The industrial use of a-cyclodextrins (alpha-CDs) has increased because their solubility is higher than those of beta-CDs. However, improving the product specificity of alpha-cyclodextrin glucanotransferases (CGTases) remains unresolved. In this study, three mutants (Y167-deletion, Y167HH, and Y167HHH) were constructed at subsite 6 of alpha-CGTase to investigate the contribution of amino acid residue 167 to the cyclization ability of alpha-CD by comparing it with Tyr167His mutant a-CGTase (previously constructed based on the wild-type gene of Bacillus sp. 602-1). As expected, the alpha:beta ratio improved with increasing number of histidine along with residue 167. The Y167HHH mutant had the highest alpha:beta ratio of 13.2 and almost produced single type a-CDs. The Y167HHH mutant enzyme was subsequently purified to homogeneity. The enzymatic properties and the optimal condition of Y167HHH mutant in converting raw starch were also investigated. This study discusses product specificity improvement by inserting specific amino acid residues in the active groove. The results indicate that the histidine-rich mutant alpha-CGTase possessed better potential in producing alpha-CDs in an industrial scale. (c) 2013 Published by Elsevier Ltd.