Enzymology Group
Enzymes are the workhorse catalysts that are involved in every aspect of bioenergy research - they catalyze the synthesis of cellulosic polymers in plants, break down the biomass into simple sugars that can be fermented, and convert sugars into ethanol and other fuels. Consequently, we have made significant investment at JBEI in understanding how these enzymes and enzyme complexes work and how we can improve them to be more efficient, robust, and tolerant of adverse processing conditions. Advances in genetic engineering and synthetic biology make it possible to make genes for hundreds and thousands of these enzymes and enzyme-variants relatively easily. However, high-throughput platforms do not exist for rapidly expressing and screening these enzymes.
Our goal is to develop high-throughput and high-content assay platforms for characterizing enzymes, their substrates, and products. We do so by utilizing state-of-the-art microfluidic and microarray chips that can be interfaced with fluorescence detectors or mass spectrometers for rapid and ultrasensitive detection. Critical to many of these assays is the availability of defined substrates. We have developed a pipeline for synthesizing native and modified oligosaccahrides that can be used with our microfluidic and microarray platforms to measure activities accurately and specifically.
A key criterion motivating our work is that the assays and platforms we develop be robust and easily transferrable to a "customer" such as a scientist in another division at JBEI, another bioenergy center, or a company. The following sections describe our technologies and how they are enabling bioenergy research.
Microfluidic Technologies for Biomass-to-Biofuels Conversion
Figure 1. High throughput screening of lignocellulolytic enzymes. (A) Sub-mg metering of ionic liquid pretreated biomass (Avicel: top & bottom rows, switchgrass: middle row) in 96-well plate using in-situ regeneration. (B) Photograph of the microfluidic device used for rapid, high-resolution, and high-sensitivity analysis of enzymatic hydrolysis of biomass. The channel depth and width are 20 μm and 70 μm, respectively. (C) Microfluidic measurement of the amount of glucose, cellobiose, and cellotriose in switchgrass hydrolysis using thermophilic cellulase cel5A. The analysis is completed in less than a minute and the detection sensitivity is ~ 1 amol.
Our overarching goal is to develop crosscutting microscale technologies for advancing biofuels development. The driving theme is to leverage novel microscale physics and chemistry to invent faster (higher throughput), better (higher sensitivity & reliability), and cheaper (lower reagent usage) biochemical assays. The hydrolysis of biomass to fermentable sugars using glycosyl hydrolases (such as cellulases and hemicellulases) is a limiting and costly step in the conversion of biomass to biofuels. Enhancement in hydrolysis efficiency is necessary and requires improvement both in enzymes and processing strategies. Advances in both areas in turn strongly depend on the progress in developing high-throughput (HTP) assays to rapidly and quantitatively screen large number of enzymes and processing conditions.
We have developed novel microscale platforms to address the throughput limitations of conventional technologies. By performing in-situ biomass regeneration in micro-volumes, we have developed a HTP approach for volumetric metering of biomass at sub-mg loading.1 In addition, we have developed a microfluidic device for rapid, precise, and high-throughput screening of lignocellulolytic enzymes and for characterization of biomass.2 Our microfluidic devices and assays have vastly superior performance compared to conventional approaches such as HPLC and colorimetric assays. For example, the run-time per sample is around 60 s (> 10X faster than HPLC) and the detection sensitivity is around 1 amol (100X smaller reagent volume than HPLC). These performance characteristics are critical for several high throughput-screening projects at JBEI. For example, we have used microfluidic electrophoresis to rapidly characterize Thermotoga maritima endoglucanase for the degradation of ionic-liquid (IL) pretreated switchgrass.
References:
1. Bharadwaj, R., A. Wong, A. et al. (2010). "High-Throughput Enzymatic Hydrolysis of Lignocellulosic Biomass via In-Situ Regeneration." Bioresource Technology, In press August 2010.
2. Bharadwaj, R., Z. Chen, et al. (2010). "Microfluidic Glycosyl Hydrolase Screening for Biomass-to-Biofuel Conversion." Analytical Chemistry 82(22): 9513-9520. (Featured cover article, see below)
Nanostructure-Initiator Mass Spectrometry (NIMS)
NIMS is being developed at JBEI for a number of high-throughput screening applications with the goal of enabling the development of low-cost biomass derived liquid fuels. The majority of efforts have focused on enzyme assays for discovering and developing high performance enzymes for biomass deconstruction into fermentable sugars. These GlycoChip technologies take advantage of the highly non-polar (lipophilic) surface of the NIMS chips to array glycan substates containing hydrophobic tags. This allows the substrate and products containing this hydrophobic tag to be selectively retained on the surface, allowing the efficient removal of cellular or environmental materials that would interfere with the subsequent NIMS analysis.
