Enzymatic Hydrolysis Technology Background

Enzymes are the relative newcomers with respect to biomass-to-ethanol processing. While the chemistry of sugar production from wood has almost 2 centuries of research and development history and 100 years of process development, enzymes for biomass hydrolysis can barely speak of 50 years of serious effort. The search for biological causes of cellulose hydrolysis did not begin in earnest until World War II. The U.S. Army mounted a basic research program to understand the causes of deterioration of military clothing and equipment in the jungles of the South Pacific — a problem that was wrecking havoc with cargo shipments during the war. This campaign resulted in the formation of the U.S. Army Natick Laboratories¹. Out of this effort to screen thousands of samples collected from the jungle came the identification of what has become one of the most important organisms in the development of cellulase enzymes — Trichoderma viride (eventually renamed Trichoderma reesei). T. reesei is the ancestor of many of the most potent enzyme-producing fungi in commercial use today.

Ironically, the research on cellulases was prompted by a need to prevent their hydrolytic attack on cellulose. Today, we turn to these enzymes in hope of increasing their hydrolytic power. This turning point in the focus of cellulase research did not occur until the early 1960s, when sugars from cellulose were recognized as a possible food source², echoing similar notions expressed by researchers in earlier days on acid hydrolysis research³. In the mid-1960s, the discovery that extracellular enzyme preparations could be made from the likes of T. reesei⁴ accelerated scientific and commercial interest in cellulases. In 1973, the army was beginning to look at cellulases as a means of converting solid waste into food and energy products⁵. By 1979, genetic enhancement of T. reesei had already produced mutant strains with up to 20 times the productivity of the original organisms isolated from New Guinea^6,7. For roughly 20 years, cellulases made from submerged culture fungal fermentations have been commercially available. In another ironic twist, the most lucrative cellulase market today is in the textile industry, where a valuable niche has been found in the production of "stone-washed" jeans.

The science of cellulases has come a long way since World War II. It has grown in conjunction with the monumental changes that have occurred in molecular biology, protein chemistry, and enzymology over the past 50 years. It is easy to forget just how extensive this change has been. In 1876, the German researcher Wilhelm Friedrich Kuhne coined the term "enzyme." Its Greek roots simply mean "in yeast." Kuhne used it to describe the "unorganized ferment from yeast and other organisms." The debate in his time was whether the catalytic activity observed in these "ferments" could exist independently of living cells⁸. By the 1920s, evidence was mounting that these enzymes were actually proteins and that proteins were actually discrete chemical entities. But, the answer to this question had to wait for sufficiently sophisticated protein purification techniques to be developed. It was not until 1951, with the elucidation of the amino acid sequence for part of insulin, that enzymes were indisputably recognized as independent protein chemicals⁹.

In many ways, however, our understanding of cellulases is in its infancy compared to other enzymes. There are some good reasons for this. Cellulase-cellulose systems involve soluble enzymes working on insoluble substrates. The jump in complexity from homogeneous enzyme-substrate systems is tremendous. It became clear fairly quickly that the enzyme known as "cellulase" was really a complex system of enzymes that work together synergistically to attack native cellulose. In 1950, this complex was crudely described as a system in which an enzyme known as C1 acts to decrystallize the cellulose, followed by a consortium of hydrolytic enzymes, known as Cx which breaks down the cellulose to sugar¹⁰. This early concept of cellulase activity has been modified, added to, and argued about for the past 40 years^11,12.

