title: Biochemical and structural characterization of two site-directed mutants of Staphylococcus xylosus lipase.
abstract: Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-Val). In the present work, in order to check the importance of the residue 309 in the specific activity, the amino acid side chain residue valine 309 was substituted by aspartate or lysine through site-directed mutagenesis. Both mutant lipases (MUT-Lys and MUT-Asp) were expressed in E. coli and the recombinant histidine-tagged lipases were purified by immobilized metal ion affinity chromatography. The enzyme activity was determined using p-nitrophenyl butyrate as substrate and secondary structure content was evaluated by circular dichroism. MUT-Lys and MUT-Asp presented significant increase of lipase activity (P < 0.05) in comparison to WT-Val, although highest activities for the three enzymes were observed at the same pH and temperature (pH 9.0 and 42 degrees C). The wild type and mutant lipases presented high thermal stability, after 30 min of incubation at 80 degrees C all enzymes retained their initial activities.
authors.affiliation: Departamento de Ciência e Tecnologia de Alimentos, Universidade Federal de Santa Catarina, Rod. Admar Gonzaga, 1346, 88034-001, Florianópolis, Brazil.
authors.forename: Deise Juliana
authors.initials: DJ
authors.lastname: Kolling
authors.forename: Jean Borges
authors.initials: JB
authors.lastname: Bertoldo
authors.forename: Fábio Cristiano Angonesi
authors.initials: FC
authors.lastname: Brod
authors.forename: Javier
authors.initials: J
authors.lastname: Vernal
authors.forename: Hernán
authors.initials: H
authors.lastname: Terenzi
authors.forename: Ana Carolina Maisonnave
authors.initials: AC
authors.lastname: Arisi
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D013210:Staphylococcus / Q000201:enzymology*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D008049:Lipase
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