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A thermostable variant of P. aeruginosa cold-adapted LipC obtained by rational design and saturation mutagenesis

MPG-Autoren
http://pubman.mpdl.mpg.de/cone/persons/resource/persons133000

Cesarini,  Silvia
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;
Faculty of Biology, University of Barcelona;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons133011

Bofill,  Christina
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;
Faculty of Biology, University of Barcelona;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons58919

Reetz,  Manfred T.
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;
Philipps-Universität Marburg, Fachbereich Chemie;

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Zitation

Cesarini, S., Bofill, C., Pastor, F. I. J., Reetz, M. T., & Diaz, P. (2012). A thermostable variant of P. aeruginosa cold-adapted LipC obtained by rational design and saturation mutagenesis. Process Biochemistry, 47, 2064-2071. doi:10.1016/j.procbio.2012.07.023.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0010-5134-6
Zusammenfassung
Cold-adapted Pseudomonas aeruginosa LipC is a secreted lipase showing differential properties compared to its well-known counterpart LipA. LipC is fundamentally a cold-acting lipase, capable of tolerating high concentrations of ions and heavy metals, and showing a shift in substrate specificity when incubated at higher temperatures. These properties make LipC an interesting enzyme, well suited for biotechnological or environmental applications, where activity at low temperatures would be required. However, a relatively low thermal resistance constitutes the main drawback for using this enzyme in long-term operational processes. To overcome the lability of LipC, we developed a rational design system to modify specific sites on the enzyme structure to obtain an improved variant of the lipase bearing higher thermal stability, but without loss of its cold-adapted properties. Eight mutant libraries plus two point mutations were constructed affecting those amino acids showing the highest flexibility on the 3D model structure. After screening more than 3000 mutant clones, a LipC variant bearing two amino acid changes and the required thermostability and cold-adapted properties was obtained. The new variant D2 H8, with a 7- fold increased thermal stability in comparison to wild type LipC, will guarantee the use and maintenance of such a lipase in a number of processes being performed at low (4–20 ◦C) temperatures.