title: Purification, Characterization, and Gene Cloning of a Cold-Adapted Endo-1,4-β-glucanase from Bellamya chinensis laeta.
abstract: An endo-1,4-β-glucanase from Bellamya chinensis laeta was purified to electrophoretically homogeneous state. The molecular weight of the purified enzyme was estimated 70,000 by SDS-PAGE. The enzyme was most active at pH 5.5 and 50 °C, and stable at around pH 10 and 50 °C. The enzyme exhibited the significant activity at 20 °C (30 % of the activity at optimal 50 °C). The enzyme was hydrolyzed cellohexaose into cellobiose, cellotriose, and cellotetraose as main products. Three cDNAs (BC-EG70a, BC-EG70b, and BC-EG70c) encoding the endo-1,4-β-glucanase were cloned by PCR-based method. Three endo-1,4-β-glucanases consisted of 1758 bp encoding 586 amino acids. The three genes were almost the same nucleotide sequences. The deduced proteins were consisted of a signal sequence, cellulose binding domain, linker, and catalytic domain. The amino acid sequence of BC-EG70a shares sequence identity degree with the endo-1,4-β-glucanases of Haliotis discus hannai (61 %), Ampullaria crossean (52 %), and Mizuhopecten yessoensis (51 %) which all belong to glycoside hydrolase family 9.
authors.affiliation: Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka, 599-8531, Japan. mueda@biochem.osakafu-u.ac.jp.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Ueda
authors.affiliation: Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka, 599-8531, Japan.
authors.forename: Tomonori
authors.initials: T
authors.lastname: Maruyama
authors.affiliation: Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka, 599-8531, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Kawasaki
authors.affiliation: Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka, 599-8531, Japan.
authors.forename: Masami
authors.initials: M
authors.lastname: Nakazawa
authors.affiliation: Laboratory of Cell Biology, Osaka University of Pharmaceutical Sciences, Osaka, 4-20-1, Nasahara, Takatsuki, Osaka, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Sakaguchi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D004795:Enzyme Stability; D049851:Gastropoda / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D013696:Temperature
chemical_list: D002480:Cellulase
keywords: Bellamya chinensis laeta; Cold-adapted; Endo-1,4-β-glucanase; Glycoside hydrolase family 9; Simultaneous saccharification and fermentation