Document Detail


Modulation of the regioselectivity of a Bacillus alpha-galactosidase by directed evolution.
MedLine Citation:
PMID:  11602805     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Alpha-galactosidase AgaB of Bacillus stearothermophilus was subjected to directed evolution in an effort to modify its regioselectivity. The wild-type enzyme displays a major 1,6 and minor 1,3 regioselectivity. We used random mutagenesis and staggered extension process (StEP) to obtain mutant enzymes displaying modified regioselectivity. We developed a screening procedure allowing first the elimination of AgaB mutants bearing the 1,6 regioselectivity and secondly the selection of those retaining a 1,3 regioselectivity. Our results show that, among the evolved enzymes that have lost most of their activity towards the 1,6 linkage both in hydrolysis and in synthesis, one (E901) has retained its 1,3 activity. However the transglycosylation level reached by this mutant is quite low versus that of the native enzyme. This work constitutes the first example of modification of glycosylhydrolase regioselectivity by directed evolution.
Authors:
M Dion; A Nisole; P Spangenberg; C André; A Glottin-Fleury; R Mattes; C Tellier; C Rabiller
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Glycoconjugate journal     Volume:  18     ISSN:  0282-0080     ISO Abbreviation:  Glycoconj. J.     Publication Date:  2001 Mar 
Date Detail:
Created Date:  2001-10-16     Completed Date:  2002-03-04     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  8603310     Medline TA:  Glycoconj J     Country:  United States    
Other Details:
Languages:  eng     Pagination:  215-23     Citation Subset:  IM    
Affiliation:
Unité de Recherches sur la Biocatalyse, FRE-CNRS 2230, Faculté des Sciences et des Techniques, 2 rue de la Houssinière, BP 92208 F-44322 Nantes Cedex 03 France. Dion@chimbio.univ-nantes.fr
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MeSH Terms
Descriptor/Qualifier:
Carbohydrate Conformation
Directed Molecular Evolution*
Disaccharides / metabolism
Geobacillus stearothermophilus / enzymology*
Glycosylation
Hexosaminidases / chemistry,  genetics,  metabolism*
Kinetics
Melibiose / metabolism
Molecular Structure
Mutagenesis
alpha-N-Acetylgalactosaminidase
Chemical
Reg. No./Substance:
0/Disaccharides; 585-99-9/Melibiose; EC 3.2.1.-/Hexosaminidases; EC 3.2.1.49/alpha-N-Acetylgalactosaminidase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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