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

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.coli BL21(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HisTrapTM excel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316.46μmol/(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%。

In order to gain theδδ-ADH from human stomach,the target gene was synthesized according to the sequence of human stomach alcohol dehydrogenaseδδ-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 ofδδ-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 HisTrapTM 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 ofδδ-ADH was 2.085 U/mg,Km was 28.43 mmol/L and Vmax was 316.46μmol/(L·min).The optimum reaction temperature ofδδ-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 ofδδ-ADH was 9.0,after a water bath in different pH buffer in 25℃for 30 min,the enzyme activity ofδδ-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 ofδδ-ADH were 65.6%、51.1%、45.4%and 44.4%,respectively。