An endo-β-1,4-mannanase, AkMan, from the common sea hare Aplysia kurodai

A mannan-degrading enzyme was isolated from the digestive fluid of the common sea hare Aplysia kurodai by ammonium sulfate fractionation followed by conventional column chromatography. The purified enzyme, named AkMan in the present paper, showed a single band with an approximate molecular mass of 40,000 Da on SDS-PAGE and preferably degraded a linear β-1,4-mannan from green algae Codium fragile producing tri- and disaccharides. The optimal temperature of AkMan was 55 °C at pH 7.0 and temperature that caused 50% inactivation of AkMan during a 20-min incubation was 52 °C. AkMan retained high activity at pH 4.0–7.5 and was not inactivated in such acidic pH range by the incubation at 40 °C for 20 min. AkMan could degrade glucomannan from konjak root and galactomannan (tara gum and guar gum) as well as the linear β-1,4-mannan, while not carboxymethyl cellulose, agarose, dextran and xylan. These results indicate that AkMan is a typical endo-β-1,4-mannanase (EC 3.2.1.78) splitting internal β-1,4-mannosyl linkages of mannan. The N-terminal and internal amino-acid sequences of AkMan shared ∼ 55% amino-acid identity to the corresponding sequences of an abalone β-1,4-mannanase, HdMan, which belongs to glycosyl hydrolase family 5 (GHF5). Thus, AkMan was also regarded as a member of GHF5 β-1,4-mannanases.