Purification, Characterization, and Gene Cloning of a Cold-Adapted Endo-1,4-β-glucanase from Bellamya chinensis laeta
2016
Ueda, Mitsuhiro | Maruyama, Tomonori | Kawasaki, Keiko | Nakazawa, Masami | Sakaguchi, Minoru
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.
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