大丽轮枝菌外切葡聚糖酶VdCBH1在毕赤酵母中的表达与酶活分析

Expression and enzyme activity analysis of exoglucanase VdCBH1 from Verticillium dahliae in Pichia pastoris

为了探究大丽轮枝菌外切葡聚糖酶VdCBH1(exoglucanase)的酶活与催化机理,本研究在生物信息学分析的基础上,以大丽轮枝菌V991菌株为材料克隆了VdCBH1基因,进一步构建了pPIC9-VdCBH1重组质粒并转入毕赤酵母中进行表达,测定了重组VdCBH1蛋白的外切葡聚糖酶活性;将预测的VdCBH1催化残基位点进行突变,随后通过SDS-PAGE分析了重组VdCBH1蛋白及其催化残基位点突变蛋白的表达情况,并检测了VdCBH1催化残基位点突变前后的酶活变化。结果表明,VdCBH1基因编码区全长1356 bp,编码451个氨基酸,蛋白理论分子量大小为48.574 kDa,理论等电点为4.29,N端前17个氨基酸为信号肽序列。保守结构域和进化树分析表明,VdCBH1蛋白含有GH7_CBH_EG保守结构域,该结构域是糖基水解酶7家族(Glycosyl hydrolase family 7,GH7)所特有的,与尖端赛多孢子菌(Scedosporium apiospermum)的外切葡聚糖酶蛋白亲缘关系最近。重组VdCBH1蛋白具有典型的外切葡聚糖酶活性,而Glu233催化残基单突变与Glu228、Asp230和Glu233催化残基三突变蛋白的酶活明显下降。这表明催化残基Glu233在外切葡聚糖酶活性中发挥更为重要的作用,Glu228、Asp230则可能在其中起协同作用。本研究为进一步揭示VdCBH1蛋白的催化机制奠定了理论基础。

On the basis of bioinformatic analysis,VdCBH1 is 1356 bp in length and predicted to encode an exoglucanase consisting of 451 amino acids with a molecular weight of 48.574 kDa,a signal peptide of the first 17 amino acids at the N-terminal,and a theoretical isoelectric point of 4.29 in Verticillium dahliae.The conserved domain and phylogenetic tree analysis showed that the VdCBH1 protein contained a GH7_CBH_EG conserved domain,which was unique to the glycosyl hydrolase 7 family(GH7)and closely related to the exoglucanase of Sedosporidium cuspidosporum.In order to explore the enzyme activity and catalytic mechanism of VdCBH1 in Verticillium dahliae,the VdCBH1 from V991 strain and its recombinants with site mutations in the predicted catalytic residue sites were cloned and constructed into the secretion expression vector pPIC9.The resultant constructs were transferred into Pichia pastoris,respectively.The expression of recombinant VdCBH1 and the mutant proteins were analyzed by SDS-PAGE,and the enzyme activity changes before and after VdCBH1 catalytic residue site mutation were detected.The recombinant VdCBH1 protein had typical exoglucanase activity,while the enzyme activity of Glu233 catalytic residue single mutant protein and Glu228,Asp230,Glu233 catalytic residue three mutant protein decreased significantly.This indicates that the catalytic residue Glu233 plays a more important role in exoglucanase activity,and Glu228 and Asp230 may play a synergistic role in it.This study laid a theoretical foundation for further revealing the catalytic mechanism of exoglucanase VdCBH1.