Construction of cellobiose phosphorylase variants with broadened acceptor specificity towards anomerically substituted glucosides.
The general application of glycoside phosphorylases such as cellobiose phosphorylase (CP) for glycoside synthesis is hindered by their relatively narrow substrate specificity. We have previously reported on the creation of Cellulomonas uda CP enzyme variants with either modified donor or acceptor specificity. Remarkably, in this study it was found that the donor mutant also displays broadened acceptor specificity towards several beta-glucosides. Triple mutants containing donor (T508I/N667A) as well as acceptor mutations (E649C or E649G) also display a broader acceptor specificity than any of the parent enzymes. Moreover, further broadening of the acceptor specificity has been achieved by site-saturation mutagenesis of residues near the active site entrance. The best enzyme variant contains the additional N156D and N163D mutations and is active towards various alkyl beta-glucosides, methyl alpha-glucoside and cellobiose. In comparison with the wild-type C. uda CP enzyme, which cannot accept anomerically substituted glucosides at all, the obtained increase in substrate specificity is significant. The described CP enzyme variants should be useful for the synthesis of cellobiosides and other glycosides with prebiotic and pharmaceutical properties.
Centre of Expertise for Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, 9000 Ghent, Belgium. Manu.DeGroeve@UGent.be
Manu R M
MR
De Groeve
Laurens
L
Remmery
Annelies
A
Van Hoorebeke
Jan
J
Stout
Tom
T
Desmet
Savvas N
SN
Savvides
Wim
W
Soetaert
D019943:Amino Acid Substitution / Q000235:genetics; D020134:Catalytic Domain; D040143:Cellulomonas / Q000201:enzymology*; D005960:Glucosides / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D005960:Glucosides; D005964:Glucosyltransferases; C034714:cellobiose phosphorylase