High-throughput Assay Development for Glycosyl Hydrolases Based on Nanostructure Initiator Mass Spectrometry (NIMS)

Cost effective conversion of biomass into sugars for biofuel production requires high-performance low-cost enzyme cocktails that are active under demanding process conditions.  Development of the requisite enzymes requires large-scale determination of both enzyme kinetics and specificity both for enzyme selection and to identify rate and yield limiting reactions that can be improved through synergistic complementation in enzyme cocktails.

 In this talk, I am going to describe our first and second generation of high throughput screening (HTS) platforms for glycosyl hydrolysases (GHs) activities determination using model substrates and bioconjugate chemistry, respectively.  Acoustic printing and nanostructure-initiator mass spectrometry are the key approaches to increase the throughput.  The critical role of synthetic organic chemistry in these assay development will be highlighted.

 The first generation high throughput NIMS (model substrates approach) is used to study the activities a GH1 library of 175 enzymes that captured maximal diversity within this protein family. In total, over 10,000 data points were generated to reveal a large functional diversity within this enzyme group. Several enzymes suitable for a targeted industrial application have been identified from this screening effort.

The second generation high throughput method can utilize a variety of substrates (including solid biomass) since a unique chemical probe was designed and synthesized and the concentration of glycan products can be quantified after enzymatic hydrolysis reactions. The sugar analysis is based on the oxime formation between the chemical probe and aldehyde (reducing ends of glycans) groups and quantitation versus ¹³C-labeled internal standards.