Desarrollo de una estrategia bioseparativa para obtención y purificación de una catalasa fúngica

In this work, the capacity of aqueous biphasic systems (ABS) as a method of pre-concentration and isolation of a catalase produced by the fungus Aspergillus niger was studied. To achieve this objective, ABS consisting of polyethylene glycol and potassium phosphate were chosen, and a solution containing the fungal catalase enzyme from the biomass formed during fungal growth was distributed in the BMS. The choice of SBA was based on the fact that they stabilise the enzyme, are environmentally friendly and their components can be recycled. In a first step, the effect of different environmental variables on the partition coefficient of fungal catalase in BMS was studied. The effect of the molecular weight of polyethylene glycol, the effect of different equilibrium concentrations of this polymer (across the binding line), the effect of temperature, pH, different ionic strengths and the presence of water structure-disrupting cations were analysed. The values of all these variables that optimise the recovery and purification rate of the process were determined. It was observed that the enzyme is preferentially contained in the phosphate-rich phase, due to its high molecular weight inducing its exclusion from the polymer-rich phase. Catalase partitioning was shown to be endothermic in most of the tested systems with positive entropy changes. This has also been observed for other macromolecules and is describing a molecular mechanism associated with the partitioning where a prior disorder of the ordered water around the polymer ethylene chains and in the hydrophobic patches of the enzyme is necessary. The salts of the Hofmaiter series, which act by modifying the water structure, also significantly modify the partitioning of the catalase, especially in PEG 600.
It was found that the system formed by PEG400 at pH 7.0 is the most suitable to pre-purify the enzyme from its natural source with a Yield of 90% and a Purification Factor of 1.5.