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Functional Expression of Horseradish Peroxidase in E. coli by Directed Evolution
Author(s) -
Lin Zhanglin,
Thorsen Todd,
Arnold Frances H.
Publication year - 1999
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp990037r
Subject(s) - directed evolution , horseradish peroxidase , escherichia coli , lac operon , peroxidase , biochemistry , mutagenesis , gene , chemistry , yeast , microbiology and biotechnology , enzyme , abts , expression vector , protein engineering , biology , mutation , mutant , recombinant dna , antioxidant , dpph
In an effort to develop a bacterial expression system for horseradish peroxidase (HRP), we inserted the gene encoding HRP into the pET‐22b(+) vector (Novagen) as a fusion to the signal peptide PelB. A similar construct for cytochrome c peroxidase (CcP) leads to high CcP activity in the supernatant. Expression of the wild‐type HRP gene in the presence of isopropyl‐β‐ d ‐thiogalactopyranoside (IPTG) yielded no detectable activity against ABTS (azinobis(ethylbenzthiazoline sulfonate) ). However, weak peroxidase activity was detected in the supernatant in the absence of IPTG. The HRP gene was subjected to directed evolution: random mutagenesis and gene recombination followed by screening in a 96‐well microplate format. From 12 000 clones screened in the first generation, one was found that showed 14‐fold higher HRP activity than wild‐type, amounting to ∼110 μg of HRP/L, which is similar to that reported from laborious in vitro refolding. No further improvement was obtained in subsequent generations of directed evolution. This level of expression has nonetheless enabled us to carry out further directed evolution to render the enzyme more thermostable and more resistant toward inactivation by H 2 O 2 . These results show that directed evolution can identify mutations that assist proteins to fold more efficiently in Escherichia coli . This approach will greatly facilitate efforts to “fine‐tune” those many enzymes that are promising industrial biocatalysts, but for which suitable bacterial or yeast expression systems are currently lacking.