Many of the phosphorylases so far studied act on disaccharides. This limits their use in polysaccharide biotechnology and the possibility of converting them into polymerising phosphorylases is highly attractive. It is likely that disaccharide phosphorylases will
Continue to yield to biochemical analysis more readily than their polymerising counterparts. The ability to convert them into polymerising enzymes therefore represents an attractive prospect for polysaccharide synthesis. Mutagenesis studies have been used
to enhance the flexibility of disaccharide phosphorylase enzymes: opening up the active site of CBP to allow glycosides of glucose to act as acceptor substrates116 and increasing the length of koji-oligosaccharides produced by kojibiose phosphorylase28 have both
been reported. Projecting ahead, comparison of phosphorylases from the same
CAZy family but with different acceptor lengths may inform mutations and the generation of chimeras to produce novel polymerising phosphorylase enzymes. Cellobiose phosphorylase and cellodextrin phosphorylase are both in the same CAZy family,GH94, so comparison between their structures may help to define the features that determine substrate length. In a similar sense, chitinbiose phosphorylase could be altered by the generation of
chimeric proteins with CDP; a nigerose phosphorylase may be
compatible with the generation of polymerising kojibiose phosphorylase chimeras, since they are in the same GH family. The lessons learnt from such studies might provide design guidelines that may be extended to those families of phosphorylase that do not
contain a polymerising activity.