| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 2075328 | Biocatalysis and Agricultural Biotechnology | 2015 | 7 Pages |
•We enhanced laccase production using different culture-dependent strategies.•We showed the important effect of carbon source in laccase production.•We purified a laccase enzyme from an Ascomycota with great specific activity.•We found an atypical laccase with low-molecular-mass.•The purified enzyme showed good resistance to ionic metals inhibitors.
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±1405 U L−1, which is 10 times higher than the activity of the control culture (1929±44 U L−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 38 kDa 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 297 μM and Vmax of 581.4 μM min−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.
