Agar plate assay for rapid screening of aryl-alcohol oxidase mutant libraries in Pichia pastoris

Directed evolution is a powerful tool for developing biocatalysts with tailor-made properties for biocatalytic applications. High-throughput activity screening of large mutant libraries generated by e.g. means of directed evolution is usually performed in 96-well microtiter plates requiring, however, time-consuming and laborious enzyme expression, cell harvesting and activity measurements. In addition, automated liquid handling systems are needed to cope with the high number of colonies to be screened. Herein, we developed an agar plate-based assay for simple and fast screening of H2O2-producing aryl-alcohol oxidase (AAO) mutant libraries in Pichia pastoris. Buffered minimal methanol agar plates were supplemented with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), horseradish peroxidase (HRP) and the target substrate. AAO activity is visualized by formation of green zones around AAO-secreting P. pastoris colonies due to ABTS oxidation by HRP which is fueled with H2O2 by AAO in course of substrate oxidation. Colonies were screened within 24 h for AAO activity using different AAO substrates like veratryl alcohol, p-anisyl alcohol or trans,trans-2,4-hexadien-1-ol and even low AAO activity towards 5-hydroxymethylfurfural could be detected within 48 h. The developed agar plate-based assay can be extended to other substrates and might also be applied for fast and substrate-specific screening of other H2O2-producing oxidases in P. pastoris.