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Applied and Environmental Microbiology, October 2009, p. 6312-6321, Vol. 75, No. 19
0099-2240/09/$08.00+0     doi:10.1128/AEM.00714-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Evolved β-Galactosidases from Geobacillus stearothermophilus with Improved Transgalactosylation Yield for Galacto-Oligosaccharide Production{triangledown} ,{dagger}

Gaël Placier,1,{ddagger} Hildegard Watzlawick,1* Claude Rabiller,2 and Ralf Mattes1

Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany,1 Biotechnologie, Biocatalyse, Biorégulation (UMR CNRS-University U3B 6204), PRES Nantes-Angers-Le Mans University, 2 rue de la Houssinière, BP 92208, F-44322-Nantes, France2

Received 26 March 2009/ Accepted 18 July 2009

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->3)-β-D-galactopyranosyl-(1->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%.

* Corresponding author. Mailing address: Institut für Industrielle Genetik, Allmandring 31, 70569 Stuttgart, Germany. Phone: 49-711-685-66981. Fax: 49-711-685-66973. E-mail: hildegard.watzlawick{at}iig.uni-stuttgart.de

{triangledown} Published ahead of print on 7 August 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: Biotech Tools s.a., Rue de Ransbeek 230, 1120 Brussels, Belgium.

Applied and Environmental Microbiology, October 2009, p. 6312-6321, Vol. 75, No. 19
0099-2240/09/$08.00+0     doi:10.1128/AEM.00714-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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