文摘
A ¦Â-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 m>Aplysia kurodaim> 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 ¡ãC and was stable at the acidic pH range from 2.0 to 6.7 and the temperature below 38 ¡ãC. The m>Km>m and m>Vm>max values for AkMnsd determined at pH 6.0 and 30 ¡ãC with m>pm>-nitrophenyl ¦Â-d-mannopyranoside were 0.10 mM and 3.75 ¦Ìmol/min/mg, respectively. AkMnsd degraded various polymer mannans as well as mannooligosaccharides liberating mannose as a major degradation product. Linear mannan from green alga m>Codium fragilem> was completely depolymerized by AkMnsd in the presence of AkMan, an endolytic ¦Â-mannanase, which we previously isolated from the same animal (Zahura et al., m>Comp. Biochem. Physiolm>. B 157, 137-148 (2010)). A cDNA encoding AkMnsd was amplified from the m>Aplysiam> 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) ¦Â-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.
