| Acetobacter aceti,a kind of aerobic gram-negative bacteria,plays an important role in food industry especially in vinegar brewing because of its good alcohol tolerance,acid tolerance and the extraordinary capacity of acetic acid production.Vinegar brewing is closely related to the metabolic enzymes which catalyze the oxidation process from alcohol to acetic acid.In acetic acid bacteria,the biosynthesis of acetic acid is maily catalyzed by membrane-bound and multiple subunit composed alcohol dehydrogenase(ADH)and aldehyde dehydrogenase(ALDH),although there is other ADH or ALDH with single peptide structure existing in the cytoplasm.How to use these metabolic enzymes in vitro to convert ethanol to acetic acid,is an interesting topic with profound social significance.Taking non-membrane-bound ADH and ALDH with single peptide structure as key focus,the gene adh P,NAD(P)-adh and ald A of A.Pasteurianus strain CICC22518 was selected as the object this study.The recombinant expression vector carrying the target gene was constructed using gene recombination technology.Escherichia coli expression system was selected to produce recombinant enzymes.The enzymes with high catalyzing activity were purified through nickel column.The results of this study was expected to provide a theoretical and practical basis for the development of enzyme which could efficiently convert ethanol to aldehyde or acetic acid in vitro.Objective: To carry out exogenous expression,purification and activity analysis of the alcohol metabolic enzymes of A.pasteurianusMethods:(1)Obtain the target gene sequence from Gen Bank database,and use Snap Gene software to analyze,the sequence of target gene was chemical synthesis;(2)Use gene recombination technology to construct expression vector;(3)Use IPTG to induce the expression of recombinant protein;(4)Based on ultrasonic cell disruption,the solubility of the recombinant protein was analyzed by SDS-PAGE;(5)The recombinant protein was purified and collected by nickel column purification;(6)Grayscale analysis and purification yield analysis were performed by Image J software;(7)The Bradford determination method was used to the protein concentration;(8)The enzyme activity was detected by potassium ferricyanide reduction method.Results:(1)The expression vectors pET28a(+)-adh P,pET28a(+)-NAD(P)-adh,pET28a(+)-ald A and pET28a(+)-NAD(P)-adh-ald A was constructed;(2)SDS-PAGE analysis showed that ADHP and NAD(P)-ADH could be expressed and accumulated as inclusion bodies in E.coli BL21(DE3),and ALDA and NAD(P)-ADH-ALDA could be over expressed and accumulated as soluble state in E.coli BL21(DE3).(3)Four recombinant protein,ADHP,NAD(P)-ADH,ALDA and NAD(P)-ADH-ALDA,were successfully recovered by nickel column purification.Data analysis showed that the recoveries were 90.13%,85.78%,94.33%and 92.89%,respectively.(4)The results of enzyme content detection and enzyme activity analysis showed that the enzyme activity of ADHP was significantly higher than that of NAD(P)-ADH-ALDA,and extremely significantly higher than that of NAD(P)-ADH,which were 98.12% and 175.21% higher,respectively,the catalytic ability was up to 277.5±7.07U/m L.The ethanol catalytic activity of the recombinant fusion enzyme NAD(P)-ADH-ALDA was significantly higher than that of NDA(P)-ADH,which was 256.667±6.24 U/m L,and the acetaldehyde catalytic activity was also 101.94% higher than that of ALDA,reaching173.333±3.12 U/m L,indicating that the enzyme activity was still retained after recombination,and the refolded spatial structure also improved the catalytic function of the enzyme.Conclusion: In this experiment,the Escherichia coli expression system was used to efficiently express the acetate metabolizing enzymes ADHP,NAD(P)-ADH,ALDA and NAD(P)-ADH-ALDA.The purified and recovered recombinant enzymes have high catalytic activity and purity.These results established the experimental foundation for the further development of the enzymes preparation for converting ethanol into acetate under in vitro conditions and related basic research. |
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