A thermostable cellobiose phosphorylase from Thermoclostridium caenicola and its application to in vitro biotransformation

Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose to glucose and α-D-glucose-1-phosphate. CBPs play an important role in the in vivo enzymatic utilization of cellulosic materials. Here we discovered a novel CBP from Thermoclostridium caenicola (TcCBP), displaying 50–75 % sequence homology with previously reported CBPs. Recombinant TcCBP was well expressed in E. coli BL21(DE3), with a 1.6-fold increase in soluble expression levels over the widely-used Clostridium thermocellum CBP (CtCBP). This enzyme exhibited broad pH adaptability, maintaining substantial activities across pH 4.0–7.5 in the synthetic direction and pH 5.0–7.5 in the phosphorolytic direction. Compared to CtCBP, TcCBP displayed the superior thermostability and a nearly 100-fold improvement in "Product-to-Enzyme Ratio" (PE value). The biosynthesis of myo-inositol from cellobiose was conducted by using this CBP along with other four thermophilic enzymes (i.e., phosphoglucomutase, inositol 1-phosphate synthase, inositol monophosphatase, and polyphosphate glucokinase) in one pot without step-by-step addition of enzymes. Approximately 96 mM of myo-inositol was produced from 50 mM of cellobiose. These results indicated that this enzyme could be a potential thermophilic enzyme used for the production of value-added biochemicals by in vitro BioTransformation.