Identification of a haloalkaliphilic and thermostable cellulase with improved ionic liquid tolerance

Identification of a haloalkaliphilic and thermostable cellulase with improved ionic liquid tolerance†

Tao Zhang,^a Supratim Datta,^b Jerry Eichler,^c Natalia Ivanova,^a Seth D. Axen,^a Cheryl A. Kerfeld,^ad Feng Chen,^a Nikos Kyrpides,^a Philip Hugenholtz,^abe Jan-Fang Cheng,^a Kenneth L. Sale,^b Blake Simmons*^b and Eddy Rubin*^a

Author affiliations

* Corresponding authors

^a DOE Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA
E-mail: EMRubin@lbl.gov

^b The Joint BioEnergy Institute, Emeryville, CA
E-mail: basimmo@sandia.gov

^c Dept. of Life Sciences, Ben Gurion University of the Negev, P.O. Box 653, Beersheva, Israel

^d Department of Plant and Microbial Biology, UC Berkeley, CA

^e Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences and Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia

Abstract

Some ionic liquids (ILs) have been shown to be very effective solvents for biomass pretreatment. It is known that some ILs can have a strong inhibitory effect on fungal cellulases, making the digestion of cellulose inefficient in the presence of ILs. The identification of IL-tolerant enzymes that could be produced as a cellulase cocktail would reduce the costs and water use requirements of the IL pretreatment process. Due to their adaptation to high salinity environments, halophilic enzymes are hypothesized to be good candidates for screening and identifying IL-resistant cellulases. Using a genome-based approach, we have identified and characterized a halophilic cellulase (Hu-CBH1) from the halophilic archaeon, Halorhabdus utahensis. Hu-CBH1 is present in a gene cluster containing multiple putative cellulolytic enzymes. Sequence and theoretical structure analysis indicate that Hu-CBH1 is highly enriched with negatively charged acidic amino acids on the surface, which may form a solvation shell that may stabilize the enzyme, through interaction with salt ions and/or water molecules. Hu-CBH1 is a heat tolerant haloalkaliphilic cellulase and is active in salt concentrations up to 5 M NaCl. In high salt buffer, Hu-CBH1 can tolerate alkali (pH 11.5) conditions and, more importantly, is tolerant to high levels (20% w/w) of ILs, including 1-allyl-3-methylimidazolium chloride ([Amim]Cl). Interestingly, the tolerances to heat, alkali and ILs are found to be salt-dependent, suggesting that the enzyme is stabilized by the presence of salt. Our results indicate that halophilic enzymes are good candidates for the screening of IL-tolerant cellulolytic enzymes.