An endo-(1→3)-β- d-glucanase from the scallop Chlamys albidus: catalytic properties, cDNA cloning and secondary-structure characterization

Abstract

An endo-(1→3)-β- d-glucanase (L 0) with molecular mass of 37 kDa was purified to homogeneity from the crystalline style of the scallop Chlamys albidus. The endo-(1→3)-β- d-glucanase was extremely thermolabile with a half-life of 10 min at 37 °C. L 0 hydrolyzed laminaran with K m ∼ 0.75 mg/mL, and catalyzed effectively transglycosylation reactions with laminaran as donor and p-nitrophenyl β d-glucoside as acceptor ( K m ∼ 2 mg/mL for laminaran) and laminaran as donor and as acceptor ( K m ∼ 5 mg/mL) yielding p-nitrophenyl β d-glucooligosaccharides ( n = 2–6) and high-molecular branching (1→3),(1→6)-β- d-glucans, respectively. Efficiency of hydrolysis and transglycosylation processes depended on the substrate structure and decreased appreciably with the increase of the percentage of β-(1→6)-glycosidic bonds, and laminaran with 10% of β-(1→6)-glycosidic bonds was the optimal substrate for both reactions. The CD spectrum of L 0 was characteristic for a protein with prevailing β secondary-structural elements. Binding L 0 with d-glucose as the best acceptor for transglycosylation was investigated by the methods of intrinsic tryptophan fluorescence and CD. Glucose in concentration sufficient to saturate the enzyme binding sites resulted in a red shift in the maximum of fluorescence emission of 1–1.5 nm and quenching the Trp fluorescence up to 50%. An apparent association constant of L 0 with glucose ( K a = 7.4 × 10 5 ± 1.1 × 10 5 M −1) and stoichiometry ( n = 13.3 ± 0.7) was calculated. The cDNA encoding L 0 was sequenced, and the enzyme was classified in glycoside hydrolases family 16 on the basis of the amino acid sequence similarity.