Enzyme Cascade with Horseradish Peroxidase Readout for High-Throughput Screening and Engineering of Human Arginase-1

Abstract

We report an enzyme cascade with horseradish peroxidase-based readout for screening human arginase-1 (hArg1) activity. We combined the four enzymes hArg1, ornithine decarboxylase, putrescine oxidase, and horseradish peroxidase in a reaction cascade that generated colorimetric or fluorescent signals in response to hArg1 activity and used this cascade to assay wild-type and variant hArg1 sequences as soluble enzymes and displayed on the surface of Escherichia coli. We screened a curated 13-member hArg1 library covering mutations that modified the electrostatic environment surrounding catalytic residues D128 and H141, and identified the R21E variant with a 13% enhanced catalytic turnover rate compared to wild type. Our scalable one-pot single-step arginase assay with continuous kinetic readout is amenable to high-throughput screening and directed evolution of arginase libraries and testing drug candidates for arginase inhibition.

Understanding activity-stability tradeoffs in biocatalysts by enzyme proximity sequencing

“…Our workflow is compatible with enzymes that can be functionally displayed on the cell surface and whose activity can be directly or indirectly (via enzymatic cascade) linked to the production of peroxide. This includes enzymes with immediate therapeutic relevance such as Arginase 64 , and Asparaginase (Supplementary Fig. 9 ), as well as biocatalysts with diagnostic or industrial applications, like Glucose Oxidase 28 .…”