胃乙醇脱氢酶δδ-ADH的原核表达及酶学性质

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

DOI：10.3969/j.issn.1673-1689.2019.07.011

中文关键词: 乙醇脱氢酶 δδ-ADH 基因克隆 酶学性质

英文关键词: alcohol dehydrogenase,δδ-ADH,gene cloning,enzymatic properties

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中文摘要:

为获得人胃乙醇脱氢酶δδ-ADH，根据GeneBank中δδ-ADH的基因序列合成目的基因ADH7，构建重组表达载体pET-32a（+）-ADH7，并转化大肠杆菌E.coliBL21（DE3）。获得的阳性转化子经异丙基硫化-β-D-半乳糖苷（IPTG）诱导表达，SDS-PAGE电泳结果显示，目的蛋白质以包涵体的形式得到大量表达。用1 mol/L尿素溶解包涵体获得有活性的粗酶液，经HisTrap^TMexcel亲和层析柱纯化获得目的蛋白质，检测δδ-ADH酶活并对其基本酶学性质进行研究。结果显示，δδ-ADH的活性为2.085 U/mg，Km为28.43 mmol/L，Vmax为316.46 μmol/（L·min）。δδ-ADH的最适反应温度为40 ℃，在30~50 ℃范围内温浴60 min，相对酶活在47%以上；最适pH为9.0，将δδ-ADH在不同pH（pH 3.0~11.0）的广泛缓冲液中于25 ℃保温30 min，在pH 6.0~11.0范围内酶活较稳定。pH 5.0时，相对酶活剩余60%；在0、500、1 000、1 500 mmol/L乙醇中37 ℃温浴30 min，相对酶活分别为65.6%、51.1%、45.4%和44.4%。

英文摘要:

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 geneADH7 in GeneBank，inserted into plasmid pET-32a（+） which was transformed intoE.coliBL21（DE3）. IPTG was used to induce the experssion of δδ-ADH inE.coliBL21（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^TMexcel 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 andVmax 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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