Engineering of cellobiose phosphorylase for glycoside synthesis

Glycosylation can significantly influence the properties of a chemical compound. In the case of pharmaceuticals, glycosylation can improve the pharmacokinetic properties of a drug and induce drug targeting to specific organs, resulting in less side effects and smaller required doses. This research is part of a larger project in which the goal is to develop a generic glycosylation technology based on glycoside phosphorylases. Here, the Cellulomonas uda cellobiose phosphorylase (CP) enzyme was used as a model. Since most natural phosphorylases have a relatively narrow substrate specificity, they should be improved by protein engineering. Therefore, a suitable high-throughput screening (HTS) system was developed and applied to an E649 saturation library to identify CP enzyme variants with acceptor specificity towards anomerically substituted glucosides. Wild-type C. uda CP has no specificity towards these acceptors. An enzyme variant containing an E649C mutation was found, displaying high activity towards methyl β-glucoside, ethyl β-glucoside and even phenyl β-glucoside as acceptor. Also the donor specificity of C. uda CP was modified, more specifically from αGlc1P towards αGal1P. Using a combination of mutagenesis methods followed by selection and screening, so-called lactose phosphorylase (LP) enzymes were created that have not yet been found in nature. Further broadening of acceptor specificity towards β-glucosides could be achieved by mutating residues near the active site entrance and screening for acceptor specificity towards octyl β-glucoside. A mutant containing five amino acid mutations was found which displays broad acceptor specificity towards various β- and α-glucosides, including alkyl β-glucosides, aryl β-glucosides and methyl α-glucoside. A flexible loop near the active site entrance of CP was found to be involved in enzyme activity, stability and specificity. Deletion of part of the loop resulted in unstable enzyme variants with low CP activity, while saturation mutagenesis of several loop residues resulted in modified substrate specificity. Finally, methyl β-cellobioside, methyl β-lactoside and lactose were produced and purified with the created enzyme variants. From these glycosides, methyl β-lactoside has most interesting properties since it was reported to have anti-cancer effects