title: Development of a High Throughput Platform for Screening Glycoside Hydrolases Based on Oxime-NIMS.
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.
authors.affiliation: US Department of Energy Joint BioEnergy Institute , Emeryville, CA , USA ; Sandia National Laboratories , Livermore, CA , USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Deng
authors.affiliation: US Department of Energy Joint BioEnergy Institute , Emeryville, CA , USA ; Sandia National Laboratories , Livermore, CA , USA.
authors.forename: Joel M
authors.initials: JM
authors.lastname: Guenther
authors.affiliation: Lawrence Berkeley National Laboratory , Berkeley, CA , USA.
authors.forename: Jian
authors.initials: J
authors.lastname: Gao
authors.affiliation: Lawrence Berkeley National Laboratory , Berkeley, CA , USA.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Bowen
authors.affiliation: US Department of Energy Joint BioEnergy Institute , Emeryville, CA , USA ; Sandia National Laboratories , Livermore, CA , USA.
authors.forename: Huu
authors.initials: H
authors.lastname: Tran
authors.affiliation: US Department of Energy Joint BioEnergy Institute , Emeryville, CA , USA ; Lawrence Berkeley National Laboratory , Berkeley, CA , USA.
authors.forename: Vimalier
authors.initials: V
authors.lastname: Reyes-Ortiz
authors.affiliation: US Department of Energy Joint BioEnergy Institute , Emeryville, CA , USA ; Lawrence Berkeley National Laboratory , Berkeley, CA , USA.
authors.forename: Xiaoliang
authors.initials: X
authors.lastname: Cheng
authors.affiliation: US Department of Energy Joint BioEnergy Institute , Emeryville, CA , USA ; Lawrence Berkeley National Laboratory , Berkeley, CA , USA.
authors.forename: Noppadon
authors.initials: N
authors.lastname: Sathitsuksanoh
authors.affiliation: US Department of Energy Joint BioEnergy Institute , Emeryville, CA , USA ; Sandia National Laboratories , Livermore, CA , USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Heins
authors.affiliation: US Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin , Madison, WI , USA.
authors.forename: Taichi E
authors.initials: TE
authors.lastname: Takasuka
authors.affiliation: US Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin , Madison, WI , USA.
authors.forename: Lai F
authors.initials: LF
authors.lastname: Bergeman
authors.affiliation: US Department of Energy Joint BioEnergy Institute , Emeryville, CA , USA.
authors.forename: Henrik
authors.initials: H
authors.lastname: Geertz-Hansen
authors.affiliation: Lawrence Berkeley National Laboratory , Berkeley, CA , USA ; Joint Genome Institute , Walnut Creek, CA , USA.
authors.forename: Samuel
authors.initials: S
authors.lastname: Deutsch
authors.affiliation: US Department of Energy Joint BioEnergy Institute , Emeryville, CA , USA ; Lawrence Berkeley National Laboratory , Berkeley, CA , USA.
authors.forename: Dominique
authors.initials: D
authors.lastname: Loqué
authors.affiliation: US Department of Energy Joint BioEnergy Institute , Emeryville, CA , USA ; Sandia National Laboratories , Livermore, CA , USA.
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Sale
authors.affiliation: US Department of Energy Joint BioEnergy Institute , Emeryville, CA , USA ; Sandia National Laboratories , Livermore, CA , USA.
authors.forename: Blake A
authors.initials: BA
authors.lastname: Simmons
authors.affiliation: US Department of Energy Joint BioEnergy Institute , Emeryville, CA , USA ; Lawrence Berkeley National Laboratory , Berkeley, CA , USA ; Department of Bioengineering, University of California Berkeley , Berkeley, CA , USA.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
authors.affiliation: US Department of Energy Joint BioEnergy Institute , Emeryville, CA , USA ; Sandia National Laboratories , Livermore, CA , USA.
authors.forename: Anup K
authors.initials: AK
authors.lastname: Singh
authors.affiliation: US Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin , Madison, WI , USA ; Department of Biochemistry, University of Wisconsin , Madison, WI , USA.
authors.forename: Brian G
authors.initials: BG
authors.lastname: Fox
authors.affiliation: US Department of Energy Joint BioEnergy Institute , Emeryville, CA , USA ; Lawrence Berkeley National Laboratory , Berkeley, CA , USA.
authors.forename: Trent R
authors.initials: TR
authors.lastname: Northen
keywords: NIMS; cellulase; enzyme assays; high throughput screening; oxime bioconjugation