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Structural Dissection of the Active Site of Thermotoga maritima β-Galactosidase Identifies Key Residues for Transglycosylating Activity

AutorTalens Perales, David ; Polaina Molina, Julio ; Marín Navarro, Julia
Palabras claveEnzyme engineering
Galacto-oligosaccharides
GH2 glycoside hydrolase
Prebiotics
Fecha de publicación21-mar-2016
EditorAmerican Chemical Society
CitaciónJournal of Agricultural and Food Chemistry 64 (14): 2917-2924 (2016)
ResumenGlycoside hydrolases, specifically β-galactosidases, can be used to synthesize galacto-oligosaccharides (GOS) due to the transglycosylating (secondary) activity of these enzymes. Site-directed mutagenesis of a thermoresistant β-galactosidase from Thermotoga maritima has been carried out to study the structural basis of transgalactosylation and to obtain enzymatic variants with better performance for GOS biosynthesis. Rational design of mutations was based on homologous sequence analysis and structural modeling. Analysis of mutant enzymes indicated that residue W959, or an alternative aromatic residue at this position, is critical for the synthesis of β-3′-galactosyl-lactose, the major GOS obtained with the wild-type enzyme. Mutants W959A and W959C, but not W959F, showed an 80% reduced synthesis of this GOS. Other substitutions, N574S, N574A, and F571L, increased the synthesis of β-3′-galactosyl-lactose about 40%. Double mutants F571L/N574S and F571L/N574A showed an increase of about 2-fold.
DescripciónManuscript of the article published in print 13 April 2016. The Supporting Information is available free of charge on the ACS Publications website at DOI:10.1021/acs.jafc.6b00222 .
Versión del editorhttp://dx.doi.org/10.1021/acs.jafc.6b00222
URIhttp://hdl.handle.net/10261/132379
DOI10.1021/acs.jafc.6b00222
ISSN0021-8561
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