title: Isolation of a thermostable variant of Lip2 lipase from Yarrowia lipolytica by directed evolution and deeper insight into the denaturation mechanisms involved.
abstract: 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°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°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.
authors.affiliation: Université de Toulouse; INSA, UPS, INP, LISBP, F-31077 Toulouse, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Bordes
authors.forename: Laurence
authors.initials: L
authors.lastname: Tarquis
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Nicaud
authors.forename: Alain
authors.initials: A
authors.lastname: Marty
mesh_terms: D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015723:Gene Library; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D016296:Mutagenesis; D009154:Mutation; D011489:Protein Denaturation; D013696:Temperature; D025062:Yarrowia / Q000201:enzymology* / Q000235:genetics
chemical_list: D005656:Fungal Proteins; C408971:LIP2 protein, Yarrowia lipolytica; D008049:Lipase