Identification by saturation mutagenesis of a single residue involved in the α-galactosidase AgaB regioselectivity

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• 作者：Michel Dion ; George Osanjo ; Corinne Andr¨¦ ; Petra Spangenberg ; Claude Rabiller and Charles Tellier
• 关键词：directed evolution ; regioselectivity ; transglycosylation ; saturation mutagenesis
• 刊名：Glycoconjugate Journal
• 出版年：2001
• 出版时间：2001
• 年：2001
• 卷：18
• 期：6
• 页码：457-464
• 全文大小：230 KB

文摘

The -galactosidase AgaB of Bacillus stearothermophilus displays a major 1,6 and a minor 1,3 regioselectivity. The wild-type enzyme was subjected to directed evolution (random mutagenesis and in vitro recombination) using a double screening strategy based on the elimination of the 1,6 regioselectivity and the analysis by TLC of the transglycosylation products. One of the AgaB mutants (E500) exhibited a new 1,2 regioselectivity and a rather high level of transglycosylation. The corresponding gene contains 10 mutations compared to the agaB gene and we demonstrated by saturation mutagenesis that the G442R substitution strongly contributes to the emergence of this new regioselectivity. Moreover, other single point mutations at this position led to new mutants displaying other kinds of regioselectivity demonstrating the importance of this position in the subtle kinetic control of transglycosylation.