Monday, May 4, 2009
5-09
Microfluidic Glycan Assays for Cellulosic Biomass
Rajiv Bharadwaj1, Kevin Chen2, Supratim Datta2, Brad M. Holmes2, Rajat Sapra3, and Anup Singh4. (1) Technology Division, Joint BioEnergy Institute, Physical Biosciences Division, Lawrence Berkeley National Laboratory, 5885 Hollis Street, Fourth Floor, Emeryville, CA 94608, (2) Deconstruction Division, Joint BioEnergy Institute, 5885 Hollis Street, Fourth Floor, Emeryville, CA 94608, (3) Energy Systems, Sandia National Laboratories, 7011 East Avenue, Livermore, CA 94551, (4) Technology Division, Joint Bioenergy Institute, 5885 Hollis Street, Emeryville, CA 94608
High-throughput, rapid, and sensitive approaches for quantitation of carbohydrates are valuable for bioenergy research in the areas of feedstock development and biomass deconstruction. Conventional assays such as Azo-CMC and the DNS assay are high-throughput but don't provide information about the glycans. HPLC-based assays provide precise information about the oligosaccharide content but the technique is low throughput. We are developing microfluidic capillary electrophoresis (μCE) devices for rapid, precise, and high-throughput characterization of glycans for enzyme screening.
We have used μCE to characterize Thermotoga maritima endoglucanase for degradation of cellulosic materials. The enzyme assays were performed on soluble substrates (cellodextrins and CMC) as well as ionic-liquid (IL) pretreated avicel and switchgrass. In μCE, the hydrolyzed glycans are tagged with a charged fluorophore at reducing ends. This enables separation based on charge-to-mass ratio as well as high-sensitivity fluorescence detection. The run-time per sample is around 60 s (> 10X faster than HPLC) and the detection sensitivity is around 1 amol. The cellulase effectively hydrolyzes cellotetrose and higher oligosaccharides. The major hydrolysis products are cellobiose, cellotriose along with small amounts of glucose. Further, results demonstrate μCE is particularly suitable for quantitative analysis of IL pretreated samples since no signal interference, arising from ILs, was observed. Whereas, the HPLC/ELSD data shows a significant overlap between the ionic-liquid and glucose peaks. We are extending the μCE technique to measure enzyme kinetics of various glycosyl hydrolases as well as glycosyltransferases. In addition, we are developing a high-throughput μCE system that integrates seamlessly with the microtiter plate assays.
We have used μCE to characterize Thermotoga maritima endoglucanase for degradation of cellulosic materials. The enzyme assays were performed on soluble substrates (cellodextrins and CMC) as well as ionic-liquid (IL) pretreated avicel and switchgrass. In μCE, the hydrolyzed glycans are tagged with a charged fluorophore at reducing ends. This enables separation based on charge-to-mass ratio as well as high-sensitivity fluorescence detection. The run-time per sample is around 60 s (> 10X faster than HPLC) and the detection sensitivity is around 1 amol. The cellulase effectively hydrolyzes cellotetrose and higher oligosaccharides. The major hydrolysis products are cellobiose, cellotriose along with small amounts of glucose. Further, results demonstrate μCE is particularly suitable for quantitative analysis of IL pretreated samples since no signal interference, arising from ILs, was observed. Whereas, the HPLC/ELSD data shows a significant overlap between the ionic-liquid and glucose peaks. We are extending the μCE technique to measure enzyme kinetics of various glycosyl hydrolases as well as glycosyltransferases. In addition, we are developing a high-throughput μCE system that integrates seamlessly with the microtiter plate assays.
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