Evolved β-Galactosidases from Geobacillus stearothermophilus with Improved Transgalactosylation Yield for Galacto-Oligosaccharide Production

A mutagenesis approach was applied to the β-galactosidase BgaB from Geobacillus stearothermophilus KVE39 in order to improve its enzymatic transglycosylation of lactose into oligosaccharides. A simple screening strategy, which was based on the reduction of the hydrolysis of a potential transglycosyl...

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Bibliographic Details
Published in:Applied and Environmental Microbiology 2009-10, Vol.75 (19), p.6312-6321
Main Authors: Placier, Gaël, Watzlawick, Hildegard, Rabiller, Claude, Mattes, Ralf
Format: Article
Language:English
Subjects:
Amino Acid Substitution - genetics
Bacillaceae - enzymology
beta-Galactosidase - genetics
beta-Galactosidase - isolation & purification
beta-Galactosidase - metabolism
Enzymology and Protein Engineering
Kinetics
Lactose - metabolism
Mutant Proteins - genetics
Mutant Proteins - isolation & purification
Mutant Proteins - metabolism
Mutation, Missense
Oligosaccharides - metabolism
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Summary:A mutagenesis approach was applied to the β-galactosidase BgaB from Geobacillus stearothermophilus KVE39 in order to improve its enzymatic transglycosylation of lactose into oligosaccharides. A simple screening strategy, which was based on the reduction of the hydrolysis of a potential transglycosylation product (lactosucrose), provided mutant enzymes possessing improved synthetic properties for the autocondensation product from nitrophenyl-galactoside and galacto-oligosaccharides (GOS) from lactose. The effects of the mutations on enzyme activity and kinetics were determined. An change of one arginine to lysine (R109K) increased the oligosaccharide yield compared to that for the wild-type BgaB. Subsequently, saturation mutagenesis at this position demonstrated that valine and tryptophan further increased the transglycosylation performance of BgaB. During the transglycosylation reaction with lactose of the evolved β-galactosidases, a major trisaccharide was formed. Its structure was characterized as β-D-galactopyranosyl-(1[rightward arrow]3)-β-D-galactopyranosyl-(1[rightward arrow]4)-D-glucopyranoside (3'-galactosyl-lactose). At the lactose concentration of 18% (wt/vol), this trisaccharide was obtained in yields of 11.5% (wt/wt) with GP21 (BgaB R109K), 21% with GP637.2 (BgaB R109V), and only 2% with the wild-type BgaB enzyme. GP643.3 (BgaB R109W) was shown to be the most efficient mutant, with a 3'-galactosyl-lactose production of 23%.
ISSN:0099-2240
1098-5336
DOI:10.1128/AEM.00714-09