Abstract

Transaminase from Vibrio fluvialis (Vfat) is a suitable enzyme for chiral amine synthesis due to its strict enantioselectivity and broad specificity for amine donors. However, like other transaminases, Vfat faces several operational challenges, particularly its stability under pH and temperature conditions— challenges could be addressed through the random mutagenesis strategy. In our research, we successfully improved two key operational properties of Vfat through random mutagenesis. A critical step in this process was developing and applying a lysis buffer, which enabled high-throughput screening in 96-well microplates. Vfat random mutagenesis library was generated through ep PCR, and the resulting library was cloned into pET28?(+)_Vfat using the MEGAWHOP method (Megaprimer PCR of Whole Plasmid). After overcoming the challenges of lysis and cloning, a library was constructed with 35 96-well microtiter plates with a mutation frequency of 2 kb?1. Variants with outstanding performance in initial activity (up to five times higher than the wild-type enzyme) and better performance at temperature challenge (six-fold increase) and in alkaline pH (retaining initial activity after incubation) were obtained. This improvement highlights the potential of random mutagenesis in this enzyme, a tool not explored in transaminases. Using a lysis buffer allowed efficient screening, thus obtaining active enzyme variants. These advances represent a significant step in using high-throughput platforms to optimize transaminases. © 2025 Elsevier B.V.