期刊
BIOCHEMICAL JOURNAL
卷 441, 期 -, 页码 487-498出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20111199
关键词
alpha-factor prepro-leader; directed evolution; enzyme promiscuity; Saccharomyces cerevisiae; versatile peroxidase
资金
- European Union [NMP2-CT-2006-026456-Biorenew, FP7-KBBE-2010-4-26537-Peroxicats, CM0701]
- National projects [BIO2010-19697, BIO2008-01533]
- NeuronBiopharma [020401070029, 020401070004]
- Biorenew grant
- Peroxicats contract
- Ministerio de Cienca e Innovacion (MICINN)
The VPs (versatile peroxidases) secreted by white-rot fungi are involved in the natural decay of lignin. In the present study, a fusion gene containing the VP from Pleurotus eryngii was subjected to six rounds of directed evolution, achieving a level of secretion in Saccharomyces cerevisiae (21 mg/l) as yet unseen for any ligninolytic peroxidase. The evolved variant for expression harboured four mutations and increased its total VP activity 129-fold. The signal leader processing by the STEI3 protease at the Golgi compartment changed as a consequence of overexpression, retaining the additional N-terminal sequence Glu-Ala-Glu-Ala that enhanced secretion. The engineered N-terminally truncated variant displayed similar biochemical properties to those of the non-truncated counterpart in terms of kinetics, stability and spectroscopic features. Additional cycles of evolution raised the T-50 8 degrees C and significantly increased the enzyme's stability at alkaline pHs. In addition, the K-m for H2O2 was enhanced up to 15-fold while the catalytic efficiency was maintained, and there was an improvement in peroxide stability (with half-lives for H2O2 of 43 min at a H2O2/enzyme molar ratio of 4000:1). Overall, the directed evolution approach described provides a set of strategies for selecting VPs with improvements in secretion, activity and stability.
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