Cyclization Reaction Catalyzed by Bacillus cereus Branching Enzyme, and the Structure of Cyclic Glucan Produced by the Enzyme from Amylose

Abstract

Branching enzyme (BE, 1,4-α-D-glucan: 1,4-α-D-glucan 6-α-D-(1,4-α-D-glucano)-transferase, EC 2.4.1.18) catalyzes transglycosylation to form α-1,6-glucosidic linkages of amylopectin or glycogen in vivo (branching reaction). It has already been demonstrated that the thermostable BEs from Bacillus stearothermophilus and Aquifex aeolicus catalyze cyclization of amylose and amylopectin in vitro in addition to branching reaction. In this study, the action of a BE from mesophile Bacillus cereus on amylose was investigated in detail. The structural gene encoding BE was cloned from B. cereus Q, and expressed in Escherichia coli. BE was purified from a recombinant plasmid-harboring E. coli. The BE was most active at 30°C and pH 7.5. The activity was lost after incubation at 50°C for 30 min. BE action on amylose resulted in a degradation of the amylose without increasing reducing power. After the glucoamylase treatment of the BE products, cyclic molecules were isolated from the products, clearly indicating that the BE catalyzed cyclization reaction as the thermostable BEs. Furthermore, we tested the effect of molecular size of substrate on the action profile of BE. It was demonstrated that the BE converted any sizes of amyloses to a highly branched glucan with the same molecular size (weight-average molecular weight, 50,000).