GH-57 familyに属する新規Branching Enzymeの同定と解析

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  • A Novel Branching Enzyme of the GH-57 Family

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Branching enzyme (BE) catalyzes formation of the branch points in glycogen and amylopectin by cleavage of the α-1,4-linkage and its subsequent transfer to the α-1,6-position. A novel BE encoded by an uncharacterized ORF (TK1436) was identified in the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. TK1436 encodes a conserved protein showing similarity to members of glycoside hydrolase family 57 (GH-57 family). TK1436 orthologs are distributed in archaea of Thermococcales, cyanobacteria, some actinobacteria and a few other bacterial species. When recombinant TK1436 protein was incubated with amylose used as the substrate, a product peak was detected by high-performance anion exchange chromatography, eluting slower than the substrate. Isoamylase treatment of the reaction mixture significantly increased the level of short-chain α-glucans, indicating that the reaction product contained many α-1,6-branching points. TK1436 protein showed an optimal pH of 7.0, an optimal temperature of 70°C, and thermostability up to 90°C as determined by the iodine-staining assay. These properties were the same when a protein devoid of the C-terminal HhH motifs (TK1436ΔH protein) was used. The average molecular weight of branched glucan after reaction with TK1436ΔH protein was over 100 times larger than that of the starting substrate. These results indicate that TK1436 encodes a structurally novel BE belonging to the GH-57 family.

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