Abstract: The commercial acetaldehyde dehydrogenase is mainly extracted from animal cell and hepatocellular mitochondria. Source is limited and the cost is high. So the micobiological fermentation was used to obtain large amounts of aldh2, which had been cloned into the expression vector pET32a with 6His tag that was advantageous to purify the recombinant protein by nickel-chelate chromatography, then the recombinant was transformed into E.coli BL21(DE3). After induced by IPTG, the recombinant protein had been expressed and their molecular masses were determined to be approximately 55 kD by SDS-PAGE. Through the optimization of inducing expression conditions, the results showed that the condition(37℃, 1.0 mmol/L IPTG, induced for 6 h)is the optimal. Because all target protein were almost expressed in the form of inclusion body, the bioactive acetaldehyde dehydrogenase was obtained by means of degeneration, purification and renaturation for the inclusion body, and measured the concentration of the renaturated protein by the Bradford method that was 77 μg/mL. Experimental data showed that the final yield of enzyme was 14.49 U/L. Activity tests showed that the protein has the acetaldehyde dehydrogenase activity of about 1.449 U/mL. By constructing a recombinant vector pET32a-ALDH2 and optimizing the expression condition, aldh2 in prokaryotic expression host get a lot of expression. In addition, the activity of renaturation protein was improved by the dialysis renaturation method.

Key words: aldh2, Cloning, Condition optimization, Activity assay