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Abstracts - Bruna Soares Dionizio

EVALUATION OF BRAZILIAN MARINE FUNGI FOR THE PRODUCTION OF OXIDASES ENZYMES

Bruna Soares Dionizio1*, André Luiz M. Porto2, Dulce Helena Ferreira de Souza1.


1 Chemistry Department, Federal University of São Carlos, São Carlos- SP, Brazil.
2 Chemistry Institute, University of São Paulo- USP, São Carlos- SP, Brazil.
*brunasdionizio@hotmail.com


Fungi isolated from the marine environment are well known as a potential genetic resource for various biotechnological applications because of their adaptations to high salinity and pH extremes1. In this work three marine fungi were selected for extra cellular ligninolytic enzymes production: Mucor racemosus CBMAI 847, Penicillium oxalicum CBMAI 1996 and Aspergillus sp. CBMAI 1198.
The confirmation of the production of oxidases by the fungi was performed through the plate activity according to the Pointing methodology2. The results showed that Mucor racemosus and Aspergillus sp. are good producer of the oxidases. Therefore, the two fungi were cultivated in liquid medium containing malt extract 2% and NaCl 3% for 7 days, then Sabouraud dextrose medium pH 5.6 was added, with or without copper sulphate and the culture was maintained under stirring during 15 days. The enzymatic activity was assayed with 1 mM ABTS substrate, 0.1 mM H2O2 and 20 mM MnSO4.H2O in 50 mM sodium acetate buffer pH 5, and the total proteins was determined by the Bradford method. The maximum activity specific for oxidase was evidenced approximately on the 7th day for M. racemosus and 5th day for A. sp.
Crude extract from M. racemosus was precipitated with 70% ammonium sulphate and the pellet was resuspended in sodium acetate buffer 50 mM pH 5 (buffer A) and applied to a HiTrap QFF column (anionic exchange chromatography) in an ÄKTA (GE) system. Elution was carried out using buffer B (buffer A plus NaCl 1M) in a 30-cv linear gradient (0–100% of buffer B) at a flow rate of 1 mL.min-1. The purification step was performed at 23°C and was followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)3 and enzymatic assays using the following substrates: ABTS (for oxidase activity), Phenol Red (for manganese peroxidase activity, MnP) and Veratryl alcohol (for Lignin peroxidase activity, LiP)1. The results showed two peaks with oxidase activity: one with no affinity with the resin and other with HiTrap QFF affinity. Both of the peaks presented high lignin peroxidase activity but also manganese peroxidase activity. Optimization of purification step for M. racemosus and identification of the extracellular enzymes produced by Aspergillus sp. are being carried out. These results demonstrate great potential for the use of these fungi in biotechnological processes.


References
1. BONUGLI-SANTOS, R. C. et al., Enzyme and Microbial Technology, 46: 32–37, 2010. 2. POINTING, S. B., Fungal Diversity 2: 17–33, 1999.
3. LAEMMLI, U. K. Nature, 227, 680–685, 1970.