Enhancement of α-cyclodextrin product specificity by enriching histidines of α-cyclodextrin glucanotransferase at remote subsite −6

• We improve product specificity by inserting histidine residues in the active groove.
• The histidine-rich mutant obtains the high α:β ratio of 13.2.
• The mutant has good ability to convert raw starch.
• The mutant can have better potential in industrial application as biocatalysts.

The industrial use of α-cyclodextrins (α-CDs) has increased because their solubility is higher than those of β-CDs. However, improving the product specificity of α-cyclodextrin glucanotransferases (CGTases) remains unresolved. In this study, three mutants (Y167-deletion, Y167HH, and Y167HHH) were constructed at subsite −6 of α-CGTase to investigate the contribution of amino acid residue 167 to the cyclization ability of α-CD by comparing it with Tyr167His mutant α-CGTase (previously constructed based on the wild-type gene of Bacillus sp. 602-1). As expected, the α:β ratio improved with increasing number of histidine along with residue 167. The Y167HHH mutant had the highest α:β ratio of 13.2 and almost produced single type α-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 α-CGTase possessed better potential in producing α-CDs in an industrial scale.