Improvement of H2O2 stability of manganese peroxidase by combinatorial mutagenesis and high-throughput screening using in vitro expression with protein disulfide isomerase

A functional expression system for a heme protein of Phanerochaete chrysosporium, manganese peroxidase (MnP), was developed using the Escherichia coli in vitro coupled transcription/translation system in the presence of hemin and fungal protein disulfide isomerase. This system has allowed the high-throughput construction and screening of a large diversity of mutant heme enzymes and has made it possible to improve the enzymatic function efficiently. Here we increased the H2O2 stability of MnP using this system; a mutant MnP library containing three randomized amino acid residues located in the H2O2-binding pocket of MnP was designed and constructed on a 384-well plate using SIMPLEX (single-molecule-PCR-linked in vitro expression). The screening of 10(4) samples independently expressed for improved H2O2 stability led to four positive mutants, the H2O2 stability of which was nine times higher than that of the wild-type.