Enzyme engineering is an important biotechnological process capable of generating tailored biocatalysts for applications in industrial chemical conversion and biopharma. Typical enhancements sought in enzyme engineering and in vitro evolution...

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.