2007 Annual Report
DISCOVERY AND ISOLATION OF A GENE ENCODING FOR A NOVEL ENZYME ACTIVITY FOR BIOMASS DEGRADATION FROM TRICHODERMA REESEI. New enzymes are needed for breaking down plant cell walls for conversion to ethanol because current enzyme blends are not always effective. The fungus T. reesei is the most common source for commercial cellulases, and novel genes related to biomass conversion are extremely important for further improvement of industrial cellulases. The enzyme discovered is a glucuronic acid esterase; glucuronic acids are present in a wide variety of biomasses and an ability to remove them is expected to directly lower yields by interfering with the release of neutral sugars. This work applies directly to National Program 307, Bioenergy and Energy Alternatives, Component I, Ethanol, because it relates to conversion of biomass into ethanol.
THE BIFUNCTIONAL BETA-D-XYLOSIDASE/ALPHA-L-ARABINOFURANOSIDASE FROM SELENOMONAS RUMINANTIUM IS THE BEST CATALYST KNOWN (KCAT, KCAT/KM) FOR PROMOTING HYDROLYSIS OF 1,4-BETA-D-XYLOOLIGOSACCHARIDES, AND IT HAS POTENTIAL FOR USE IN SACCHARIFICATION PROCESSES. Highly active biomass conversion enzymes are required for economic and efficient saccharification of agricultural biomass. Active-site amino acid residues that are involved in substrate distortion and increasing enzyme activity have been identified. As well, active-site amino acid residues that are involved in preferring xylose glycosides over arabinose glycosides have been identified. Enzyme-inhibitor complexes that could be formed in saccharification processes have been demonstrated. This work applies directly to National Program 307, Bioenergy and Energy Alternatives, Component I, Ethanol, because it relates to conversion of biomass into ethanol.
IMPROVING FERMENTABILITY OF BIOMASS SUGARS. Inhibitors arising during conversion of biomass to sugars are impediments to fermentative production of ethanol. Over three dozen chemical by-products were detected in hydrolyzed corn and evaluated for removal by a biological treatment system. The fermentability of hydrolysates was improved by biological abatement, due to removal of a number of inhibitory compounds present in acid-pretreated corn stover. This work is being expanded to include an alkaline-pretreated feedstock, which has a different array of inhibitory compounds. The inhibitor abatement methods remove side-products of pretreatment and can greatly enhance the yield and rate of production for biofuels. This work applies directly to National Program 307, Bioenergy and Energy Alternatives, Component I, Ethanol, because it relates to conversion of biomass into ethanol.
ISOLATION OF HYDROLYTIC ENZYMES FROM RHIZOPUS ORYZAE SPECIES. The filamentous fungus Rhizopus oryzae contains a large and diverse complement of biomass degrading enzymes that are of interest for improving the efficacy and efficiency of biomass degradation. Three unique previously unidentified glucoamylase genes (glucoamylase aids in the breakdown of starch) from two different Rhizopus strains were isolated, expressed, and initial biochemical characterization performed. Two of the enzymes were highly active, while the third enzyme is inactive, even though it is a truncated version of a previously isolated enzyme. Indepth biochemical characterization of the active enzymes is in progress with an eye toward the potential use of these enzymes as processing aids in biomass degradation. This work applies directly to National Program 307, Bioenergy and Energy Alternatives, Component I, Ethanol, because it relates to conversion of biomass into ethanol.
Dien, B.S., Jung, H.G., Vogel, K.P., Casler, M.D., Lamb, J.F., Iten, L.B., Mitchell, R., Sarath, G. 2006. Chemical composition and response to dilute-acid pretreatment and enzymatic saccharification of alfalfa, reed canarygrass, and switchgrass. Biomass and Bioenergy. 30:880-891.
Dien, B.S., Li, X., Iten, L.B., Jordan, D.B., Nichols, N.N., O Bryan, P.J., Cotta, M.A. 2006. Enzymatic saccharification of hot-water pretreated corn fiber for production of monosaccharides. Enzyme and Microbial Technology. 39:1137-1144.
Mertens, J.A., Skory, C.D. 2007. Isolation and characterization of a second glucoamylase gene without a starch binding domain from Rhizopus oryzae. Enzyme and Microbial Technology. 40:874-880.
Bischoff, K.M., Rooney, A.P., Li, X., Liu, S., Hughes, S.R. 2006. Purification and characterization of a family 5 endoglucanase from a moderately thermophilic strain of Bacillus licheniformis. Biotechnology Letters. 28:1761-1765.
Jordan, D.B., Li, X.-L., Dunlap, C.A., Whitehead, T.R., Cotta, M.A. 2007. Structure-function relationships of a catalytically efficient beta-D-xylosidase. Applied Biochemistry and Biotechnology. 141:51-76. Jordan, D.B., Li, X., Dunlap, C.A., Whitehead, T.R., Cotta, M.A. 2007. Beta-D-xylosidase from Selenomonas ruminantium of glycoside hydrolase family 43. Applied Biochemistry and Biotechnology. 136-140:93-104.
Lopez, M.J., Vargas-Garcia, M., Suarez-Estrella, F., Nichols, N.N., Dien, B.S., Moreno, J. 2007. Lignocellulose-degrading enzymes produced by the ascomycete Coniochaeta ligniaria and related species: application for a lignocellulosic substrate treatment. Enzyme and Microbial Technology. 40:794-800.
Li, X., Skory, C.D., Ximenes, E.A., Jordan, D.B., Dien, B.S., Hughes, S.R., Cotta, M.A. 2007. Expression of an AT-rich xylanase gene from the anaerobic fungus Orpinomyces sp. strain PC-2 in and secretion of the heterologous enzyme by Hypocrea jecorina. Applied Microbiology and Biotechnology. 74:1264-1275.
Ximenes, E., Dien, B.S., Ladisch, M.R., Mosier, N., Cotta, M.A., Li, X. 2007. Enzyme production by industrially relevant fungi cultured on coproduct from corn dry grind ethanol plants. Applied Biochemistry and Biotechnology. 136-140:171-183.
Mertens, J.A., Skory, C.D. 2007. Isolation and characterization of two genes that encode active glucoamylase without a starch binding domain from Rhizopus oryzae. Current Microbiology. 54:462-466.
Chen, H., Hopper, S.L., Li, X., Ljungdahl, L.G., Cerniglia, C.E. 2006. Isolation of extremely AT-rich genomic DNA and analysis of genes encoding carbohydrate-degrading enzymes from Orpinomyces sp. strain PC-2. Current Microbiology. 53:396-400.
Chen, H., Li, X., Xu, H., Ljungdahl, L.G., Cerniglia, C.E. 2006. High level expression and characterization of the cyclophilin B gene from the anaerobic fungus Orpinomyces sp. strain PC-2. Protein and Peptide Letters. 13:727-732.
Hughes, S.R., Dowd, P.F., Hector, R.E., Riedmuller, S.B., Bartolett, S., Mertens, J.A., Qureshi, N., Liu, S., Bischoff, K.M., Li, X., Jackson Jr, J.S., Sterner, D., Panavas, T., Cotta, M.A., Farrelly, P.J., Butt, T. 2007. High-throughput fully automated construction of a multiplex library of mutagenized open reading frames for an insecticidal peptide using a plasmid-based functional proteomic robotic workcell with improved vacuum system. Journal of Laboratory Automation. 12(4):202-212.