A B S T R A C TIn this recent study, Tyr93 of Bacillus sp. A2-5a (BA2-5a) cyclodextrin glucanotransferase (CGTase) was replaced with Phe residue by site-directed mutagenesis to study its role on the product specificity and kinetic properties. Molecular docking approach was also applied to acquire a detailed analysis of enzyme-substrate interaction. The Y93F was overproduced in Escherichia coli BL21 (DE3) and purified by Ni-NTA resin. The purified Y93F CGTase was 76.39 kDa based on 10% SDS-PAGE analysis and showed both β-cyclization and starch hydrolysis activities in a zymography assay. There was no major structural conformational change in the Y93F since, its optimum and stability of temperature and pH were the same as the wild type. The substitution did not alter the cooperativity property of the enzyme. Our work provided the new evidence that Y93F substitution caused no α-cyclodextrin (CD) formation in BA2-5a CGTase. In addition, the Y93F produced more β-CD and decreased its k cat and k cat /K m . In conclusion, Y93F substitution of BA2-5a CGTase alters the enzyme kinetic property and product specificity.