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

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.