The Roles of Tryptophan and Histidine Residues in the Catalytic Activities $\beta$-Cyclodextrin Glucanotransferase from Bacillus firmus var. alkalophilus

In order to investigate the critical amino acid residues involved in the catalytic activities of $\beta$-cyclodextrin glucanotransferase ($\beta$-CGTase) excreted by Bacillus firmus var. alkalophilus, the amino acid residues in $\beta$-CGTase were modified by various site-specific amino acid modifying reagents. The cyclizing and amylolytic activities of $\beta$-CGTase were all seriously reduced after treatment with Woodward's reagent K (WRK) modifying aspartic/glutamic acid, N-bromosuccinimde (NBS) modifying tryptophan, and diethylpyrocarbonate (DEPC) modifying histidine residues. The roles of tryptophan and histidine residues in $\beta$-CGTase were further investigated by measuring the protection effect of various substrates during chemical modification, comparing protein mobility in native and affinity polyacrylamide gel electrophoresis containing soluble starch, and comparing the $K_m$ and $V_{max}$ values of native and modified enzymes. Tryptophan residues were identified as affecting substrate-binding ability rather than influencing catalytic activities. On the other hand, histidine residues influenced catalytic ability rather than substrate-binding ability, plus histidine modification had an effect on shifting the optimum pH and pH stability.