title: Heterologous expression in Pichia pastoris and characterization of a β-glucosidase from the xylophagous cockroach Panesthia angustipennis spadica displaying high specific activity for cellobiose.
abstract: 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 Vmaxand kcat/Kmvalues 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.
authors.affiliation: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Yihai
authors.initials: Y
authors.lastname: Li
authors.affiliation: National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan; Tropical Biosphere Research Center, COMB, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan.
authors.forename: Gaku
authors.initials: G
authors.lastname: Arakawa
authors.affiliation: Tropical Biosphere Research Center, COMB, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan.
authors.forename: Gaku
authors.initials: G
authors.lastname: Tokuda
authors.affiliation: National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan; Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (reorganized from 2 in April, 2016), Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Watanabe
authors.affiliation: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Electronic address: arioka@mail.ecc.u-tokyo.ac.jp.
authors.forename: Manabu
authors.initials: M
authors.lastname: Arioka
mesh_terms: D000818:Animals; D056804:Biofuels; D002475:Cellobiose / Q000378:metabolism*; D003058:Cockroaches / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D000431:Ethanol / Q000378:metabolism; D017344:Genes, Insect; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D056804:Biofuels; D019476:Insect Proteins; D011994:Recombinant Proteins; D002475:Cellobiose; D000431:Ethanol; D001617:beta-Glucosidase
keywords: Glucose tolerance; Heterologous expression; Pichia pastoris; Xylophagous cockroach; β-Glucosidase