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

Authors :
Yamada R
Higo T
Yoshikawa C
China H
Yasuda M
Ogino H
Source :
Biotechnology progress [Biotechnol Prog] 2015 Jul-Aug; Vol. 31 (4), pp. 917-24. Date of Electronic Publication: 2015 Jun 12.
Publication Year :
2015

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.<br /> (© 2015 American Institute of Chemical Engineers.)

Details

Language :
English
ISSN :
1520-6033
Volume :
31
Issue :
4
Database :
MEDLINE
Journal :
Biotechnology progress
Publication Type :
Academic Journal
Accession number :
26033926
Full Text :
https://doi.org/10.1002/btpr.2117