K.pneumoniae XJPD-Li甘油脱水酶基因的定点突变及其性能研究

Site-directed Mutagenesis of Glycerol Dehydratase from K.pneumoniae XJPD-Li

针对菌株K.pneumoniae XJPD-Li高效转化生产1,3-丙二醇的特性,将其甘油脱水酶与已知报道的甘油脱水酶(U60992)的序列比对分析,根据差异位点设计了Nβ47I的定点突变,利用反向PCR定点突变技术构建了甘油脱水酶(dhaBCE)的突变体质粒M1821,并在E.coli BL21(DE3)中高效表达。SDS-PAGE结果显示,诱导表达后在相对分子质量66、24、16KD出现三条特征条带,与预期结果一致。突变体M1821甘油脱水酶的比酶活为38.7U/mg,催化反应最适pH为8.0,最适温度为40℃,与未突变重组甘油脱水酶比较,最适温度降低了约5℃。

Through the alignment of gene and amino acid sequence, glycerol dehydratase （ dhaBCE ） from K. pneumoniae XJPD-Li shows a significant identity with GDHt from K. pneumoniae（U60992）. According to the differences of amino acid residue, Nβ471 mutant was designed and constructed by one step inverse PCR. The recombinant mutant plasmid M1821 was expressed in E. coli BL21 （DE3）. SDS-PAGE shows three bands with molecular weight of 66,24 and 16KD. The special activity of glycerol dehydratase from the mutation M1821 is 38.7 U/rag, and the optimal reaction temperature（Tin） and pH are 40℃ and 8.0, respectively. There are no obvious differences between mutation M1821 and recombinant GDHt in enzymes activity and optimal pH. The Tm of mutant （ M1821 ） is 5℃ lower than that of GHDt from K. pneumoniae XJPD-Li.