Isolation and cloning of an endo-beta-1,4-mannanase from Pacific abalone Haliotis discus hannai.

An endo-beta-1,4-mannanase was isolated from digestive fluid of Pacific abalone, Haliotis discus hannai, by successive chromatographies on TOYPEARL CM-650M, hydroxyapatite, and TOYOPEARL HW50F. The abalone mannanase, named HdMan in the present paper, showed a molecular mass of approximately 39,000 Da on SDS-PAGE, and exhibited high hydrolyic activity on both galactomannan from locust bean gum and glucomannan from konjac at an optimal pH and temperature of 7.5 and 45 degrees C, respectively. HdMan could degrade either beta-1,4-mannan or beta-1,4-mannooligosaccharides to mannotriose and mannobiose similarly to beta-1,4-mannanases from Pomacea, Littorina, and Mytilus. In addition, HdMan could disperse the fronds of a red alga Porphyra yezoensis into cell masses consisting of 10-20 cells that are available for cell engineering of this alga. cDNAs encoding HdMan were amplified by polymerase chain reaction from an abalone-hepatopancreas cDNA library. From the nucleotide sequences of the cDNAs, the sequence of 1232 bp in total was determined and the amino-acid sequence of 377 residues was deduced from the translational region of 1134 bp locating at nucleotide positions 15-1148. The N-terminal region of 17 residues except for the initiation Met, was regarded as the signal peptide of HdMan because it was absent in the HdMan protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretory proteins. Accordingly, mature HdMan was considered to consist of 359 residues with the calculated molecular mass of 39,627.2 Da. HdMan is classified into glycoside hydrolase family 5 (GHF5) on the basis of sequence homology to GHF5 enzymes.