High throughput Enzyme activity screening
Developing cost effective methods for deconstructing plant biomass into fermentable sugars is critical to viable cellulosic biofuels. Unfortunately, the potential combinations of plant feedstocks, pre-treatment options and hydrolytic enzymes greatly complicate the efficient and effective determination of optimal deconstruction strategies. To address this challenge we have developed a nanoliter-scale acoustic sample deposition and nanostructure-initiator mass spectrometry (NIMS) analysis platform to rapidly detect and characterize glyco- and lignolytic enzyme activities and substrate specificities. We are applying this approach for analysis of in vitro and cell based expression systems to quickly screen large libraries of enzymes against a wide range of substrates including native plant biomasses. This effort will serve as the foundation in the development of this new technology that will have several applications, including enzyme “cocktail” engineering for enhanced performance in industrially relevant biorefining operating environments for the production of sugars from biomass.
High throughput mass spectrometry based metabolite screening
Metabolite profiling using mass spectrometry provides an attractive approach for the interrogation of microbial metabolic capabilities to discover and optimize microbial enzymes and for the production of biofuels. A challenge to this approach is the disconnect between the rate of clone production vs. specific functional analysis. Typically, pooled assays or selections are required to down select libraries prior to chemically specific analysis using mass spectrometry. Our group is now extending the acoustic printing NIMS screening platform for direct analysis of important classes of metabolites for biofuel production. This will enable a direct read-out to support the development of high performance microbes for cost effective fuel production.
Learn more about Trent Northen’s research here: www.northenlab.org
For more information, visit High Throughput Biochemistry at JBEI.
- Utilize MS Chip based assays to support the development of high performance lignocellulolytic enzymes (cellulases, hemicellulases and lignanases) screened across a matrix of conditions (T, pH, IL stability) to understand the protein features that impart ionic liquid tolerance
- Develop chip based assays to perform high throughput biofuel and bioproduct production
JBEI’s Director of Array-based Assays, Trent Northen, has been recognized with an R&D 100 Award as the co-inventor of the Nanostructure-Initiator Mass Spectrometry (NIMS) technology.
NIMS is a highly sensitive method used to study metabolites and the products of enzymatic reactions in biological systems.
HT-NIMS screening, co-developed at JBEI, is a high-speed mass spectrometry technology that researchers can use to discover the function of large numbers of biologically active molecules. At speeds 100 times faster than that of conventional probes, it can rapidly screen tens of thousands of enzymatic biomass deconstruction reactions that could be used to turn grass into biofuel.
A workhorse of biotechnology, mass spectrometry (MS) offers unparalleled accuracy, but its potential as a screening tool has been limited by its slow throughput. HT-NIMS dramatically speeds up the process.
Tiny samples are deposited in rows and columns on a slide of silicon created a microarray of as many as 10,000 discrete sites. Each site is shot with a laser and analyzed in a split second. In the time it takes conventional MS to characterize one sample, HT-NIMS can profile hundreds, cost-effectively.
HT-NIMS is being used at JBEI to screen for enzymes that can be used to modify lignocellulose for the production of advanced biofuels that could replace gasoline on a gallon-for-gallon basis. MORE>
- “A high-throughput mass spectrometric enzyme activity assay enabling the discovery of cytochrome P450 biocatalysts,”Angew Chem Int Ed Engl. (2019)
- “Lessons from two Design-Build-Test-Learn cycles of dodecanol production in Escherichia coli aided by machine learning,” ACS Synth Biol. (2019)
- “Rapid Characterization of the Activities of Lignin-Modifying Enzymes Based on Nanostructure-Initiator Mass Spectrometry (NIMS),” Biotechnology for Biofuels (2018)
- “Determination of glycoside hydrolase specificities during hydrolysis of plant cell walls using glycome profiling,” Biotechnology for Biofuels (2017)
- “Comprehensive in Vitro Analysis of Acyltransferase Domain Exchanges in Modular Polyketide Synthases and Its Application for Short-Chain Ketone Production”, ACS Synth. Biol. (2016)
- “Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis”, mBio (2016)
- “Substrate Binding Effects of Carbohydrate Binding Modules on the Catalytic Activity of a Multifunctional Cellulase”, Biophysical Journal (2015)
- “High-Throughput Platforms for Metabolomics”, Curr Op in Chemical Biology (2015)
- “Use of Nanostructure-Initiator Mass Spectrometry to Deduce Selectivity of Reaction in Glycoside Hydrolases”, Frontiers in Bioengineering and Biotechnology (2015)
- “Development of a High Throughput Platform for Screening Glycoside Hydrolases Based on Oxime-NIMS”, Frontiers in Bioengineering and Biotechnology (2015)
- “Rapid Kinetic Characterization of Glycosyl hydrolases (GHs) based on Oxime Derivatization and Nanostructure-Initiator Mass Spectrometry (NIMS)”, ACS Chemical Biology (2014)
- “Phylogenomic Guided Identification of Industrially Relevant GH1 ?-Glucosidases Through Coupled DNA Synthesis and Nanostructure-Initiator Mass Spectrometry”, ACS Chemical Biology (2014)
- “Encoding substrates with mass tags to resolve stereospecific reactions using Nimzyme”, RCM (2012)
- “Acoustic deposition with NIMS as a high-throughput enzyme activity assay”, Analytical and Bioanalytical Chemistry (2012)
- “Colloid-based multiplexed screening for plant biomass-degrading glycoside hydrolase activities in microbial communities.”, Energy & Environmental Science (2011)