title: Identification and enzymatic characterization of an endo-1,3-β-glucanase from Euglena gracilis.
abstract: Euglena produces paramylon as a storage polysaccharide, and is thought to require β-1,3-glucan degrading enzymes to release and utilize the accumulated carbohydrate. To investigate β-1,3-glucan degradation in Euglena, endo-1,3-β-glucanases were partially purified from Euglena gracilis by hydrophobic, gel filtration and anion-exchange chromatography. Tryptic digests and mass-spectrometric analysis identified three proteins in the purified fraction as a member of glycoside hydrolase family (GH) 17 and two members of GH81. These genes were cloned from an Euglena cDNA pool by PCR. EgCel17A fused with a histidine-tag at the carboxy terminus was heterologously produced by Aspergillus oryzae and purified by immobilized metal affinity chromatography. Purified EgCel17A had a molecular weight of about 40kDa by SDS-PAGE, which was identical to that deduced from its amino acid sequence. The enzyme showed hydrolytic activity towards β-1,3-glucans such as laminarin and paramylon. Maximum activity of laminarin degradation by EgCel17A was attained at pH 4.0-5.5 and 60°C after 1h incubation or 50°C after 20h incubation. The enzyme had a Km of 0.21mg/ml and a Vmax of 40.5units/mg protein for laminarin degradation at pH 5.0 and 50°C. Furthermore, EgCel17A catalyzed a transglycosylation reaction by which reaction products with a higher molecular weight than the supplied substrates were initially generated; however, ultimately the substrates were degraded into glucose, laminaribiose and laminaritriose. EgCel17A effectively produced soluble β-1,3-glucans from alkaline-treated Euglena freeze-dried powder containing paramylon. Thus, EgCel17 is the first functional endo-1,3-β-glucanase to be identified from E. gracilis.
authors.affiliation: Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan. Electronic address: takumitakeda27@gmail.com.
authors.forename: Takumi
authors.initials: T
authors.lastname: Takeda
authors.affiliation: Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Nakano
authors.affiliation: Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan.
authors.forename: Machiko
authors.initials: M
authors.lastname: Takahashi
authors.affiliation: Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan.
authors.forename: Naotake
authors.initials: N
authors.lastname: Konno
authors.affiliation: Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Sakamoto
authors.affiliation: euglena Co., Ltd., 2-6-1, Koraku, Bunkyo-ku, Tokyo 112-0004, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Arashida
authors.affiliation: euglena Co., Ltd., 2-6-1, Koraku, Bunkyo-ku, Tokyo 112-0004, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Marukawa
authors.affiliation: euglena Co., Ltd., 2-6-1, Koraku, Bunkyo-ku, Tokyo 112-0004, Japan.
authors.forename: Eriko
authors.initials: E
authors.lastname: Yoshida
authors.affiliation: Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ishikawa
authors.affiliation: euglena Co., Ltd., 2-6-1, Koraku, Bunkyo-ku, Tokyo 112-0004, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Suzuki
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D005056:Euglena gracilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000302:isolation & purification / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D008970:Molecular Weight; D047071:beta-Glucans / Q000032:analysis
chemical_list: D047071:beta-Glucans; C033363:beta-1,3-glucan; D006026:Glycoside Hydrolases; D004693:Glucan Endo-1,3-beta-D-Glucosidase
keywords: Endo-1,3-β-glucanase; Euglena; Euglenaceae; Glycoside hydrolase family 17; Hydrolysis; Paramylon; Transglycosylation