A novel oligoalginate lyase from abalone, Haliotis discus hannai, that releases disaccharide from alginate polymer in an exolytic manner

Abstract: We previously reported the isolation and cDNA cloning of an endolytic alginate lyase, HdAly, from abalone Haliotis discus hannai [Carbohydr. Res. 2003, 338, 2841–2852]. Although HdAly preferentially degraded mannuronate-rich substrates, it was incapable of degrading unsaturated oligomannuronates smaller than tetrasaccharide. In the present study, we used conventional chromatographic techniques to isolate a novel unsaturated-trisaccharide-degrading enzyme, named HdAlex, from the digestive fluid of the abalone. The HdAlex showed a molecular weight of 32,000 on SDS-PAGE and could degrade not only unsaturated trisaccharide but also alginate and mannuronate-rich polymers at an optimal pH and temperature of 7.1 and 42°C, respectively. Upon digestion of alginate polymer, HdAlex decreased the viscosity of the alginate at a slower rate than did HdAly, producing only unsaturated disaccharide without any intermediate oligosaccharides. These results indicate that HdAlex degrades the alginate polymer in an exolytic manner. Because HdAlex split saturated trisaccharide producing unsaturated disaccharide, we considered that this enzyme cleaved the alginate at the second glycoside linkage from the reducing terminus. The primary structure of HdAlex was deduced with cDNAs amplified from an abalone hepatopancreas cDNA library by the polymerase chain reaction. The translational region of 822 bp in the total 887-bp sequence of HdAlex cDNA encoded an amino-acid sequence of 273 residues. The N-terminal sequence of 16 residues, excluding the initiation methionine, was regarded as the signal peptide of this enzyme. The amino-acid sequence of the remaining 256 residues shared 62–67% identities with those of the polysaccharide lyase family-14 (PL14) enzymes such as HdAly and turban-shell alginate lyase SP2. To our knowledge, HdAlex is the first exolytic oligoalginate lyase belonging to PL14. [Copyright &y& Elsevier]