(587r) Comparing the Biophysical Parameters of Two Thermostable and IL-Tolerant Bacterial Cellulases From Three Recombinant Hosts | AIChE

(587r) Comparing the Biophysical Parameters of Two Thermostable and IL-Tolerant Bacterial Cellulases From Three Recombinant Hosts


Feldman, T. - Presenter, Joint BioEnergy Institute
Gladden, J. M., Joint BioEnergy Institute
Park, J. I., Joint BioEnergy Institute
Sale, K. L., Joint BioEnergy Institute
Baker, S. E., Pacific Northwest National Laboratory
Simmons, B., Lawrence Berkeley National Laboratory
Magnuson, J. K., Pacific Northwest National Laboratory
Lynn, J., Joint BioEnergy Institute
Srikrishnan, S., University of California, Irvine

The development of biofuels from lignocellulosic biomass is a challenging endeavor, both for technical and economic reasons, and efforts have focused on developing technologies that increase efficiency and reduce costs. Cellulase enzymes have received particular attention as they are a central component of most bioprocessing platforms but are currently expensive. Several interesting enzymes have been investigated as alternatives to the current commercial cellulases. For example, Park et al. (2012), demonstrated that a combination of two thermostable bacterial enzymes expressed in Escherichia coli could saccharify biomass at high temperatures and in the presence of ionic liquids, making them attractive targets for developing alternative cellulase cocktails. This simple cocktail is composed of a cellobiohydrolase from Caldicellulosiruptor saccharolyticus (E.C., UniProt ID A4XIF7, with its GH10 domain removed) and a β-glucosidase from Thermotoga petrophila (EC, UniProt ID A5IL97). One major barrier to developing designer cellulase cocktails composed of these and other interesting enzymes is their high-titer production in heterologous hosts. Since E. coli is unlikely to be a viable commercial host for high level enzyme production, alternative host organisms must be investigated to determine whether these enzymes will express and retain their functionality.  To explore this topic, the two enzymes were expressed in E. coli and two fungal systems (Pichia pastoris, and Aspergillus niger), allowing a direct comparison of the biophysical properties on expression from each diverse host. The genes encoding the enzymes were optimized for expression in each host, and the recombinant enzymes were characterized for optimal temperature, pH, and thermostability. In addition, the IL-tolerance of the recombinant enzymes was tested. The results of this study will help elucidate the effect of host strain selection on protein stability and functionality of recombinant enzymes.