Solid-phase agar plate assay for screening amine transaminases

Abstract

Agar plate assays represent a useful method for high-throughput prescreening of larger enzyme libraries derived from for example error-prone PCR or multiple site-saturation mutagenesis to decrease screening effort by separating promising variants from less active, inactive, or neutral variants. In order to do so, colonies are directly applied for enzyme expression and screening on adsorbent and microporous membranes instead of elaborately preparing cell lysates in 96-well plates. This way, 400–800 enzyme variants can be prescreened on a single membrane, 10,000–20,000 variants per week and per single researcher respectively (25 membranes per week). The following chapter gives a detailed protocol of how to screen transaminase libraries in solid phase, but it also intends to provide inspiration to establish a direct or coupled agar plate assay for screening variable enzymatic activities by interchanging assay enzymes and adapting assay conditions to individual needs.