Biochemical and Structural Characterization of Two Site-Directed Mutants of <Emphasis Type="Italic">Staphylococcus xylosus</Emphasis> Lipase |
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Authors: | Deise Juliana Kolling Jean Borges Bertoldo Fábio Cristiano Angonesi Brod Javier Vernal Hernán Terenzi Ana Carolina Maisonnave Arisi |
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Institution: | 1.Departamento de Ciência e Tecnologia de Alimentos,Universidade Federal de Santa Catarina,Florianópolis,Brazil;2.Departamento de Bioquímica,Universidade Federal de Santa Catarina,Florianópolis,Brazil |
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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°C). The wild type and mutant lipases presented high thermal stability, after 30 min of incubation at 80°C all
enzymes retained their initial activities. |
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