Heterologous expression in Pichia pastoris and characterization of a β-glucosidase from the xylophagous cockroach Panesthia angustipennis spadica displaying high specific activity for cellobiose

• Published in: Enzyme and microbial technology Vol. 97; pp. 104 - 113
• Main Authors: Li, Yihai, Arakawa, Gaku, Tokuda, Gaku, Watanabe, Hirofumi, Arioka, Manabu
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2017
• Subjects: Animals; beta-Glucosidase - genetics; beta-Glucosidase - metabolism; Biofuels; Cellobiose - metabolism; Cockroaches - enzymology; Cockroaches - genetics; Enzyme Stability; Ethanol - metabolism; Genes, Insect; Glucose tolerance; Heterologous expression; Insect Proteins - genetics; Insect Proteins - metabolism; Kinetics; Pichia - enzymology; Pichia - genetics; Pichia pastoris; Protein Processing, Post-Translational; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Substrate Specificity; Xylophagous cockroach; β-Glucosidase; aa; pNPG; CBB; EG; Xylophagous cockroach; Endo H; CBH; TLC; Pichia pastoris; Glucose tolerance; β-Glucosidase; pNP; GPF; Heterologous expression
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2016.11.004

•A β-glucosidase, PaBG1b, from the xylophagous cockroach Panesthia angustipennis spadica was heterologously expressed and characterized.•PaBG1b demonstrated high specific activity and catalytic efficiency towards cellobiose with Vmax and kcat/Km values of 436.7±6.3U/mg and 109.8mM−1s−1, respectively.•Although the glucose tolerance of PaBG1b was moderate (Ki=200.3±1.1mM), it was not inhibited by cellobiose up to the highest concentration tested (100mM), which makes it a suitable candidate for commercial bioethanol production from cellulose. A β-glucosidase (BG), PaBG1b, from the xylophagous cockroach Panesthia angustipennis spadica was heterologously expressed in the methylotrophic yeast Pichia pastoris, purified, and biochemically characterized. Post-translational modification and N-terminal sequencing analysis demonstrated that the expression product was comprised of two polypeptides with different N-terminal sequences, presumably due to the presence of lysine-arginine (KR) sequence in the putative mature region. Substrate specificity analysis showed that PaBG1b hydrolyzed a broad range of substrates including cellohexaose, with the preference for aryl β-d-fucosyl linkage and laminaribiose. Although the glucose tolerance of PaBG1b was moderate (Ki=200.3±1.1mM), PaBG1b demonstrated high specific activity and catalytic efficiency towards cellobiose with Vmax and kcat/Km values of 436.7±6.3U/mg and 109.8mM−1s−1, respectively. In addition, PaBG1b was not inhibited by cellobiose up to the highest concentration tested (100mM). Collectively, our work demonstrates that PaBG1b is a potentially valuable BG for commercial bioethanol production from cellulose.