Expression and Enzymatic Properties of Stomach Alcohol Dehydrogenase δδ-ADH in Escherichia coli

doi:10.3969/j.issn.1673-1689.2019.07.011

Home > Archive>Volume 38, Issue 7, 2019 >78-85. DOI:10.3969/j.issn.1673-1689.2019.07.011

Expression and Enzymatic Properties of Stomach Alcohol Dehydrogenase δδ-ADH in Escherichia coli
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                        10.3969/j.issn.1673-1689.2019.07.011
                    
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In order to gain the δδ-ADH from human stomach，the target gene was synthesized according to the sequence of human stomach alcohol dehydrogenase δδ-ADH gene ADH7 in GeneBank，inserted into plasmid pET-32a（+） which was transformed into E.coli BL21（DE3）. IPTG was used to induce the experssion of δδ-ADH in E.coli BL21（DE3）. δδ-ADH still existed in the form of inclusion body even after optimizing the expression conditions. Active crude enzyme solution was obtained after the inclusion body was solubilized with 1 mol/L urea and the target protein was purified by HisTrap^TM excel affinity chromatography column. Then the specific activity of the enzyme and its essential enzymatic properties were studied. The results showed that the specific activity of δδ-ADH was 2.085 U/mg，Km was 28.43 mmol/L and Vmax was 316.46 μmol/（L·min）. The optimum reaction temperature of δδ-ADH was 40 ℃，and its specific activity decreased to a value which was about half of the maximal activity after a water bath in 30~50 ℃ for 60 min. The optimum pH of δδ-ADH was 9.0，after a water bath in different pH buffer in 25 ℃ for 30 min，the enzyme activity of δδ-ADH kept almost consistent at pH 6.0~11.0，while the relative enzyme activity was only 60% remained at pH 5.0. After a water bath in different concentrations of alcohol（0、500、1 000、1 500 mmol/L）in 37 ℃，the relative enzyme activities of δδ-ADH were 65.6%、51.1%、45.4% and 44.4%，respectively.

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CHANG Kaixia, SUN Junyong, LI Xiaomin, WU Dianhui, ZHU Dewei, LU Jian. Expression and Enzymatic Properties of Stomach Alcohol Dehydrogenase δδ-ADH in Escherichia coli[J]. Journal of Food Science and Biotechnology,2019,38(7):78-85.

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