拟青霉内切β-1,3(4)-葡聚糖酶基因克隆、表达及重组酶性质

Cloning, heterologous gene expression and biochemical characterization of the endo-β-1,3(4)-glucanase from Paecilomyces sp. FLH30

摘要:

采用改造的酿酒酵母表达载体pYES2-GSL构建了拟青霉Paecilomyces sp. FLH30表达cDNA文库, 刚果红平板法筛到一内切β-1,3(4)-葡聚糖酶基因(PsBg16A), 全长cDNA为1 217 bp, 完整开放阅读框(ORF) 951 bp, 编码316个氨基酸, 属于糖苷水解酶16家族。去信号肽的PsBg16A克隆入表达载体pET28a(+)并在E. coli BL21成功表达, 经16 °C 20%乳糖诱导24 h, 酶活达482 U/mL。重组PsBg16A可水解大麦葡聚糖、地衣多糖和昆布多糖, 显示PsBg16A为典型的β-1,3(4)-葡聚糖酶(EC 3.2.1.6), 重组酶最适pH和温度分别为7.0和65 °C, 对大麦葡聚糖、地衣多糖和昆布多糖的Km 分别为3.12、4.86和10.32 g/L。

Abstract:

Saccharomyces cerevisiae cDNA express library of Paecilomyces sp. FLH30 was constructed by re-construction of the vector pYES2-GSL, and full-length cDNA encoding an endo-β-1,3(4)-glucanase gene (PsBg16A) was screened using Congo red-staining method. The full-length cDNA of PsBg16A is 1 217 bp and has an open reading frame of 951 bp. The PsBg16A encodes a 316-residue precursor protein with a putative signal peptide, and the deduced amino acid sequence of revealed that this enzyme belongs to glycoside hydrolase family 16. PsBg16A without signal peptide was cloned into a vector pET28a(+) and was expressed successfully in E. coli BL21(DE3), and the enzyme activity reached 482 U/mL induced by lactose at 16 °C. The purified recombinant PsBgl6A with optimum pH at 7.0 and optimum temperature at 65 °C, can randomly hydrolyze barely β-glucan, lichenin and laminarin. The results suggested that the enzyme is a typical endo-β-1,3(4)-glucanase (EC 3.2.1.6) with broad substrate specificity for β-glucans, and Km for β-glucan, lichenin and laminarin were 2.92, 4.35 and 9.88 g/L, respectively.