Engineering of cellobiose phosphorylase for glycoside synthesis.

Disaccharide phosphorylases are increasingly applied for glycoside synthesis, since they are very regiospecific and use cheap and easy to obtain donor substrates. A promising enzyme is cellobiose phosphorylase (CP), which was discovered more than 50 years ago. Many other bacterial CP enzymes have since then been characterized, cloned and applied for glycoside synthesis.

Probing the active site of cellodextrin phosphorylase from Clostridium stercorarium: kinetic characterization, ligand docking, and site-directed mutagenesis.

Cellodextrin phosphorylase from Clostridium stercorarium has been recombinantly expressed in Escherichia coli for the first time. Kinetic characterization of the purified enzyme has revealed that aryl and alkyl β-glucosides can be efficiently glycosylated, an activity that has not yet been described for this enzyme class. To obtain a better understanding of the factors that determine the enzyme's specificity, homology modeling and ligand docking were applied.

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.

Enzymatic production of alpha-D-galactose 1-phosphate by lactose phosphorolysis.

alpha-D-Galactose 1-phosphate (alphaGal1P) is an important building block for the synthesis of nucleotide sugars that are substrates for glycosyltransferases. We have previously reported the creation of novel lactose phosphorylase enzymes that are useful for the synthesis of alphaGal1P from the cheap and abundant lactose. Here we describe the application of such a lactose phosphorylase in a production system using permeabilized Escherichia coli cells.

Creating lactose phosphorylase enzymes by directed evolution of cellobiose phosphorylase.

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.

Cloning, sequence analysis and heterologous expression of the Myrothecium gramineum orotidine-5'-monophosphate decarboxylase gene.

A 2918 bp sequence coding for the orotidine-5'-monophosphate decarboxylase enzyme (OMPD) was isolated from the genome of Myrothecium gramineum. This sequence was analysed and, remarkably, it is the first OMPD gene of a Sordariomycete that has an intron. The gene codes for an enzyme of 282 amino acids.