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

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.

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 Creators:
Cesarini, Silvia1, 2, Author              
Bofill, Christina1, 2, Author              
Pastor, F. I. Javier2, Author
Reetz, Manfred T.1, 3, Author              
Diaz, Pilar2, Author
Affiliations:
1Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, DE, ou_1445588              
2Faculty of Biology, University of Barcelona, Barcelona, ES, ou_persistent22              
3Philipps-Universität Marburg, Fachbereich Chemie, ou_persistent22              

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Free keywords: Cold-adapted lipase Rational design Saturation mutagenesis Enzyme improvement Pseudomonas
 Abstract: 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.

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 Dates: 2012-12
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1016/j.procbio.2012.07.023
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Title: Process Biochemistry
Source Genre: Journal
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Pages: - Volume / Issue: 47 Sequence Number: - Start / End Page: 2064 - 2071 Identifier: ISSN: 0032-9592
CoNE: https://pure.mpg.de/cone/journals/resource/110992357403106