Obtaining and Purification of Crude Extracts with Cellulase and Laccase Enzymatic Activity from an Isolated Strain of Fusarium Spp

The growing demand of biotechnological applications requires the use of microorganisms of interest, because of its production of molecules with applicability in industrial processes, or its favorable interaction with other microorganisms; in order to maintain living organisms for a certain time, and preserving their own morphological characteristics (Voyron et al., 2009) protocols should be implemented to generate low variations in phenotypic and genotypic aspects, with less impact on its added value. The genus Fusarium is a type of filamentous fungus which is distributed in a wide number of ecosystems. Due to its characteristics, it is part of the soil microbiota and can also be found in association with plants and other organisms. Filamentous fungi supplement their nutritional needs from biomass degradation, which makes them organisms of interest for its production of enzymes for cell wall degradation. Cellulose is one of the most important renewable sources of carbon, which is mostly synthesized by plants, with an annual production of 7.2 x 10¹¹tons (David Krogh, Rock, Åkesson, & Nielsen, 2005 ).

In this paper optimization for fusarium spp.specific medium was performed. In order to favor the Laccase production, methods of inoculation with PDA solid medium, then in PDB liquid medium, and finally a modified Kirk culture medium for 8 days and 200 rpm at 30 ° C, were applied. This extract was vacuum filtered with a pore size of 0.2 micron, and finally stored at low temperatures for measuring its enzymatic activity for Laccase, in international units, and Cellulase in F.P.U units. The strain preservation was carried out by immersion in sterile mineral oil at room temperature.

After production of the extract, the process of separation and purification of the enzyme was performed using three-phase partition method. For this experiment, was made central composite design, which had input factors of temperature, tert-butyl alcohol (extract: tert-butanol) concentration and ammonium sulfate saturation (% PV), depositing the phase of interest in a phosphate buffer, maintaining its pH at 7 for one hour. Finally, the extract was measured in FPU Cellulase activity, using the DNS method.

The extract was produced with Xylose as a carbon source, peptone as nitrogen source and methanol as inductor, presenting as higher enzymatic activity 42 U/L. By purifying the Laccase from the produced extract, a pure enzyme with fold purification 1.2164 ± 0.1830 of performance and 114.13 ± 7.69% was obtained. For this same extract, Cellulase activity was tested, obtaining a 0,08 U/L; this result was verified for specific activity in FPU, showing that Cellulase activity was not significant, hence a growth medium for this strain was suggested in order to increase the Cellulase activity in the extract.

References

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