Engineering Endoglucanase Enzymes for Higher Thermostability

The Science

Endoglucanase enzyme complexes break down the internal structure of cellulose, disrupting its crystalline structure and leading to glucose, the desired end product needed for fermentation to ethanol. Like all enzymes, endoglucanases only function within a certain temperature range; however, high temperatures are often part of the biomass breakdown process. Research at DOE’s Bioenergy Research Center (BESC) at Oak Ridge is pushing the upper boundary of the temperature range for endoglucanases from the microbe Clostridium phytofermentans. Percival Zhang and colleagues studied directed mutational evolution of mutant proteins from the endoglucanase Cel5A family. They found mutants that are actually more active at 60°C, with the exact activity dependant on the specific cellulose substrate used. These results suggest that there may be a more complex relationship between endoglucanase activity and soluble or solid cellulose substrates then was previously thought. Further research will seek additional improvements of endogluconases for potential application to biofuel production.

Summary

A family 5 glycoside hydrolase from Clostridium phytofermentans was cloned and engineered through a cellulase cell surface display system in Escherichia coli. The presence of cell surface anchoring, a cellulose binding module, or a His tag greatly influenced the activities of wild-type and mutant enzymes on soluble and solid cellulosic substrates, suggesting the high complexity of cellulase engineering. The best mutant had 92%, 36%, and 46% longer half-lives at 60°C on carboxymethyl cellulose, regenerated amorphous cellulose, and Avicel, respectively.

Principal Investigator

Percival Zhang
BioEnergy Science Center

References

Liu, W., X.-Z. Zhang, Z. Zhang, and Y. H. P. Zhang. “Engineering of Clostridium phytofermentans Endoglucanase Cel5A for Improved Thermostability,” Applied and Environmental Microbiology 76, 4914–17. DOI:10.1128/AEM.00958-10.