This Small Business Innovation Research Phase I project helps unlock the potential of forests to provide sustainable, carbon-neutral raw material for much of the nation's energy and chemical needs. Current technology cannot efficiently separate cellulose, hemicellulose and lignin (the major components of wood as well as cellulosic biomass). Cellulose can be used to make paper, lignin can be burned to provide power, and hemicellulose can be used via fermentation to produce biofuels and platform chemicals. The major difficulty in fractionating wood is breaking ether bonds between lignin and hemicellulose. Research Objectives include: demonstration of novelty by comparison to known enzymes that are active against xylan, design of a pilot scale manufacturing process for the new microorganism and enzyme, determination of the enzyme's polypeptide structure through zymography, and identification via protein microsequencing. Anticipated results are: an enzyme that is available in pilot scale quantities for testing and the data necessary to create a recombinant enzyme suitable for manufacture on an industrial scale.
The broader impact/commercial potential of this project is to develop a novel, environmentally friendly production method for pulp products and to create new feedstocks for biofuels and platform chemicals. In pulp mills, Tethys's enzyme will allow previously wasted hemicellulose and hemicellulose-derived sugar to be recovered. Hemicellulose can be added back to pulp to increase yield or be converted, via existing fermentation technology, into building block chemicals and biofuels. Additionally, the presence of Tethys's enzyme in the pulp will loosen lignin, allowing better delignification with the use of less energy and chemicals compared to the current state of the art. Important outcomes of this research if successful, will be (i) wood, a renewable resource, will be more effectively used to meet a significant portion of America's energy and platform chemical needs; (ii) energy and industrial chemicals used in pulp and paper mills and cellulosic biorefineries will be reduced, and the resulting effluents improved; and (iii) America's mills and cellulosic biorefineries (and the rural towns where they are located) will receive an economic boost from reduced costs and increased revenues from higher yields.
Tethys Research LLC is an enzyme discovery company that identifies and develops novel enzymes to separate lignin from cellulose and hemicellulose in woody biomass. Cellulose and hemicellulose are chains of sugars. The longest of those chains can be used for paper production, or all the chains can be depolymerized into sugars which then can be fermented—using existing technology—into biofuels or platform chemicals. At present, the major impediment to the use of woody biomass for biofuels and platform chemicals is the difficulty of separating the polysaccharides (cellulose and hemicellulose, which together make up ~65% of woody biomass) from lignin. Lignin and hemicellulose are primarily linked by ether bonds, a type of chemical bond that is difficult to break. Current bioprocessing employs hazardous and environmentally damaging chemicals that only partially accomplish lignin separation and results in damaged hemicellulose and cellulose, leading to decreased paper or sugar yield. To counter these problems, Tethys is developing novel enzymes that specifically cleave hemicellulose-lignin ether bonds without causing collateral damage. By replacing chemical separation, Tethys’s enzymes will improve efficiency and reduce the environmental footprint at both paper mills and lignocellulosic biorefineries that produce biofuels and other renewable bio-based chemicals. They will enhance sugar recovery from lignocellulosic feedstocks while concomitantly decreasing formation of fermentation inhibitors for biorefineries. Further, the use of Tethys’s enzymes will generate a cleaner lignin fraction for chemical processing into fuel or chemicals. The enzymes will also work in concert with current and developing pre-extraction technology to potentiate the development of a hemicellulose product stream for pulp mills. Other laboratories that have been working on separation technology for woody biomass have concentrated on glycosidic lignin-hemicellulose bonds. Glycosidic bonds are the type of bonds that link one sugar to another in the chains of cellulose and hemicellulose. But wood biosynthesis strongly implies that non-glycosidic links are the major type of bonds between lignin and hemicellulose. Tethys has specifically searched for enzymes that cleave non-glycosidic bonds between lignin and hemicellulose. Tethys calls these new enzymes "HLEs" (hemicellulose:lignin etherases). HLEs split ether bonds between non-glycosidic carbons of sugars in polysaccharides and other moieties, in this case the phenyl groups of lignin. The enzymes will be used to increase the yield and quality of sugars obtained through conversion of lignocellulosic biomass in order to ferment those sugars into biofuels and/or other chemicals. The resulting lignin would also be cleaner and easier to use as a chemical feedstock than lignin from current practice. In addition, these enzymes will be used to design a new pulping process to improve the quality of paper and reduce environmental costs of pulping. In previous work, Tethys discovered an enzyme that would cleave the non-glycosidic phenyl ether bonds between lignin and the mannan hemicelluloses that predominate in softwoods. Tethys also bioprospected for microbes that cleave the non-glycosidic phenyl ether bonds between lignin and the xylan hemicelluloses predominant in hardwoods, agricultural residue, and energy crops like switchgrass. In this project, Tethys successfully discovered new enzyme activities from the hardwood-directed microbes. In particular, efforts were concentrated on the activity from a newly discovered microbe, E518. Tethys proved that the activity from E518 was due to a novel enzyme, not a known enzyme such as hemicellulase or xylosidase, both glycosidases. The enzyme from E518 is particularly desirable as a biomass deconstruction enzyme for cellulosic bioenergy production or as a bleaching enzyme for pulp and paper operations. Its activity is very rapid compared to glycosidases and it is remarkably stable to heating and freeze-thaw cycles. Because of its stability, Tethys concludes that commercial production of the enzyme is highly feasible. However, Microbe E518 grows very slowly and the enzyme would be more efficiently produced if its gene were expressed in another microbe, one engineered for the production of large quantities of a heterologous protein. Consequently, Tethys is currently engaged in cloning the microbial gene of the enzyme in order to engineer it into a more convenient expression system for large-scale testing.
