Relief of Xylose Binding to Cellobiose Phosphorylase by a Single Distal Mutation.

Cellobiose phosphorylase (CBP) cleaves cellobiose-abundant in plant biomass-to glucose and glucose 1-phosphate. However, the pentose sugar xylose, also abundant in plant biomass, acts as a mixed-inhibitor and a substrate for the reverse reaction, limiting the industrial potential of CBP. Preventing xylose, which lacks only a single hydroxymethyl group relative to glucose, from binding to the CBP active site poses a spatial challenge for protein engineering, since simple steric occlusion cannot be used to block xylose binding without also preventing glucose binding.

Overcoming inefficient cellobiose fermentation by cellobiose phosphorylase in the presence of xylose.

Background: Cellobiose and xylose co-fermentation holds promise for efficiently producing biofuels from plant biomass. Cellobiose phosphorylase (CBP), an intracellular enzyme generally found in anaerobic bacteria, cleaves cellobiose to glucose and glucose-1-phosphate, providing energetic advantages under the anaerobic conditions required for large-scale biofuel production. However, the efficiency of CBP to cleave cellobiose in the presence of xylose is unknown.