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Characterization of a β-D-mannosidase from a marine gastropod, Aplysia kurodai

Zahura, Umme Afsari, Rahman, Mohammad Matiur, Inoue, Akira, Ojima, Takao
Comparative biochemistry and physiology 2012 v.162 no.1-3 pp. 24-33
Codium, amino acid sequences, ammonium sulfate, beta-mannosidase, complementary DNA, fractionation, hepatopancreas, mannans, mannose, molecular weight, pH, polymerase chain reaction, polymers, temperature
A β-D-mannosidase (EC with a molecular mass of approximately 100kDa was purified from the digestive fluid of a marine gastropod Aplysia kurodai by ammonium sulfate fractionation followed by column chromatographies on TOYOPEARL Butyl-650M, TOYOPEARL DEAE-650M, 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 0.10mM and 3.75μmol/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 2985bp and encoded an amino-acid sequence of 931 residues with the calculated molecular mass of 101,970Da. 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.