Optimization of the production, purification and characterization of a laccase from the native fungus Xylaria sp.

Jesus D. Castaño, Carlos Cruz, Esperanza Torres

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24 Scopus citations

Abstract

The production of a laccase enzyme secreted by the Ascomycota fungus Xylaria sp. was improved under submerged fermentation using a combination of the one factor-at-a-time method for different carbon and nitrogen sources, a Taguchi orthogonal array and inducers. The laccase activity of the optimized culture was 20,535±1405UL-1, which is 10 times higher than the activity of the control culture (1929±44UL-1) and a high value compared with other fungi. From this culture, a laccase was purified through diafiltration and anion exchange and size exclusion chromatography, with a purification factor of 7.45 and a 0.51% yield. According to a two-dimensional (2D) electrophoretic analysis, the molecular mass of the protein was 38kDa and had a pI of 4.9. Analysis of the protein using mass spectrometry revealed the presence of the peptides GPASAPYDEDK and LVNTAIDTMFK, which coincide with other reported laccases. The purified enzyme had an optimal activity at pH 3.0-4.0 and 50-66°C. Using 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as substrate, a Km of 297μM and Vmax of 581.4μMmin-1 were determined. The enzyme was stable at temperatures between 4-30°C and pH values of 6-8, and it was inhibited by the Fe2+ ion but induced by the Cu2+ ion. Additionally, the enzyme was slightly inhibited by ethylenediaminetetraacetic acid (EDTA) but strongly inhibited by sodium azide, dithiothreitol (DTT) and potassium cyanide (KCN). The observed biochemical characteristics found indicate that Xylaria sp. laccase has the potential for use in biotechnological processes related to bioremediation.

Original languageEnglish (US)
Pages (from-to)710-716
Number of pages7
JournalBiocatalysis and Agricultural Biotechnology
Volume4
Issue number4
DOIs
StatePublished - Oct 1 2015
Externally publishedYes

Bibliographical note

Funding Information:
This work was funded by Colciencias (Contract- 632-2011 ) and was conducted under MAVDT Collection permit 0255, March 12 2014. To Chicaque Natural Park, and UNIPALMA for providing the oil palm empty fruit bunches. The authors also acknowledge the National University of Colombia, the Colombian Center for Genomics and Bioinformatics of Extreme Environments (GeBiX) for funding this investigation and the Department of Botany & Plant Pathology of Purdue University for the MALDI-TOF-TOF analysis. We thank Rosa Mejía Calderón for preserving the samples, and Cristhian Camilo Crespo for assisting in the fieldwork and sample identification.

Publisher Copyright:
© 2015 Elsevier Ltd.

Keywords

  • Chromatography
  • Inducer
  • Laccase enhancement
  • Multi-copper oxidase
  • Orthogonal design
  • Submerged fermentation

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