title: Random mutagenesis and selection of organic solvent-stable haloperoxidase from Streptomyces aureofaciens.
abstract: 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.
authors.affiliation: Dept. of Chemical Engineering, Osaka Prefecture University, Sakai, Osaka, 599-8531,, Japan.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Yamada
authors.affiliation: Dept. of Chemical Engineering, Osaka Prefecture University, Sakai, Osaka, 599-8531,, Japan.
authors.forename: Tatsutoshi
authors.initials: T
authors.lastname: Higo
authors.affiliation: Dept. of Chemical Engineering, Osaka Prefecture University, Sakai, Osaka, 599-8531,, Japan.
authors.forename: Chisa
authors.initials: C
authors.lastname: Yoshikawa
authors.affiliation: Dept. of Chemical Engineering, Osaka Prefecture University, Sakai, Osaka, 599-8531,, Japan.
authors.forename: Hideyasu
authors.initials: H
authors.lastname: China
authors.affiliation: Dept. of Chemical Engineering, Osaka Prefecture University, Sakai, Osaka, 599-8531,, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Yasuda
authors.affiliation: Dept. of Chemical Engineering, Osaka Prefecture University, Sakai, Osaka, 599-8531,, Japan.
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Ogino
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D019143:Evolution, Molecular; D016296:Mutagenesis; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012997:Solvents; D013304:Streptomyces aureofaciens / Q000201:enzymology* / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D012997:Solvents; D010544:Peroxidases
keywords: directed evolution; haloperoxidase; organic solvent stability; random mutagenesis; thermostability