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Original Article
Cloning, expression and decoding of the cold adaptation of a new widely represented thermolabile subtilisin-like protease
Article first published online: 27 DEC 2012
DOI: 10.1111/jam.12033
© 2012 The Society for Applied Microbiology
Additional Information
How to Cite
Acevedo, J.P., Rodriguez, V., Saavedra, M., Muñoz, M., Salazar, O., Asenjo, J.A. and Andrews, B.A. (2013), Cloning, expression and decoding of the cold adaptation of a new widely represented thermolabile subtilisin-like protease. Journal of Applied Microbiology, 114: 352–363. doi: 10.1111/jam.12033
Publication History
- Issue published online: 14 JAN 2013
- Article first published online: 27 DEC 2012
- Accepted manuscript online: 8 OCT 2012 11:24PM EST
- Manuscript Accepted: 27 SEP 2012
- Manuscript Revised: 23 SEP 2012
- Manuscript Received: 31 JUL 2012
Funded by
- FONDEF. Grant Number: DO4I-1374
- Millenium Scientific Initiative
- Conicyt. Grant Number: P05-001-F
Keywords:
- cold adaption;
- Pseudoalteromonas sp.;
- subtilisin-like protease
Abstract
Aims
Cloning, expression and characterization of a new cold-adapted protease with potential biotechnological application, isolated from Antarctic bacteria.
Method and Results
A subtilisin-like gene was isolated from several Antarctic bacterial genus using CODPEHOP-designed primers and a genome walking method. This gene encodes a precursor protein, which undergoes an autocatalytic cleavage resulting in a 34·6 kDa active cold-adapted protease with a maximum activity at 25–35°C and optimum pH of 8·0–9·0. It showed a higher catalytic efficiency at lower temperatures compared to its mesophilic counterpart. Heat-induced inactivation resulted in a very low melting point. Local packing analysis using the homology model indicated Ala284 as an important cold-adaptation determinant, which was corroborated by the site-directed mutagenesis.
Conclusions
A new thermolabile subtilisin-like protease has been successfully cloned and analysed, and an important hot spot in the evolution of the cold adaptation and substrate specificity of this enzyme was identified and tested.
Significance and Impact of the Study
This work reports a new cold-adapted protease with a vast representation amongst Antarctic genus, suggesting therefore its evolutionary success in this cold environment. Likewise, important sites for genetic potentiation have been identified, which are extrapolated to other enzymes of the same kind.