海洋弧菌QY105中褐藻胶寡糖酶OalA的基因克隆、重组表达与性质研究

Cloning,expression and characterization of a new oligoalginate lyase OalA from marine bacteriumVibrio sp.QY105

目的褐藻胶寡糖酶可将褐藻胶多糖和寡糖降解为单糖,但现有的褐藻胶寡糖酶数量极少且活力较差。本文旨在从海洋弧菌QY105中克隆寡糖酶OalA的编码基因,重组表达并研究其酶学性质。方法利用简并PCR和SiteFinding-PCR从海洋弧菌QY105中克隆得到褐藻胶寡糖酶OalA的编码基因,在大肠杆菌中进行重组表达,对重组酶进行分离纯化及酶学性质研究。结果褐藻胶寡糖酶OalA基因全长2082bp,编码1条含有693个氨基酸残基的多肽链,理论分子量为79.17kDa,理论等电点为4.98,属于多糖裂解酶第15家族(PL^(-1)5)。重组OalA比活力为9.2U·mg^(-1),最适温度30℃,在10℃的酶活力达到最高酶活的45%,最适pH7.6,在低于30℃和pH6~10范围内稳定,偏好降解polyM。结论OalA具有低温酶的特点,且对polyM具有偏好性,可用于褐藻胶单糖制备、PL^(-1)5家族结构与功能相互关系研究等领域。

Objective Oligoalginate lyases could release monosaccharides from alginate and alginate oligosaccharides.Few oligosaccharide lyases had been found and characterized.The purpose of this study was to clone,express and characterize a new oligoalginate lyase OalA from marine bacteriumVibrio sp.QY105.Methods Degenerate PCR and SiteFinding-PCR had been used to clone OalA encoding gene.Gene oalAhad been expressed in Escherichia coli.Results Gene oalAencoded a protein with 693 amino acid residues which belonged to polysaccharide lyase family 15.The molecular weight of OalA was predicted to be 79.17 kDa with a theoretical pI of 4.98.Recombinant OalA was most active at 30 ℃ and pH7.6with specific activity of 9.2U·mg-1,and showed 45%of the highest activity at 10℃.The enzyme was stable below 30 ℃ and in the range of pH6-10.The activity of OalA towards polyM was higher than the activity towards alginate and polyG.Conclusion OalA could degrade alginate at low temperature and preferred polyM which might be used in preparation of alginate monosaccharides and the study of structure-function relationship.