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Researchers developing salt-tolerant enzymes tailored to ionic liquid processes for production of advanced cellulosic biofuels

Halorhabdus utahensis, the source of the salt-tolerant enzyme. Source: JGI. Click to enlarge.

Researchers from the US Department of Energy (DOE) Joint Genome Institute (JGI) and the Joint BioEnergy Institute (JBEI) at DOE’s Lawrence Berkeley National Laboratory have used a genome-based approach to identify and characterize a halophilic (salt-tolerant) cellulase (Hu-CBH1) from the halophilic archaeon, Halorhabdus utahensis.

Such salt-tolerant enzymes, particularly cellulases, offer significant advantages for industrial utility over conventional enzymes, they said. The group plans to expand this research to develop a full complement of enzymes that is tailored for the ionic liquid process technology with the goal of demonstrating a complete biomass-to-sugar process, one they hope can enable the commercial viability of advanced biofuels.

A paper on their work is published online in the RSC journal Green Chemistry.

Ionic liquids (ILs) have been shown to be effective solvents for biomass pretreatment and enhancing the yield of sugars liberated from it for the production of advanced biofuels. However, while ionic liquids are useful for breaking down biomass, they can also inhibit fungal cellulases used to produce sugars after pretreatment, thereby making digestion of cellulose inefficient in their presence. Ionic liquids are a liquid form of salt that will inactivate enzymes by interfering with the folding of polypeptides: the building-blocks of proteins.

Several improvements are needed in the ionic liquid pretreatment process technology before it is cost effective with other pretreatments that are based on the pulp and paper processing technologies that utilize dilute acids and bases. One of the most important areas for cost reduction is reducing the number of washes required after IL pretreatment. Unfortunately, commercial fungal cellulases are inhibited by some ILs and, therefore, require extensive washing after IL pretreatment. Therefore, it is crucial to identify IL-resistant enzymes for digesting cellulose in the presence of ionic liquids to decrease the number of washes required and increase the yields of monomeric sugars.

...We have identified a gene cluster that contains multiple cellulolytic enzymes from the halophilic archaeon, Halorhabdus utahensis. We cloned and expressed one cellobiohydrolase in a different haloarchaeal host, Haloferax volcanii. We named this gene as Halorhabdus utahensis CBH1, or Hu-CBH1, in short. Using cellulase activity assay, we found that this enzyme is a haloalkaliphilic and heat tolerant cellobiohydrolase. The protein is enriched in acidic amino acids and presents strong negative charges on its surface.

Interestingly, we determined that salt is essential for the stability and function of the protein and that it can tolerate up to 20% (w/w) of ILs, including 1-ethyl-3-methylimidazolium acetate ([Emim]Ac), 1-ethyl-3-methylimidazolium chloride ([Emim]Cl), 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) and 1-allyl- 3-methylimidazolium chloride ([Amim]Cl).

—Zhang et al.

Halorhabdus utahensis was isolated from the natural environment at the Great Salt Lake and sequenced at the DOE JGI as part of the Genomic Encyclopedia of Bacteria and Archaea (GEBA) project.

The researchers suggest that their results indicate that halophilic enzymes are good candidates for the screening of IL-tolerant cellulolytic enzymes, and that understanding the mechanism of salt-tolerance may help to engineer novel cellulolytic enzymes adapted to ionic liquid environments.

This is one of the only reports of salt-tolerant cellulases, and the only one that represents a true genome-to-function relevant to ionic liquids from a halophilic environment. This strategy enhances the possibility of identifying true obligatory halophilic enzymes.

This project has established a very important link between genomic science and the realization of enzymes that can handle very demanding chemical environments, such as those present in a biorefinery.

—Blake Simmons



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