title: Characterization of a β-D-mannosidase from a marine gastropod, Aplysia kurodai.
abstract: A β-D-mannosidase (EC 3.2.1.25) with a molecular mass of approximately 100 kDa was purified from the digestive fluid of a marine gastropod Aplysia kurodai by ammonium sulfate fractionation followed by column chromatographies on TOYOPEARL Butyl-650 M, TOYOPEARL DEAE-650 M, and Superdex 200 10/300 GL. This enzyme, named AkMnsd in the present study, showed optimal activities at pH 4.5 and 40 °C and was stable at the acidic pH range from 2.0 to 6.7 and the temperature below 38 °C. The Km and Vmax values for AkMnsd determined at pH 6.0 and 30 °C with p-nitrophenyl β-d-mannopyranoside were 0.10 mM and 3.75 μmol/min/mg, respectively. AkMnsd degraded various polymer mannans as well as mannooligosaccharides liberating mannose as a major degradation product. Linear mannan from green alga Codium fragile was completely depolymerized by AkMnsd in the presence of AkMan, an endolytic β-mannanase, which we previously isolated from the same animal (Zahura et al., Comp. Biochem. Physiol. B 157, 137-148 (2010)). A cDNA encoding AkMnsd was amplified from the Aplysia hepatopancreas cDNA by the PCR using degenerated primers designed on the basis of N-terminal and internal amino-acid sequences of AkMnsd. The cloned AkMnsd cDNA consisted of 2985 bp and encoded an amino-acid sequence of 931 residues with the calculated molecular mass of 101,970 Da. The deduced sequence of AkMnsd showed 20-43% amino-acid identity to those of glycoside-hydrolase-family 2 (GHF2) β-mannosidases. The catalytically important amino-acid residues determined in GHF2 enzymes were completely conserved in AkMnsd. Thus, AkMnsd is regarded as a new member of GHF2 mannosidase from marine gastropod.
authors.affiliation: Laboratory of Marine Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, Minato-cho 3-1-1, Hakodate 041-8611, Japan.
authors.forename: Umme Afsari
authors.initials: UA
authors.lastname: Zahura
authors.forename: Mohammad Matiur
authors.initials: MM
authors.lastname: Rahman
authors.forename: Akira
authors.initials: A
authors.lastname: Inoue
authors.forename: Takao
authors.initials: T
authors.lastname: Ojima
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001048:Aplysia / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013696:Temperature; D044902:beta-Mannosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D044902:beta-Mannosidase