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Poster Presentation 2-10
Development of Automated Enzyme Assays for Analysis of Pentose Utilization Enzymes in Cellular Extracts
Christina Eddy1, Steven Decker1, Qiang Gao2, Dhinakar S. Kompala2, James D. McMillan1, Min Zhang1
1Biotechnology Division for Fuels and Chemicals, National Renewable Energy Laboratory, 1617 Cole Blvd., Golden, CO 80401
2Department of Chemical Engineering, University of Colorado, Boulder, CO 80309-0424
Telephone: (303) 384-7768; Fax: (303) 384-7752; E-mail: Christina_eddy@nrel.gov
Metabolic engineering and strain development rely on the analysis of enzymes and metabolites to guide the design of new experiments and strains. Many of the enzymes required for fermentation of hexose and pentose sugars to ethanol are assayed by monitoring the rate of disappearance or appearance of NADH. We have developed an automated kinetic assay method, which uses a Zymark SCICLONE Liquid Handling System, a Mitsubishi robotic arm, and a SpectraMax microplate reader from Molecular Devices. The method was tested by measuring the activities of enzymes required for pentose utilization, including transaldolase, transketolase and xylose isomerase, in cellular extracts prepared from Zymomonas mobilis and Saccharomyces cerevisiae. Results are comparable to those achieved using a manual method, but higher productivity and reduced variability can be achieved. The automated method is adaptable for measurement of any enzyme or metabolite that can be monitored by the oxidation of NADH or reduction of NAD.
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