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PEDS Advance Access originally published online on May 31, 2009
Protein Engineering Design and Selection 2009 22(7):393-399; doi:10.1093/protein/gzp017
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© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Creating lactose phosphorylase enzymes by directed evolution of cellobiose phosphorylase

Manu R.M. De Groeve1, Miet De Baere, Lieve Hoflack, Tom Desmet, Erick J. Vandamme and Wim Soetaert

Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, 9000 Ghent, Belgium

1 To whom correspondence should be addressed. E-mail: manu.degroeve{at}ugent.be

Disaccharide phosphorylases are interesting enzymes for the production of sugar phosphates from cheap starting materials and for the synthesis of novel glycosides. Cellobiose phosphorylase (CP) from Cellulomonas uda was subjected to directed evolution in order to create enzyme variants with significantly increased lactose phosphorylase (LP) activity, useful for the production of {alpha}-D-galactose 1-phosphate. In a first round, random mutagenesis was performed on part of the CP gene and the resultant library was selected on minimal lactose medium. One clone containing six amino acid mutations was found with increased LP activity compared with the wild-type CP enzyme. The negative and neutral mutations were eliminated by site-directed mutagenesis and the resultant enzyme variant containing two amino acid substitutions (T508A/N667T) showed more LP activity than the parent mutant. Saturation mutagenesis of the beneficial sites and screening for improved mutants allowed us to identify the T508I/N667A mutant which has 7.5 times higher specific activity on lactose than the wild-type. The kinetic parameters of the mutants were determined and showed that the increased LP activity was caused by a higher kcat value. This is the first report of an engineered CP with modified substrate specificity.

Keywords: cellobiose phosphorylase/directed evolution/lactose phosphorylase/screening/selection

Received February 27, 2009; revised April 29, 2009; accepted May 1, 2009.


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