Though many researchers still talk in terms of the original model of a nonhydrolytic C₁ enzyme and a set of C_x hydrolytic enzymes, our current picture of how these enzymes work together is much more complex. Three major classes of cellulase enzymes are recognized today:

Endoglucanases, which act randomly on soluble and insoluble glucose chains
Exoglucanases, which include glucanhydrolases that preferentially liberate glucose monomers from the end of the cellulose chain and cellobiohydrolases that preferentially liberate cellobiose (glucose dimers) from the end of the cellulose chain
ß-glucosidases, which liberate D-glucose from cellobiose dimers and soluble cellodextrins

For a long time, researchers have recognized that these three classes of enzymes work together synergistically in a complex interplay that results in efficient decrystallization and hydrolysis of native cellulose. In reaching out to "non-scientific" audiences, promoters of cellulase research often oversimplify the basic description of how these enzymes work together to efficiently attack cellulose¹³. The danger in such oversimplifications is that they may mislead many about the unknowns and the difficulties we still face in developing a new generation of cost-effective enzymes. While our understanding of cellulase's modes of action has improved, we have much more to learn before we can efficiently develop enzyme cocktails with increased activity.

References

¹Augustine, N.R. "Key Note Address: Technology transfer from Military Requirements to Public Need." Biotechnology and Bioengineering Symposium, No. 6, John Wiley & Sons, 1976, pp 1-8.

²Reese, R.T. "History of the Cellulase Program at the U.S. Army Natick Development Center." Biotechnology and Bioengineering, No. 6, pp 9-20.

³Peterson, W.H.; Snell,J.F.; Frazier, W.C. "Fodder Yeast from Wood Sugar." Industrial and Engineering Chemistry, Vol 37, No. 1, 1945, pp 30-35.

⁴Mandels, M.; Reese, E.T. "Fungal Cellulases and the Microbial Decomposition of Cellulosic Fabric." Developments in Industrial Microbiology, Vol. 5, Society for Industrial Microbiology, Washington, D.C., 1964, pp 5-20.

⁵Brandt, D.; Hontz, L.; Mandels, M. AIChE Symposium Series, No. 69, 1973, p 127.

⁶Mandels, M.; Weber, J.; Parizek, R., Applied Microbiology, Vol 21, 1971, p 152.

⁷Montenecourt, B.S.; Eveleigh, D.E. "Selective Screening Methods for the Isolation of High Yielding Cellulase Mutants of Trichoderma reese." In Advances in Chemistry Series: Hydrolysis of cellulose: Mechanism of Enzymatic and Acid Catalysis, No. 181, American Chemical Society, Washington, D.C., 1979, pp 289-301.

⁸Gutfreund, H. "Wilhelm Friedrich Kuhne: An Appreciation." In FEBS Letters: Enzymes: One Hundred Years (Gutfreund, H., ed) , Vol. 62 Supplement, 1976, pp 1-23. Kuhne sided with those who believed "enzymes" with observable catalytic activity could exist independently of living cells. The opposing view was led by none other than Louis Pasteur.

⁹Perham, R.N. "The Protein Chemistry of Enzymes." In FEBS Letters: Enzymes: One Hundred Years (Gutfreund, H., ed) , Vol. 62 Supplement, 1976, pp 20-36.

¹⁰Reese, E.T.; Siu, R.G.H.; Levinson, H.S. "The Biological Degradation of Soluble Cellulose Derivatives and Its Relationship to the Mechanism of Cellulose Hydrolysis." Journal of Bacteriology, Vol. 59, 1950, pp 485-497.

¹¹Lee, Y.-H.; Fan, L.T. "Properties and Mode of Action of Cellulase." Advances in Biochemical Engineering, Vol. 17, Springer-Verlag, New York, 1980, pp101-129.

¹²Kuhad, R.C.; Singh, A.; Ericksson,K.-E. "Microorganisms and Enzymes Involved in the Degradation of Plant Fiber Cell Walls." In Advances in Biochemical Engineering : Biotechnology in the Pulp and Paper Industry (Eriksson, K.-E., ed). Springer-Verlag, New York, 1997, pp 45-125

¹³See, for example, Wyman, C., "Overview of the Simultaneous Saccharification and Fermentation Process for Ethanol Production from Cellulosic Biomass." In Ethanol Annual Report FY 1990, (Texeira, R.; Goodman, B., Eds). Solar Energy Research Institute (now the National Renewable Energy Laboratory), Golden, CO, 1991.