Comparative study of the cyclization reactions of three bacterial cyclomaltodextrin glucanotransferases

The actions of cyclomaltodextrin glucanotransferases (CGTase; EC 2.4.1.19) from alkalophilic Bacillus sp. strain A2-5a (A2-5a CGTase), Bacillus macera ns (Bmac CGTase), and Bacillus stearothermophilus (Bste CGTase) on amylose were investigated. All three enzymes produced large cyclic alpha -1,4-gluca ns (cycloamyloses) at the early stage of the reaction, but these were subse quently converted into smaller cycloamyloses. However, the rates of this co nversion differed among the three enzymes. The product specificity of each CGTase in the cyclization reaction was determined by measuring the amount o f each cycloamylose from CD6 to CD31 (CDn, a cycloamylose with a degree of polymerization of n). A2-5a CGTase produced 10 times more CD7, while Bmac C GTase produced 34 times more CD6 than other cycloamyloses. Bste CGTase prod uced 12 and 34 times more CD6 and CD7 than other cycloamyloses, respectivel y. The substrate specificities of the linearization reactions of CD6, CD7, CD8, and larger cycloamyloses (a mixture of CD22 to CD50) were investigated , and we found that CD7 and CD8 are extremely poor substrates for both hydr olytic and transglycosidic linearization (coupling) reactions while larger cycloamyloses are linearized at a much higher rate. By repeating these cycl ization and linearization reactions, the larger cycloamyloses initially pro duced are converted into smaller cycloamyloses and finally into mainly CD6, CD7, and CD8. These three enzymes also differ in their hydrolytic activiti es, which seem to accelerate the conversion of larger cycloamyloses into sm aller cycloamyloses.