and fixed to volume, sterilized at 121°C for 20 minutes, set aside. LB solid medium (plate petri dish): Add 20g / L agar powder to the LB liquid medium, sterilize at 121°C, cool down, and introduce it into the petri dish to make a flat plate.

[0034] The engineered strain E.coli BL21(DE3) containing related genes was inoculated into 5mL LB liquid medium containing 50μg / mL kanamycin and cultured at 37°C with shaking for 12h. Transfer to 500mL fresh LB liquid medium containing 50μg / mL Kan, and shake culture to OD at 37℃ 600 When it reaches about 0.8, add IPTG to its concentration of 0.3mM, and induce culture at 28℃ for 20h. After the cultivation, the culture solution was centrifuged at 10000 rpm for 10 min, the supernatant was discarded, the bacterial ...

Example Embodiment

[0041] Example 2 Construction of Escherichia coli genetically engineered bacteria overexpressing molecular chaperone GroES-EL

[0042] Using CRISPR gene editing technology, the weak promoter σ26-σ32 (gene sequence SEQ ID NO.4) at the front end of the GroES-EL gene expression molecular chaperone in the E. coli engineered bacteria genome was replaced with a stronger constitutive promoter J23104 ( From http: / / parts.igem.org / Promoters / Catalog / Anderson, the gene sequence is SEQID NO.5).

[0043] The specific steps are as follows:

[0044] (1) Preparation of electrocompetent E. coli cells carrying pCas plasmid

[0045] 1) Preparation of transcompetent cells: Pick a single colony of Escherichia coli BL21(DE3) and culture it in a 5mL LB liquid medium test tube with a kanamycin concentration of 50μg / mL for 6-8h, and then use 2% inoculum Inoculate in a 50mL Erlenmeyer flask, incubate at 37°C, 200rpm for 2h, and place the triangle plate on ice to cool for 30min. Take 10 mL of the bacterial sol...

Example Embodiment

[0097] Example 3 Construction of CbADH wild-type and mutant recombinant bacteria

[0098] Entrust Beijing Kinco Xinye Biotechnology Co., Ltd. to provide codon optimization and gene synthesis services, and synthesize CbADH wild-type (NCBI accession number WP_077844196.1) and mutant genes (SEQ ID NO.3) into pET-28a(+ ) On the plasmid with the sequence encoding the 6×His tag (located at the C-terminus of the protein) to facilitate protein purification and place the target gene between the restriction sites Nco I and Xho I. Obtain pET-28a(+)-CbADH-WT and pET-28a(+)-CbADH-22M recombinant plasmids, and respectively transform them into the E. coli genetically engineered bacteria obtained in Example 2 (enhanced expression molecular chaperone GroES-EL )in.

[0099] The amino acid sequence of the wild-type CbADH protein expressed by the wild-type gene sequence is shown in SEQ ID NO. 1, and the amino acid sequence of the mutant CbADH protein expressed by the mutant gene sequence is shown in ...

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Abstract

The invention discloses an alcohol dehydrogenase mutant and application thereof. The amino acid sequence of the alcohol dehydrogenase mutant is as shown in SEQ ID NO. 2. The alcohol dehydrogenase mutant is obtained by point mutation of 22 sites of a wild type enzyme, and the activity expression capability in escherichia coli is remarkably improved. By taking isopropanol as a substrate, the catalytic activity and thermal stability of the alcohol dehydrogenase mutant disclosed by the invention are remarkably improved. A brand-new escherichia coli genetically engineered bacterium is constructed by strengthening expression of molecular chaperone GroES-EL protein in escherichia coli and used for expressing the alcohol dehydrogenase mutant, so that the problem of inclusion bodies is basically solved, and the fermentation enzyme activity is further improved to 38.20 U/mL.

Description

technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to an alcohol dehydrogenase mutant and its application. Background technique [0002] Alcohol dehydrogenase (ADH) is an enzyme that widely exists in bacteria and eukaryotes. Alcohol dehydrogenase can catalyze the oxidative dehydrogenation of alcohol under certain conditions to generate the corresponding ketone, and at the same time convert the oxidized coenzyme NAD(P) + Converted to reduced coenzyme NAD(P)H; ketones can also be reduced to corresponding alcohols while consuming the same amount of reduced coenzyme NAD(P)H. Therefore, alcohol dehydrogenase has a wide range of applications in the fields of coenzyme recycling and asymmetric synthesis of chiral alcohols. Alcohol dehydrogenase (CbADH) from Clostridium beijerinckii is a NADPH-dependent alcohol dehydrogenase. When isopropanol is used as a substrate to regenerate NADPH, its activity per unit protein is the highes...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P19/36C12P7/16C12P7/04C12R1/19
CPCC12N9/0006C12N15/70C12P7/04C12P7/16C12P19/36C12Y101/01001Y02E50/10
Inventor 杨立荣周海胜邓通吴坚平张红玉
Owner ZHEJIANG UNIV
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