【摘 要】
：
本研究以Delfiia sp.T3-6为材料,通过硫酸铵分级沉淀、DEAE阴离子交换层析、疏水层析及Sephadex G200凝胶层析从粗酶液中纯化出单一条带,活力回收0.28％,纯化倍数为20倍,比
【机 构】
：
江西农业大学生物科学与工程学院,江西南昌330045河南科技大学食品与生物工程学院河南洛阳471003南京农业大学生命科学学院农业部农业环境微生物工程重点实验室江苏南京210095
【出 处】
：
第十六次全国环境微生物学学术研讨会
论文部分内容阅读
本研究以Delfiia sp.T3-6为材料,通过硫酸铵分级沉淀、DEAE阴离子交换层析、疏水层析及Sephadex G200凝胶层析从粗酶液中纯化出单一条带,活力回收0.28％,纯化倍数为20倍,比酶活为4126.43U/mg,SDS--PAGE电泳确定该蛋白表观分子量约为32.52kD.通过蛋白质N端测序和肽指纹测序得到三条氨基酸序列,根据氨基酸序列从Delftia sp.T3-6中成功的克隆到一个新的酰胺酶功能基因DamH,DamH基因ORF长度903bp,编码300个氨基酸序列；经NCBI比对,该酶是一个具有酰胺酶活性的酯酶.构建了该基因的表达载体pET29a-damH并在E.coli BL21(DE3)进行诱导表达,通过Ni-NTA亲和层析将重组蛋白进行纯化后进行酶学特性研究,酶反应最适温度为35℃,最适酶反应pH=6.0,Mn2+离子对酶活有促进作用,Cu2+、Zn2+、Ni2+、Fe2+对酶活有抑制作用；PMSF、SDS强烈抑制酶的活性.
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