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Updated: May 7, 2026

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins
Published on: October 4, 2017
Mutation of tyrosine167histidine at remote substrate binding subsite -6 in α-cyclodextrin glycosyltransferase
Binghong Song1, Yang Yue, Ting Xie
1State Key Laboratories of Transducer Technology, National Engineering Lab for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China, 2005sbh@163.com.
Abstract:
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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