(719d) High Throughput Multiplexed GlycoChip Enzymatic Assays for Biofuels Development

Authors: 
Northen, T., Joint BioEnergy Institute
Reindl, W., Joint BioEnergy Institute
Deng, K., Joint BioEnergy Institute
Gladden, J., Joint BioEnergy Institute
Singer, S., Joint BioEnergy Institute
Singh, A., Joint BioEnergy Institute
Simmons, B., Joint BioEnergy Institute
Adams, P., Joint BioEnergy Institute
Hazen, T., Joint BioEnergy Institute
Keasling, J. D., Technical University of Denmark


Critical to the development of lignocellulosic biofuel processes is the efficient deconstruction of this highly recalcitrant material into fermentable sugars. At JBEI this is being approached by both optimizing the feedstock and the deconstruction processes. In both cases, new technologies are required for identification of unknown activities (e.g. glucosyltransferase) and the high throughput screening of ?targeted' reactions i.e. glycosylhydrolase. The high sensitivity and resolution of mass spectrometry make it well suited for both targeted and untargeted analysis. However, the low throughput of conventional GC/MS and LC/MS precludes implementation for screening purposes. To overcome this, we have developed GlycoChip enzyme assays based on Nanostructure-Initiator Mass Spectrometry (NIMS) ionization approach. In this ?Nimzyme' assay the enzyme substrates are immobilized on the mass spectrometry surface using fluorous phase interactions. This ?soft' immobilization allows efficient desorption/ionization while also allowing surface washing to reduce signal suppression from complex biological samples as a result of the preferential retention of the tagged products and reactants.

A multiplexed Nimzyme assay for simultaneous detection and characterization of beta-glucosidases and beta-xylanase activities has been developed based on substrates carrying a fluorous tag. The assay can be used to screen for the optimal pH and temperature of an enzyme or to monitor an enzymatic reaction over time. Additionally our Nimzyme assay is stable in the presence of even high concentrations of ionic liquid. In future work this assay will be transferred to an automated platform to allow for high throughput functional analysis of both enzyme libraries and environmental samples. Together these assays will help to identify and optimize the conversion of lignocellulose into biofuels.

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