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United States Department of Agriculture

Agricultural Research Service

Research Project: DEVELOPING BIOCONVERSION PROCESSES FOR HIGH-VALUE CARBOHYDRATE PRODUCTS

Location: Renewable Product Technology Research Unit

2007 Annual Report


1a.Objectives (from AD-416)
To develop biocatalytic methods for the conversion of crop derived carbohydrates to high value polysaccharides or oligosaccharides. The project will be composed of two major objectives. .
1)Develop biocatalytic methods for the conversion of starch, corn coproducts, beet sugar, or cane sugar to value-added oligosaccharides. .
2)Develop green chemistry ionic liquid-based methods for the biocatalytic production of value-added oligosaccharides. Their common feature is the use of agriculturally derived carbohydrates for the production of high-value products which utilize some of the structural features of the original carbohydrates.


1b.Approach (from AD-416)
Glycansucrases. Our research in this area will focus on the use of alternansucrase to synthesize oligosaccharides via transfer of glucosyl units from sucrose to mono- or oligo-saccharide acceptors. As alternansucrase is better at catalyzing acceptor reactions than commercial dextransucrase, yielding a better variety of mixed-linkage products, we will first focus on this enzyme. The newly synthesized oligosaccharides are expected to support the growth of specific beneficial microbes in the gastrointestinal tract. We will develop these and other products and determine their structures. The role of oligomer structure and size (degree of polymerization) in fermentability and prebiotic activity will be investigated. This is expected to yield not only new, more strain-specific prebiotics, but will also give rise to a better understanding of the mechanism of prebiotic action. Corn coproducts. This research will utilize abundant, low-value agricultural biomass, particularly corn fiber arising as a coproduct of corn wet milling for production of starch, sweeteners, and ethanol. This is attractive as a model corn residue because it accumulates in enormous volumes in milling facilities and does not need to be collected and transported from fields as do corncobs and stover. Research will also utilize DDGS, an abundant low-value coproduct of dry grind fuel ethanol production. Both corn fiber and DDGS are rich in arabinoxylan, a complex polysaccharide with a backbone of B- (1,4) linked xylose and various side chains and other modifications. Combinations of specific enzymes will be used to cut corn fiber xylan at these linkages to produce a collection of novel oligosaccharides. Transglycosylations in ionic liquids. The approach will be to use commercial enzymes in non-aqueous ionic liquids to produce new prebiotic oligosaccharides and cyclic oligosaccharides that are unattainable in water-based systems. Various ionic liquids, enzymes, and conditions will be tested in order to optimize the non-aqueous transglycosylations. Oligosaccharides produced from this part of the project plan will be tested for prebiotic activity in vitro and in vivo.


4.Accomplishments
MECHANISM OF ACCEPTOR REACTIONS BY ALTERNANSUCRASE. Enzymes such as alternansucrase allow us to convert low-value sugar into value-added carbohydrates for food and feed use, but we need a better understanding of how they work in order to predict and modify their action. We determined the structures of alternansucrase acceptor reactions with maltose from corn for oligosaccharides of up to eight sugar units. These structures give us insight into the reaction mechanism of the enzyme. This allows the prediction of enzyme reaction pathways with other substrates and a foundation for future reaction engineering and enzyme engineering. This accomplishment directly addresses National Program 306 - Quality and Utilization of Agricultural Products (100%), in particular Component 2, New Processes, New Uses, and Value-Added Foods and Biobased Products. Specifically, it relates to Problem Area 2b - New Uses for Agricultural By-products.

SUGARS FROM CORN GERM. Practical new methods are needed to produce fermentable sugars from low-value agricultural residues such as defatted corn germ. We found that defatted germ was efficiently digested without a pretreatment regardless of oil extraction method. This work has potential scientific impact for researchers developing new uses and value-added products from agricultural commodities and byproducts. This accomplishment directly addresses National Program 306 - Quality and Utilization of Agricultural Products (100%), in particular Component 2, New Processes, New Uses, and Value-Added Foods and Biobased Products. Specifically, it relates to Problem Area 2b - New Uses for Agricultural By-products.

