Alcohol dehydrogenase mutant and application thereof

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...

Examples

Embodiment 1

[0031] Example 1 Microbial Culture and Enzyme Activity Determination

[0032] 1.1 Culture of microorganisms

[0033] Composition of LB liquid medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water and then constant volume, sterilized at 121°C for 20min, ready for use. LB solid medium (plate petri dish): add 20g / L agar powder on the basis of LB liquid medium, sterilize at 121°C, cool down, and introduce into a petri dish to make a flat plate.

[0034] The engineered bacteria E.coli BL21(DE3) containing related genes were inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin, and cultured with shaking at 37°C for 12 hours. Transfer to 500mL fresh LB liquid medium also containing 50μg / mL Kan, shake culture at 37°C until OD 600 When it reaches about 0.8, add IPTG to its concentration of 0.3mM, and induce culture at 28°C for 20h. After the cultivation, the culture solution was centrifuged at 10,000 rpm for 10 min, the supernatant was dis...

Embodiment 2

[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 is SEQ ID NO.4) at the front end of the expression molecular chaperone GroES-EL gene in the genome of Escherichia coli engineering bacteria was replaced with a higher strength constitutive promoter J23104 ( Sourced from http: / / parts.igem.org / Promoters / Catalog / Anderson, the gene sequence is SEQID NO.5).

[0043] The specific operation steps are as follows:

[0044] (1) Preparation of Escherichia coli electroporation competent cells carrying pCas plasmid

[0045] 1) Preparation of competent cells for transduction: Pick a single colony of E. coli E coli BL21 (DE3) and culture it in a 5 mL LB liquid medium test tube with a kanamycin concentration of 50 μg / mL for 6-8 hours, then use 2% inoculum Inoculate in a 50mL Erlenmeyer flask, incubate at 37°C, 200rpm for 2h, and p...

Embodiment 3

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

[0098] Entrusted Beijing Qingke Xinye Biotechnology Co., Ltd. to provide codon optimization and gene synthesis services, and synthesized CbADH wild-type (NCBI accession number WP_077844196.1) and mutant genes (SEQ ID NO.3) in pET-28a (+ ) plasmid with the sequence for coding 6×His tag (located at the C-terminus of the protein), which is convenient for protein purification, and the target gene is placed between the enzyme cutting sites Nco I and Xho I. Obtain pET-28a (+)-CbADH-WT and pET-28a (+)-CbADH-22M recombinant plasmid, and it is respectively transformed into the Escherichia coli genetic engineering bacterium (enhanced expression molecular chaperone GroES-EL) obtained in embodiment 2 )middle.

[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, the amino acid sequence of the mutant CbADH protein expressed by the m...

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...

Examples

Embodiment 1

[0031] Example 1 Microbial Culture and Enzyme Activity Determination

[0032] 1.1 Culture of microorganisms

[0033] Composition of LB liquid medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water and then constant volume, sterilized at 121°C for 20min, ready for use. LB solid medium (plate petri dish): add 20g / L agar powder on the basis of LB liquid medium, sterilize at 121°C, cool down, and introduce into a petri dish to make a flat plate.

[0034] The engineered bacteria E.coli BL21(DE3) containing related genes were inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin, and cultured with shaking at 37°C for 12 hours. Transfer to 500mL fresh LB liquid medium also containing 50μg / mL Kan, shake culture at 37°C until OD 600 When it reaches about 0.8, add IPTG to its concentration of 0.3mM, and induce culture at 28°C for 20h. After the cultivation, the culture solution was centrifuged at 10,000 rpm for 10 min, the supernatant was dis...

Embodiment 2

[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 is SEQ ID NO.4) at the front end of the expression molecular chaperone GroES-EL gene in the genome of Escherichia coli engineering bacteria was replaced with a higher strength constitutive promoter J23104 ( Sourced from http: / / parts.igem.org / Promoters / Catalog / Anderson, the gene sequence is SEQID NO.5).

[0043] The specific operation steps are as follows:

[0044] (1) Preparation of Escherichia coli electroporation competent cells carrying pCas plasmid

[0045] 1) Preparation of competent cells for transduction: Pick a single colony of E. coli E coli BL21 (DE3) and culture it in a 5 mL LB liquid medium test tube with a kanamycin concentration of 50 μg / mL for 6-8 hours, then use 2% inoculum Inoculate in a 50mL Erlenmeyer flask, incubate at 37°C, 200rpm for 2h, and p...

Embodiment 3

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

[0098] Entrusted Beijing Qingke Xinye Biotechnology Co., Ltd. to provide codon optimization and gene synthesis services, and synthesized CbADH wild-type (NCBI accession number WP_077844196.1) and mutant genes (SEQ ID NO.3) in pET-28a (+ ) plasmid with the sequence for coding 6×His tag (located at the C-terminus of the protein), which is convenient for protein purification, and the target gene is placed between the enzyme cutting sites Nco I and Xho I. Obtain pET-28a (+)-CbADH-WT and pET-28a (+)-CbADH-22M recombinant plasmid, and it is respectively transformed into the Escherichia coli genetic engineering bacterium (enhanced expression molecular chaperone GroES-EL) obtained in embodiment 2 )middle.

[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, the amino acid sequence of the mutant CbADH protein expressed by the m...

Images