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- Title
Random mutagenesis and selection of organic solvent-stable haloperoxidase from Streptomyces aureofaciens.
- Authors
Yamada, Ryosuke; Higo, Tatsutoshi; Yoshikawa, Chisa; China, Hideyasu; Yasuda, Masahiro; Ogino, Hiroyasu
- 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. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:917-924, 2015
- Subjects
MUTAGENESIS; ORGANIC solvents; STREPTOMYCES aureofaciens; OXYGENASES; HALOGENATION; POLYMERASE chain reaction
- Publication
Biotechnology Progress, 2015, Vol 31, Issue 4, p917
- ISSN
8756-7938
- Publication type
Article
- DOI
10.1002/btpr.2117