Improved Stability of Baeyer–Villiger Mono-Oxygenase from Pseudomonas fluorescens by Substitution of Cysteine Residues

Baeyer–Villiger mono-oxygenases (BVMOs) are enzymes that could insert an oxygen into carbon skeletons adjacent to carbonyl groups to form lactone or ester. Recently, a new method of sebacic acid production from oleic acid based on Pseudomonas fluorescens DSM 50106 (PfBVMO) catalysis was reported. However, the instable property of PfBVMO limited the further investigation and application of this enzyme. In this study, we aimed to improve the stability of PfBVMO by sitedirected mutagenesis. The sequence of PfBVMO indicates probable factors from the susceptible oxidizing cysteine residue leading to the instability of PfBVMO. Activity and stability of mutants and the wild-type were characterized to investigate the correlation of cysteine residues and activity. The mutant enzyme with entire cysteine residues replaced by serine residues, showed significantly higher stability than the wild type. The result would facilitate further work on the enzyme modification and crystal structure determination. Meanwhile, the results of this study provided potential for other enzyme stability evolution.