An endo-(1-->3)-beta-D-glucanase from the scallop Chlamys albidus: catalytic properties, cDNA cloning and secondary-structure characterization.
An endo-(1-->3)-beta-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)-beta-d-glucanase was extremely thermolabile with a half-life of 10 min at 37 degrees C. L(0) hydrolyzed laminaran with K(m) approximately 0.75 mg/mL, and catalyzed effectively transglycosylation reactions with laminaran as donor and p-nitrophenyl betad-glucoside as acceptor (K(m) approximately 2mg/mL for laminaran) and laminaran as donor and as acceptor (K(m) approximately 5mg/mL) yielding p-nitrophenyl betad-glucooligosaccharides (n=2-6) and high-molecular branching (1-->3),(1-->6)-beta-d-glucans, respectively. Efficiency of hydrolysis and transglycosylation processes depended on the substrate structure and decreased appreciably with the increase of the percentage of beta-(1-->6)-glycosidic bonds, and laminaran with 10% of beta-(1-->6)-glycosidic bonds was the optimal substrate for both reactions. The CD spectrum of L(0) was characteristic for a protein with prevailing beta 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 x 10(5)+/-1.1 x 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.
Kovalchuk
Svetlana N
SN
Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, Vladivostok, Russia. kovalchuk@piboc.dvo.ru
Bakunina
Irina Yu
IY
Burtseva
Yulia V
YV
Emelyanenko
Viktor I
VI
Kim
Natalia Yu
NY
Guzev
Konstantin V
KV
Kozhemyako
Valeri B
VB
Rasskazov
Valeri A
VA
Zvyagintseva
Tatyana N
TN
D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D044644:Endo-1,3(4)-beta-Glucanase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D049897:Pectinidae / Q000201:enzymology*
D018076:DNA, Complementary; D044644:Endo-1,3(4)-beta-Glucanase