The conversion of cellulosic materials to simple organics
Late in 2007, Dr. Keith Kyler approached me with the idea of collaborating on a project. He had developed a highly effective, environmentally friendly and efficient method for delignifying cellulosic materials, and was looking to develop lower cost enzymes to convert that cellulose into sugar. He knew I had some familiarity with biotechnology and protein purification, and proposed that we look into producting engineered cellulase for his process.
At this same time, Dr. Jaeju Ko was making significant progress in the development and application of THEMATICS to the prediction of enzyme active sites. Becoming involved with cellulases was attractive because it would allow Dr. Ko and me to collaborate, creating and characterizing mutants to refine THEMATICS and possibly evolve it into a tool for protein redesign. Plus, this is a problem of considerable importance - cellulosic materials (essentially, biomass) are the largest renewable feedstock available, and an efficient way to utilitize cellulosic materials will free humanity from our current dependence on fossil fuels. Each year, about 100 billion metric tons of carbon is removed from the atmosphere due to photosynthesis; currently, we generate about 6.5 billion metric tons of carbon from burning fossil fuels. Clearly, we can use the carbon in biomass to replace that from fossil fuel, IF we can find appropriate processes. This importance is reflected in the fact that the American Chemical Society has created a Cellulose and Renewable Materials Division. A recent article from Chemical and Engineering News on this topic is available.
This is a challenging problem. First, cellulosic materials are quite complex. Here's some elementary references on the structure of wood.
| a chapter from The Forest Department's Wood Handbook | PDF File |
| This entire books is available online |
at Google Books |
| lecture slides by Matthew McBroom, Ph. D., CF; Assistant Professor, Forest Hydrology, Arthur Temple College of Forestry and Agriculture, Stephen F. Austin State University | PDF File |
| course supplement by Art Ragauskas, Ph.D., Professor, School of Chemistry and Biochemistry, University of Western Ontario | PDF File |
Cellulose itself is also complex - as you can tell from the Wikipedia entry for cellulose. A reprint of a treatise on cellulose is available here, again on Google Books. Although fascinating, be aware that this book was first published in 1938, and revised in 1942. Cellulose is not really a polymer of glucose, but rather a linear polymer of cellobiose, which is a dimer of two glucose molecules. Further, cellulose strands have regions of crystallinity, with extensive interchain hydrogen-bonding, interspersed with region of relative disorder (so-called amorphorous regions). It appears that the number of repeating units and the relative amount of crystallinity varies according to the source of the cellulose.
"Cellulase" is likewise complex. As befitting the complexity of cellulosic materials, cellulase is not a single enzyme, but is typically a combination of four classes of enzymes: endoglucanases (EGs, 1,4-β -D-glucan-4-glucanohydrolase), cellobiohydrolases, (CBHs, 1,4-β -D-glucan cellobiohydrolase) exoglucohydrolases (EXGs, 1,4-β -D-glucan-4-glucan glucohydrolase), and β -D-glucoside glucohydrolases ( β -G).
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The actions of these are shown diagrammatically in the Figure, which is taken from the supplementary information accompanying the report, "Hydrolytic Activity of Free and Immobilized Celluse," by I. R. M. Tebeka, A. G. L. Silva, and D. F. S. Petri, Langmuir, published on the web January 5, 2009 DOI: 10.1021/la802882s. Note that once an EG has created a break in the cellulose chain, because of the bonding between the individual cellobiose molecules, it has created two distinct ends. Consequently, there are two classes of CBHs, one for each "end." CBHs are generally considered to be highly processive, "chewing" off many cellobiose molecules for each attachment. The EGs, on the other hand, seem to snip the cellulose chain once, release, reattach elsewhere and snip again. This is sensible, as each snip generates ends on which the CBHs can work. Note shown here, but also of interest are swollenins, more recently discovered proteins that seem to cause the crystalline regions of cellulose to swell and thus renders them more susceptible to the action of the EGs. |
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There are numerous issues to explore here, in part because of the heterogeneity of the substrate and the complexity and variety of the enzymes involved. For example, the kinetics of each enzyme have been measured on a variety of cellulosic substrates, but only a few enzymes have been studied using purified cello-oligosaccharides. And, as might be expected, many studies use derivatized cellulosics, tagged with chromophores or other labels to render detection simpler or more sensitive. Thus, isolating cello-oligosaccharides and studying the kinetics using appropriate chromatographic techniques is of interest to me.
Closely related is the issue of exactly how the presence of the substrate induces the expression of the cellulase enzymes. Clearly, the substrate is too big to diffuse into the cell. It is generally considered that low levels of the enzymes are constitutively expressed, and these generate soluble products from cellulosic materials, which in turn induce expression of more enzyme. Once we have secured reasonable amounts of purified cello-oliosaccharides, we can explore this issue.