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Biochemical and Structural Characterization of Two Site-Directed Mutants of Staphylococcus xylosus Lipase

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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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Acknowledgements

The present work was financially supported by CNPq process 552508/2007-1, and Rede Proteoma de Santa Catarina (FAPESC/FINEP/MCT). DJK, JBB and FCAB were recipients of fellowships from CAPES or CNPq, Brazil.

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Authors and Affiliations

  1. Departamento de Ciência e Tecnologia de Alimentos, Universidade Federal de Santa Catarina, Rod. Admar Gonzaga, 1346, 88034-001, Florianópolis, Brazil

    Deise Juliana Kolling, Fábio Cristiano Angonesi Brod & Ana Carolina Maisonnave Arisi

  2. Departamento de Bioquímica, Universidade Federal de Santa Catarina, 88040-900, Florianópolis, Brazil

    Jean Borges Bertoldo, Javier Vernal & Hernán Terenzi

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  1. Deise Juliana Kolling
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  2. Jean Borges Bertoldo
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  3. Fábio Cristiano Angonesi Brod
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  4. Javier Vernal
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  5. Hernán Terenzi
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  6. Ana Carolina Maisonnave Arisi
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Correspondence to Ana Carolina Maisonnave Arisi.

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Kolling, D.J., Bertoldo, J.B., Brod, F.C.A. et al. Biochemical and Structural Characterization of Two Site-Directed Mutants of Staphylococcus xylosus Lipase. Mol Biotechnol 46, 168–175 (2010). https://doi.org/10.1007/s12033-010-9282-5

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