Isolation of a thermostable variant of Lip2 lipase from Yarrowia lipolytica by directed evolution and deeper insight into the denaturation mechanisms involved
In: Journal of biotechnology, Jg. 156 (2011-02-06), Heft 2
Online
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Zugriff:
International audience; Lip2 lipase from Yarrowia lipolytica is a very promising lipase with many potential applications (e. g. resolution of racemic mixtures, production of fine chemicals). Unfortunately this potential is impeded by a very low thermostability for temperatures higher than 40 degrees C. Error-prone PCR and screening of the library in a high-performance yeast expression system (Y. lipolytica) enabled a thermostable variant to be identified. This variant presents only one mutation, the free cysteine 244 is changed into an alanine. At 60 degrees C, the half-life time of the purified variant was 127-fold increased compared to the WT enzyme (from 1.5 min to 3 h). Saturation mutagenesis experiment at position 244 demonstrated that the presence of a cysteine at this position was responsible for the thermal denaturation. It was demonstrated that WT Lip2 and the thermostable variant are both inactivated through aggregation mechanisms, but that the kinetics and the nature of the aggregation were different. For the WT enzyme, rapid intermolecular disulphide bridge interchanges triggered by the free cysteine 244 mediates aggregation. For the variant C244A, aggregation still occurred but much slower than for the WT lipase and was mainly driven by hydrophobic forces.
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Isolation of a thermostable variant of Lip2 lipase from Yarrowia lipolytica by directed evolution and deeper insight into the denaturation mechanisms involved
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Autor/in / Beteiligte Person: | Marty, Alain ; Nicaud, Jean-Marc ; Bordes, Florence ; Tarquis, Laurence ; Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP) ; Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse) ; Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA) ; Microbiologie et Génétique Moléculaire (MGM) ; Institut National de la Recherche Agronomique (INRA)-Institut National Agronomique Paris-Grignon (INA P-G)-Centre National de la Recherche Scientifique (CNRS) ; The high-throughput screening work was carried out at the Laboratory for BioSystems & Process Engineering (Toulouse, France) with the equipments of the ICEO facility, dedicated to the screening and the discovery of new and original enzymes. ICEO is supported by grants from the Region Midi-Pyrenees, the European Regional Development Fund and the Institut National de la Recherche Agronomique (INRA). ; Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse) ; Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS) ; Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS) |
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Zeitschrift: | Journal of biotechnology, Jg. 156 (2011-02-06), Heft 2 |
Veröffentlichung: | 2011 |
Medientyp: | unknown |
ISSN: | 1873-4863 (print) ; 0168-1656 (print) |
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