title: A Solanum torvum GH3 β-glucosidase expressed in Pichia pastoris catalyzes the hydrolysis of furostanol glycoside.
abstract: Plant β-glucosidases are usually members of the glucosyl hydrolase 1 (GH1) or 3 (GH3) families. Previously, a β-glucosidase (torvosidase) was purified from Solanum torvum leaves that specifically catalyzed hydrolysis of two furostanol 26-O-β-glucosides, torvosides A and H. Furostanol glycoside 26-O-β-glucosides have been reported as natural substrates of some plant GH1 enzymes. However, torvosidase was classified as a GH3 β-glucosidase, but could not hydrolyze β-oligoglucosides, the natural substrates of GH3 enzymes. Here, the full-length cDNA encoding S. torvum β-glucosidase (SBgl3) was isolated by the rapid amplification of cDNA ends method. The 1887bp ORF encoded 629 amino acids and showed high homology to other plant GH3 β-glucosidases. Internal peptide sequences of purified native Sbgl3 determined by LC-MS/MS matched the deduced amino acid sequence of the Sbgl3 cDNA, suggesting that it encoded the natural enzyme. Recombinant SBgl3 with a polyhistidine tag (SBgl3His) was successfully expressed in Pichia pastoris. The purified SBgl3His showed the same substrate specificity as natural SBgl3, hydrolyzing torvoside A with much higher catalytic efficiency than other substrates. It also had similar biochemical properties and kinetic parameters to the natural enzyme, with slight differences, possibly attributable to post-translational glycosylation. Quantitative real-time PCR (qRT-PCR) showed that SBgl3 was highly expressed in leaves and germinated seeds, suggesting a role in leaf and seedling development. To our knowledge, a recombinant GH3 β-glucosidase that hydrolyzes furostanol 26-O-β-glucosides, has not been previously reported in contrast to substrates of GH1 enzymes.
authors.affiliation: Department of Tropical Nutrition and Food Science, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand.
authors.forename: Rungarun
authors.initials: R
authors.lastname: Suthangkornkul
authors.affiliation: Department of Tropical Nutrition and Food Science, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand.
authors.forename: Pornpisut
authors.initials: P
authors.lastname: Sriworanun
authors.affiliation: Division of Fundamental AgriScience Research, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakai
authors.affiliation: Division of Fundamental AgriScience Research, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Okuyama
authors.affiliation: Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
authors.forename: Jisnuson
authors.initials: J
authors.lastname: Svasti
authors.affiliation: Division of Fundamental AgriScience Research, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
authors.forename: Atsuo
authors.initials: A
authors.lastname: Kimura
authors.affiliation: National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathumthani 12120, Thailand; Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Mahidol University, Bangkok 10400, Thailand.
authors.forename: Saengchan
authors.initials: S
authors.lastname: Senapin
authors.affiliation: Department of Tropical Nutrition and Food Science, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand. Electronic address: dumrongkiet.art@mahidol.ac.th.
authors.forename: Dumrongkiet
authors.initials: D
authors.lastname: Arthan
mesh_terms: D006027:Glycosides / Q000378:metabolism*; D006868:Hydrolysis; D010843:Pichia / Q000235:genetics*; D032322:Solanum / Q000378:metabolism*; D013261:Sterols / Q000378:metabolism*; D001617:beta-Glucosidase / Q000378:metabolism*
chemical_list: D006027:Glycosides; D013261:Sterols; C574436:furostanol glycoside; D001617:beta-Glucosidase
keywords: Family 3; Pichia pastoris; Solanaceae; Solanum torvum; Torvoside A; β-Glucosidase