α-Cyclodextrin glycosyltransferase (α-CGTase) can convert starch into α-cyclodextrin with various proportions of β-cyclodextrin and/or γ-cyclodextrin in the products. To improve the α-cyclodextrin-forming specificity, directed evolution on the wild-type α-CGTase was performed by constructing mutant library with error-prone PCR method. The positive mutant strains were selected in combination of starch plate screening with HPLC detection of the products. An α-CGTase from the mutant strain (assigned No. 95) was found to be able to increase the α:β ratio in product mixture from 3.4 to 7.8 in comparison with the wild-type α-CGTase. Sequence alignment indicated that two mutations occurred in the No. 95 mutant α-CGTase, which were Y167H and A536V. Reverse mutation revealed that Y167H was responsible for this change. A series of 167 site-substituted mutants could improve the α:β ratio to different extents as indicated by saturated mutagenesis, with Y167H as the best substitution. In conclusion, Y167 was confirmed to be one of the main subsites in the -6 domain of α-CGTase that is responsible for the α:β ratio in the product mixture. Y167H is most preferable among all types of mutant enzymes tested at this site. The reconstructed Y167H (i.e., No. 95) α-CGTase showed better potential for α-cyclodextrin production on industrial scale.
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