Improve The Activity Of The Human Alcohol Dehydrogenase2 By Applying The Site-directed Mutagenesis

Alcohol dehydrogenase (ADH) is a metallic enzyme which contains zinc.It is most noted as being the enzyme primarily responsible for catalyzing the first reaction in the metabolism of ethanol.Studying on ADH plays an important role in protecting against damage of liver tissue aroused by alcohol. ADH shown its great potential for developing and researching the product of antialcoholism and protecting liver. With rapid development of the site-directed mutagenesis technique, people can achieve the aim at changing the structure and function of protein by applying the site-directed mutagenesis.Residue near the active center of ADH2 restricts its application,so the replacement of the target site would enlarge the pocket and change the specificity and affinity of the substrate, and the activity of ADH2 can be improved obviously. We changed the ADH2 gene over the megaprimer PCR method. Three primers were synthesized. one was a primer with the needed mutation; the other two containing appropriate enzyme sites for construction of the PCR fragment into a suitable plasmid were located at the flanks of the mutation primer. After the amplification of the PCR fragment using the mutation primer and the reverse flanking primer,another PCR was performed using the previous PCR mutation segment as primer and the other flanking primer.As a result, the point mutations had been completed successfully. The phenylalanine of the 94th amino acid was replaced by alanine,and the threonine of the 371st amino acid was replaced by cysteine.ADH2 was recombined with pTYB11 vector at EcoR I and Xho I sites, and recombinant DNA (prokaryotic system) was transformed into ER2566. The result of the best induced expression indicates that the human ADH2 fusion protein is induced by 0.3mmol·L^-1 IPTG for 4h at 16℃. After the positive recombinant screened was induced at the best condition, ADH2 was got and expressed accounted for 15.85% of totle proteins.Recombinant ADH2 was purified by IMPACT^TM-CN Protein Purification System and the molecular weights of the target proteins were 40 KD. During the mensuration of the concentrations of purified proteins, we got protein with the concentration about 0.25～0.33 mg·mL^-1.The enzyme activities of human ADH2 was detected by being monitored the A_340nm values at 25℃in 0.1 mol·L^-1 sodium phosphate, pH 7.5. We calculated its activity with YADH as standard protein,the activity of ADH2 was 8.9 U·mg^-1,obviously, the activity of ADH2 had been improved significantly.In this experiment,the point mutations had been completed successfully and the activity of ADH2 also improved significantly.This could provide the material for researching their bioactivities and lay a foundation of developing and researching the product of antialcoholism and protecting liver.