Random mutagenesis and selection of organic solvent‐stable haloperoxidase from Streptomyces aureofaciens

Haloperoxidases are useful oxygenases involved in halogenation of a range of water‐insoluble organic compounds and can be used without additional high‐cost cofactors. In particular, organic solvent‐stable haloperoxidases are desirable for enzymatic halogenations in the presence of organic solvents. In this study, we adopted a directed evolution approach by error‐prone polymerase chain reaction to improve the organic solvent‐stability of the homodimeric BPO‐A1 haloperoxidase from Streptomyces aureofaciens. Among 1,000 mutant BPO‐A1 haloperoxidases, an organic solvent‐stable mutant OST48 with P123L and P241A mutations and a high active mutant OST959 with H53Y and G162R mutations were selected. The residual activity of mutant OST48 after incubation in 40% (v/v) 1‐propanol for 1 h was 1.8‐fold higher than that of wild‐type BPO‐A1. In addition, the OST48 mutant showed higher stability in methanol, ethanol, dimethyl sulfoxide, and N,N‐dimethylformamide than wild‐type BPO‐A1 haloperoxidase. Moreover, after incubation at 80°C for 1 h, the residual activity of mutant OST959 was 4.6‐fold higher than that of wild‐type BPO‐A1. Based on the evaluation of single amino acid‐substituted mutant models, stabilization of the hydrophobic core derived from P123L mutation and increased numbers of hydrogen bonds derived from G162R mutation led to higher organic solvent‐stability and thermostability, respectively. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:917–924, 2015