The same NIMS technology is being used to screen microbial lysates for targeted molecular production. For example, molecules containing long hydrocarbon tails are both of great interest as biofuel molecules and bind to the NIMS chip for high throughput analysis. Using this approach, JBEI is developing methods for screening libraries to optimize biofuel production.
Microscale Platform for Cell-Free Cellulase Expression and Screening
Cellulases play an important role in the bioconversion of lignocellulosic biomass to liquid biofuels by hydrolyzing the cellulose polymers in biomass to fermentable sugars, which are subsequently converted to ethanol by microorganisms. This breakdown of cellulose to glucose is achieved by enzymatic activity of three groups of cellulase enzymes: endoglucanases, cellobiohydrolases and β-glucosidases. In order to reduce the costs associated with the enzymatic hydrolysis process, several research efforts have been dedicated to the identification and generation of improved cellulases with industrially desired properties. Such cellulase engineering efforts generate large DNA libraries that need to be expressed and screened. Therefore, there is a critical need to develop high-throughput analytical approaches for rapid expression and activity screening of cellulases.
To address this need, we have developed a microscale platform, based on cell-free protein expression, which seamlessly integrates cellulase expression and activity screening without the need for any protein purification procedures. Starting from cellulase DNA templates, our approach achieves transcription, translation, and activity screening of generated cellulases within 2-3 hours. Furthermore, in our platform, expression and screening is achieved in 2-3 μL volumes, thereby significantly reducing reagent usage and costs. Most importantly, our platform is compatible with a wide temperature range and enables screening even at elevated temperatures (up to 95°C) with minimal evaporation. Therefore, this microscale approach is ideally suited for rapid, first-pass activity screening of large libraries of cellulase enzymes from thermophilic microorganisms.
For more information, please see the following publication:
- Chandrasekaran, A., R. Bharadwaj, et al. (2010). "A Microscale Platform for Integrated Cell-Free Expression and Activity Screening of Cellulases." Journal of Proteome Research 9(11): 5677-5683.
High-Throughput Microtiter Plate-Based Assays For Lignins
In addition to developing miniaturized chips described above, we also use readily-available high-throughput platforms such as microtiter plates to develop assays for bioenergy research. These assays are developed with two goals: (1) rapid turnaround by using commercially available components; and, (2) ready adaptation to existing robotic instrumentation. We have developed high throughput screening (HTS) microplate assays for lignin compositional analyses, analyses of lignin carbohydrate complex (LCC), lignin modifying enzymes (LMEs), and redox shuttle mediators (RSM). We developed an absorbance-based assay for coniferyl alchol, a major monolignol of primary, S1 and S2 cell walls, exploiting hitherto unrecognized spectroscopic properties. We also developed fluorescence-based assays for quantifying laccases that have been adapted to a high-throughput pipeline for screening mutant libraries of laccases.
The following papers describe these assays:
- Achyuthan, K. E., J. L. McClain, et al. (2009). "Orthogonal, Spectroscopic High Throughput Screening of Laccase-Catalyzed p-Cresol Oxidation." Combinatorial Chemistry & High Throughput Screening 12(7): 678-689.
- J.C. Harper et al, ACS Appl. Mater. Interfac., 1: 1591-1598, 2009.
- Achyuthan, K. E., P. D. Adams, et al. (2009). "Spectroscopic Analyses of the Biofuels-Critical Phytochemical Coniferyl Alcohol and Its Enzyme-Catalyzed Oxidation Products." Molecules 14(11): 4758-4778.
- Achyuthan, K. E., P. D. Adams, et al. (2010). "Hitherto Unrecognized Fluorescence Properties of Coniferyl Alcohol." Molecules 15(3): 1645-1667.
- K.E. Achyuthan et al, Molecules, 2010 (invited review).
People
- Anup Singh, Director
- Rajiv Bharadwaj, Deputy Director
- Trent Northen, Research Scientist
- Kai Deng, Research Scientist
- Aarthi Chandrasekaran, Postdoctoral Scientist
- Chieh (Jay) Chang, Postdoctoral Scientist
- April Wong, Research Associate
In the News
- Trent Northen, Research Scientist in the Enzymology Group at JBEI wins the prestigious PECASE award
- Enzymology group paper featured on the cover of Analytical Chemistry.
- Sandia Lab News features microfluidic work being done at JBEI.
