Biochemical and structural characterization of two site-directed mutants of Staphylococcus xylosus lipase.
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.
Kolling
Deise Juliana
DJ
Departamento de Ciência e Tecnologia de Alimentos, Universidade Federal de Santa Catarina, Rod. Admar Gonzaga, 1346, 88034-001, Florianópolis, Brazil.
Bertoldo
Jean Borges
JB
Brod
Fábio Cristiano Angonesi
FC
Vernal
Javier
J
Terenzi
Hernán
H
Arisi
Ana Carolina Maisonnave
AC
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
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D008049:Lipase
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