Author
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Wagschal, Kurt |
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Lee, Charles |
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Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 1/12/2012 Publication Date: 1/20/2012 Citation: Wagschal, K.C., Lee, C.C. 2012. Microplate-based active/inactive 1 screen for biomass degrading enzyme library purification and gene discovery. Analytical Biochemistry. 89: 83-85. Interpretive Summary: This work describes an active/inactive microplate screen for enzyme activities which are critical for the enzymatic deconstruction of biomass for fuels and chemicals. Enzymes that occur naturally in the environment, or enzymes engineered in the laboratory are screened using fluorophore-tagged substrates directly using a minimum of specialized equipment and supplies. The method is useful for both enzyme engineering and gene discovery for these and other enzyme activities for which suitable fluorophore-tagged substrates exist. Technical Abstract: We present here a whole-cell and permeabilized E. coli cell 1' active/inactive microplate screen for ß-D-xylosidase, xylanase, endocellulase, and ferulic acid esterase enzyme activities which are critical for the enzymatic deconstruction of biomass for fuels and chemicals. Transformants from genomic or mutagenesis-derived libraries are screened using fluorophore-tagged substrate/enzyme activity pairs that are assayed directly in the protein expression host growth media using a minimum of specialized equipment and supplies. The method is useful for mutagenized enzyme library purification and gene discovery for these and other enzyme activities for which suitable fluorophore-tagged substrates exist.Our method for enzyme library purification has enabled more wide-spread enzyme engineering of enzymes involved in biomass breakdown, since the method does not require expensive liquid handling equipment and uses minimal equipment. |
