Molecular cloning, overexpression and purification of a β-glucanase from Streptomyces sp. J103 in Escherichia coli

Abstract

β-glucans are polysaccharides comprising β-1,3 and/or β-1,4 glycosidic bonds. There are four main types of endo-acting enzymes capable of depolymerizing β-1,3-1,4-glucan (Planas 2000 McCarthy et al. 2003): i) β-1,3(4)-glucanases (EC 3.2.1.6) which can randomly hydrolyze β-1,3 and β-1,4-glycosidic bonds in β-glucan ii) β-1,3-glucanases (EC 3.2.1.39, also known as laminarinases) which can randomly hydrolyze only β-1,3-glycosidic bonds in β-glucan iii) β -1,3-1-4-glucanases (EC 3.2.1.72) which can degrade only β-1,4-linkages adjacent to the reducing terminal side of β-1,3-linkage in mixed β-glucans and iv) β-1,4-glucanases (EC 3.2.1.4) which hydrolyze β-1,4-glycosidic linkages, prominently present in cellulose. In this study, we isolated Streptomyces sp. strain J103 from coastal area of Incheon, Republic of Korea. Genomic DNA was sequenced by next generation sequencing (NGS). Predicted β-glucanase encoding gene (designated as Sglu68) was identified by NCBI BLAST. The ORF of Sglu68 comprised of 1929 bp which encodes for a putative 634 amino acid protein with a predicted molecular mass of 68 kDa. The predicted signal peptide was observed in the N-terminal. Moreover, the predicted cellulase domain, Fibronectin type 3 domain (FN3) and carbohydrate binding domain III (CBM3) were observed in the C-terminal region. The mature Sglu86 was cloned and overexpressed as a maltose binding protein (MBP) tagged .2.1.6) which can randomly hydrolyze β-1,3 and β-1,4-glycosidic bonds in β-glucan ii) β-1,3-glucanases (EC 3.2.1.39, also known as laminarinases) which can randomly hydrolyze only β-1,3-glycosidic bonds in β-glucan iii) β -1,3-1-4-glucanases (EC 3.2.1.72) which can degrade only β-1,4-linkages adjacent to the reducing terminal side of β-1,3-linkage in mixed β-glucans and iv) β-1,4-glucanases (EC 3.2.1.4) which hydrolyze β-1,4-glycosidic linkages, prominently present in cellulose. In this study, we isolated Streptomyces sp. strain J103 from coastal area of Incheon, Republic of Korea. Genomic DNA was sequenced by next generation sequencing (NGS). Predicted β-glucanase encoding gene (designated as Sglu68) was identified by NCBI BLAST. The ORF of Sglu68 comprised of 1929 bp which encodes for a putative 634 amino acid protein with a predicted molecular mass of 68 kDa. The predicted signal peptide was observed in the N-terminal. Moreover, the predicted cellulase domain, Fibronectin type 3 domain (FN3) and carbohydrate binding domain III (CBM3) were observed in the C-terminal region. The mature Sglu86 was cloned and overexpressed as a maltose binding protein (MBP) tagged