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Abstract
Cost-effective hydrolysis of biomass into sugars for biofuel production requires high-performance low-cost glycoside hydrolase (GH) cocktails that are active under demanding process conditions. Improving the performance of GH cocktails depends on knowledge of many critical parameters, including individual enzyme stabilities, optimal reaction conditions, kinetics, and specificity of reaction. With this information, rate- and/or yield-limiting reactions can be potentially improved through substitution, synergistic complementation, or protein engineering. Given the wide range of substrates and methods used for GH characterization, it is difficult to compare results across a myriad of approaches to identify high performance and synergistic combinations of enzymes. Here, we describe a platform for systematic screening of GH activities using automatic biomass handling, bioconjugate chemistry, robotic liquid handling, and nanostructure-initiator mass spectrometry (NIMS). Twelve well-characterized substrates spanning the types of glycosidic linkages found in plant cell walls are included in the experimental workflow. To test the application of this platform and substrate panel, we studied the reactivity of three engineered cellulases and their synergy of combination across a range of reaction conditions and enzyme concentrations. We anticipate that large-scale screening using the standardized platform and substrates will generate critical datasets to enable direct comparison of enzyme activities for cocktail design.
Highlights
Lignocellulosic biomass (Carroll and Somerville, 2009) is a renewable source of energy, capable of providing the nation with clean, renewable transportation fuels
To address some of these limitations, we developed a mass spectrometry-based enzyme assay platform called Nimzyme (Northen et al, 2008; Reindl et al, 2011; Greving et al, 2012)
We developed an oxime-Nimzyme probe (Deng et al, 2014) to directly study enzyme hydrolysis of plant biomass
Summary
Lignocellulosic biomass (Carroll and Somerville, 2009) is a renewable source of energy, capable of providing the nation with clean, renewable transportation fuels. There are many sources of inedible plant biomass that can serve as feedstocks for biofuel production, including agricultural wastes [corn stover, switchgrass (SG), wood trimming], municipal solid wastes, and emerging bioenergy crops. These various feedstocks have different glycan composition, bond linkages, and individual sugar contents, which complicates the development of cost-effective saccharification approaches. The content and structure of glycans and lignin from the same biomass may respond in different ways to the prerequisite pretreatments (Li et al, 2010) These variations contribute to the observation that there is no universal enzyme or enzyme cocktail for all substrates and biofuels processes
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