Purification, cDNA cloning and homology modeling of endo-1,3-β- d-glucanase from scallop Mizuhopecten yessoensis

Abstract

The retaining endo-1,3-β- d-glucanase (LV) with molecular mass of 36 kDa was purified to homogeneity from the crystalline styles of scallop Mizuhopecten yessoensis. The purified enzyme catalyzed hydrolysis of laminaran as endo-enzyme forming glucose, laminaribiose and higher oligosaccharides as products ( K m ∼ 600 μg/mL). The 1,3-β- d-glucanase effectively catalyzed transglycosylation reaction that is typical of endo-enzymes too. Optima of pH and temperature were at 4.5 and 45 °C, respectively. cDNA encoding the endo-1,3-β- d-glucanase was cloned by PCR-based methods. It contained an open reading frame that encoded 339-amino acids protein. The predicted endo-1,3-β- d-glucanase amino acid sequence included a characteristic domain of the glycosyl hydrolases family 16 and revealed closest homology with 1,3-β- d-glucanases from bivalve Pseudocardium sachalinensis, sea urchin Strongylocentrotus purpuratus and invertebrates lipopolysaccharide and β-1,3-glucan-binding proteins. The fold of the LV was more closely related to κ-carrageenase, agarase and 1,3;1,4-β- d-glucanase from glycosyl hydrolases family 16. Homology model of the endo-1,3-β- d-glucanase from M. yessoensis was obtained with MOE on the base of the crystal structure of κ-carrageenase from P. carrageonovora as template. Putative three-dimensional structures of the LV complexes with substrate laminarihexaose or glucanase inhibitor halistanol sulfate showed that the binding sites of the halistanol sulfate and laminarihexaose are located in the enzyme catalytic site and overlapped.