IMPROVED METHODS OF TESTING THE POSITIVE EFFECTS OF PREBIOTIC OLIGOSACHARIDES ON HUMAN GUT BIFIDOBACTERIA. The enumeration of the probiotic human gut bacterium Bifidobacterium is difficult, and is currently based on demonstration of "bifid shunt" activity. The "bifid shunt" is a unique Bifidobacterium metabolism for the degradation of complex sugars. We have developed a new mass spectrometry-based assay to demonstrate that the bifid shunt is the predominant pathway for the degradation of carbohydrates by Bifidobacterium. This evidence for bifid shunt usage may provide researchers with new ways to measure the prebiotic potential of oligosaccharides. This accomplishment directly addresses National Program 306 - Quality and Utilization of Agricultural Products (100%), in particular Component 2, New Processes, New Uses, and Value-Added Foods and Biobased Products. Specifically, it relates to Problem Area 2b - New Uses for Agricultural By-products.

DEVELOP METHODS FOR SEPARATION AND ANALYSIS OF OLIGO-SACCHARIDES. Predicting how high-value carbohydrates are metabolized requires detailed knowledge of their chemistry. Carbohydrate products may differ by size, by which sugars are present, and by how they are joined together. Two new methods have been developed: (1) evaluate the number of hydroxyl groups on carbohydrates, giving insight into their size and branching; and (2) develop "locked ring" sugars to improve dye-labeling and immobilization of water-soluble oligosaccharides. This work has potential impact for researchers developing new uses and value-added sugars from agricultural commodities and byproducts. This accomplishment directly addresses National Program 306 - Quality and Utilization of Agricultural Products (100%), in particular Component 2, New Processes, New Uses, and Value-Added Foods and Biobased Products. Specifically, it relates to Problem Area 2b - New Uses for Agricultural By-products.


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

Number of new CRADAs and MTAs1
Number of active CRADAs and MTAs2
Number of invention disclosures submitted1
Number of patent applications filed1
Number of U.S. patents granted2
Number of new commercial licenses granted1
Number of non-peer reviewed presentations and proceedings12
Number of newspaper articles and other presentations for non-science audiences5

Review Publications
Sanz, M., Cote, G.L., Gibson, G., Rastall, R.A. 2006. Influence of glycosidic linkages and molecular weight on the fermentation of maltose-based oligosaccharides by gut bacteria. Journal of Agricultural and Food Chemistry. 54:9779-9784.

Leathers, T.D., Price, N.P. 2007. Effect of oil extraction method on the enzymatic digestibility of corn germ arabinoxylan. Cereal Chemistry. 84(3):243-245.

Skinner-Nemec, K.A., Nichols, N.N., Leathers, T.D. 2007. Biofilm formation by bacterial contaminants of fuel ethanol production. Biotechnology Letters. 29:379-383.

Adeuya, A., Price, N.P. 2007. Enumeration of carbohydrate hydroxyl groups by silylation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Communications in Mass Spectrometry. 21:2095-2100.

Prasongsuk, S., Berhow, M.A., Dunlap, C.A., Weisleder, D., Leathers, T.D., Eveleigh, D.E., Punnapayak, H. 2007. Pullulan production by tropical isolates of Aureobasidium pullulans. Journal of Industrial Microbiology and Biotechnology. 34(1):55-61.

Weimer, P.J., Price, N.P., Kroukamp, O., Joubert, L.M., Wolfaardt, G.M., Van Zyl, W.H. 2006. Studies of the extracellular glycocalyx of the anaerobic ruminal bacterium Ruminococcus albus 7. Applied and Environmental Microbiology. 72(12):7559-7566.

Price, N.P., Momany, F.A., Adeuya, A. 2007. Differential EI fragmentation pathways for peracetylated C-glycoside ketones as a consequence of bicyclic ketal ring structures. Journal of Mass Spectrometry. 43:53-62.

Price, N.P., Rooney, A.P., Swezey, J.L., Perry, E., Cohan, F.M. 2007. Mass spectrometric analysis of lipopeptide from Bacillus strains isolated from diverse geographical locations. Federation of European Microbiological Societies Microbiology Letters. 271(1):83-9.

Price, N.P. 2006. Ionic liquids: the prospects of enzyme-catalyzed glycosylations without water. In: Hou, C.T., Shaw, J-F., editors. Biocatalysis and biotechnology for functional foods and industrial products. 1st edition. New York, NY: CRC Press, Taylors and Francis Group. p. 451-466.

Last Modified: 4/16/2014
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