Thermotoga neapolitana中α-半乳糖苷酶基因的克隆表达与酶学性质研究

发布时间：2018-09-01 19:28

【摘要】：将新阿波罗栖热袍菌(Thermotoga neapolitana)的耐热α-半乳糖苷酶基因经PCR扩增,以p ET-28a为表达载体,转化至大肠杆菌(Escherichia coli)BL21(DE3)中进行表达,用0.8 mmol/L的异丙基-β-D-硫代半乳糖苷(IPTG)诱导,采用超声波细胞破碎菌体得到α-半乳糖苷酶。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)的方法获得α-半乳糖苷酶的分子质量约为35 ku。并对α-半乳糖苷酶的酶学性质进行研究,结果表明:该α-半乳糖苷酶的最适反应温度为85℃;最适反应p H值为5.0;在金属离子对酶活影响的研究中发现Al~(3+)、Na~+、K~+、Ca~(2+)、Mg~(2+)、Zn~(2+)、Co~(2+)对酶活起到促进作用,Cu~(2+)、Fe~(2+)、Ag~(2+)、Mn~(2+)、Ni~(2+)的终浓度达到100 mmol/L时对酶活有较强的抑制作用;具有较好的热稳定性,在95℃处理38 h后仍具有50%的活性。
[Abstract]:The heat-resistant 伪 -galactosidase gene of (Thermotoga neapolitana) was amplified by PCR and transformed into E. coli (Escherichia coli) BL21 (DE3) with p ET-28a as expression vector. It was induced by isopropyl- 尾 -Dthiogalactoside (IPTG) of 0.8 mmol/L. 伪-galactosidase was obtained by supersonic cell fragmentation. The molecular weight of 伪 -galactosidase was about 35 ku. by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzymatic properties of 伪 -galactosidase were studied. The results showed that the optimum reaction temperature of 伪 -galactosidase was 85 鈩，

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