Isolation of a Cellobiohydrolase of Clostridium thermocellum Capable of Degrading Natural Crystalline Substrates

A cellobiohydrolase (CBH3) of Clostridium thermocellum was isolated from the recombinant strains of Escherichia coli . The enzyme was shown to have a Mr of 78 kDa and isoelectric point of 4.75. The hydrophilic nature of the enzyme was confirmed by its unretarded elution on gel filtration column. The enzyme had a binding affinity for the microcrystalline substrate (Avicel). The pH and temperature optimum were determined as 6.5 and 60°C. The enzyme was found to be active on p-nitrophenyl cellobioside, carboxymethyl cellulose, Avicel, amorphous cellulose, lichenan and xylan. The most distinguishing feature of CBH3 was the degradation of a variety of natural crystalline substrates, with maximum activity on filter paper. The enzyme was tolerant to high levels of cellobiose, susceptible to heavy metals and was protected by DTT and calcium at higher temperatures. This is the first report of a highly active cellobiohydrolase produced in C. thermocellum.