The pharmaceutical oseltamivir (Tamifluä) is one of only two agents effective against H1N1 type influenza virus. Currently it is prepared by a twelve-step process beginning with shikimic acid.

Shikimate is an intermediate on the metabolic pathway used by plants to synthesize the aromatic amino acids phenylalanine, tyrosine, and tryptophan. Further along in that same pathway, tyrosine is converted to the monomers that are used in synthesizing lignin. For use in making oseltamivir, it is extracted from the Chinese star anise plant, where it bioaccumulates during the short growing season of this annual plant. The supply is limited and consequently the price has ranged from $300 to $700 per kilogram, depending on the imminence ofa new influenza outbreak.

Gedivine Nilmini, a PhD candidate in our group, has found that shikimate also accumulates in the needles of conifers, especially white pine, an important commercial species in Maine. When conifers are logged for timber or pulp, the foliage is generally left in the fields to rot. Hence a process for extracting the shikimate would provide a valuable addlitional resource for the forest products industry. Nilmini has developed large scale extraction and separation procedures for both shikimate and quinate, the immediate precursor of shikimate.

Hemicelluloses are heterogeneous sugar oligomers that can be extracted from wood prior to pulping in moderate quantity without affecting the yield or quality of the pulp.

Antioxidants, molecules that can trap reactive oxygen radicals, are a widely employed group of food supplements ("nutraceuticals"). Their utility in this role has not been established, although the benefits of foods containing antioxidants are well known. Plants synthesize many of these types of molecules as defenses against attack by fungi.

Regan LeBlanc (MS candidate) and several undergraduates and high school students in our group have been evaluating the bark of coniferes as sources of antioxidants. We have been particularly focused on a group of phenolics including resveratrol, which supposedly confers the health benefits of red wine as part of the Mediterranean diet.

Red spruce bark has proved to be an especially rich source of resveratrol, quercitin, and catechin. This mixture has greater antioxidant power in standard tests such as DPPH radical scavenging than any of the commercial antioxidant mixtures we have tested. Other barks also contain useful quantities of phenolics and flavonoids.

Hemicelluloses are a group of heterogeneous sugar oligomers that form one of the major constituents of wood. As part of the biorefinery concept, it has been proposed that a portion of the hemicellulose be extracted from wood before processing the remainder into pulp for paper.

We are interested in developing enzymatic methods for processing the hemicellulose into sugars that could be used to make fuels or be converted into new polymers. Two general steps are required: (1) removal of associated lignin and lignin fragments; and (2) hydrolysis of the sugar oligomers. Two types of enzymes are being evaluated by MS candidate Gamini Rupasinghe: oxidants like laccases or dioxygenase to remove the lignin, and xylanase to hydrolyze the oligomers.

Several families of oxidative enzymes are capable of degrading lignin. One of these is laccase, a four-copper fungal enzyme. Others are the iron-based dioxygenases.

The Laccase from M. albomyces	The Extradiol dioxygenase from S. paucimobilis

Laccases act by depolymerizing lignin; dioxygenases destroy the aromatic rings, producing muconic acid and carbon dioxide.

For hydrolysis, two structural families of xylanases are being investigated:

Family 10 Xylanase from S. lividans	Family 11 Xylanase from A. niger, substrate bound

We are attempting to immobilize the enzymes on glass or polymer beads to allow easy separation and reuse of the enzyme.

Recent Publications

Thomas Elder and R. C. Fort Jr., “Reactivity of Lignin – Correlation with Molecular Orbital Calculations”, in Lignin and Lignans, ed. C. Heitner, Taylor and Francis, Boca Raton, FL; in press

Raymond C. Fort Jr., Barbara J. W. Cole, Raju Kovur, and Andru O’Farrill “Oxidation of Lignin-carbohydrate Models. I. Oxidation of Aryl Glucosides by the Laccase from Trametes versicolor”, Proceedings of the 15th International Symposium on Wood, Fiber and Pulping Chemistry, Oslo, Norway, June, 2009

Barbara J. W.Cole, Regan LeBlanc, Genivine Nilmini, Joseph M. Genco, Raymond C. Fort, Jr., Melody Rhine, and Sorel Edes, “Bark and Foliage Extracts as Antioxidants and Other High Value Materials”, Proceedings of the 15th International Symposium on Wood, Fiber and Pulping Chemistry, Oslo, Norway, June, 2009

D. Montgomery, M. Nguyen, B. J. W. Cole, and R. C. Fort Jr, “Docking of Lignin Models and Lignin-Carbohydrate Compounds to the Laccase from T. Versicolor”, Proceedings of the 10th International Congress on Biotechnology in the Pulp and Paper Industry, Madison, WI, June 2007 L. B. Graham, J. M. Genco, B. J. W. Cole, and R. C. Fort Jr, “Comparison of Acid and Enzymatic Hydrolysis of Birch Xylan”, Proceedings of the 10th International Congress on Biotechnology in the Pulp and Paper Industry, Madison, WI, June 2007.

D. F. Guay, B. J. W. Cole, R. C. Fort Jr., M. C. Hausman, J. M. Genco, and T. J. Elder, "Mechanisms of Oxidative Degradation of Carbohydrates During Oxygen Delignification. III. Reaction of Photochemically Generated Hydroxyl Radicals with 1,5-Anhydrocellobitol and Cellulose", J. Pulp and Paper Sci., 2002, 28(7), 217

D. F. Guay, B. J. W. Cole, R. C. Fort, Jr., M. C. Hausman, J. M. Genco, T. J. Elder, and K. R. Overly, "Mechanisms of Oxidative Degradation of Carbohydrates During Oxygen Delignification. II. Reaction of Photochemically Generated Hydroxyl Radicals with Methyl b-D-cellobioside", J. Wood Chem. Technol., 2001, 21(1), 67.

D. F. Guay, B. J. W. Cole, R. C. Fort, Jr., J. M. Genco, and M. C. Hausman, "Mechanisms of Oxidative Degradation of Carbohydrates During Oxygen Delignification. I. Reaction of Photochemically Generated Hydroxyl Radicals with Methyl b-D-glucoside", J. Wood Chem. Technol., 2000, 20(3), 375.