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

Autor:	M, Dion, G, Osanjo, C, André, P, Spangenberg, C, Rabiller, C, Tellier
Rok vydání:	2002
Předmět:	Glycosylation Molecular Sequence Data Disaccharides Substrate Specificity alpha-N-Acetylgalactosaminidase Geobacillus stearothermophilus Kinetics Structure-Activity Relationship Hexosaminidases Mutagenesis Cloning Molecular Directed Molecular Evolution Codon Nuclear Magnetic Resonance Biomolecular
Zdroj:	Glycoconjugate journal. 18(6)
ISSN:	0282-0080
Popis:	The alpha-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.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=pmid________::09db893afda7578bcac5afaf71b23070 https://pubmed.ncbi.nlm.nih.gov/12084981 Zobrazit plný text záznamu Full text from SpringerLink