Development of a bioprocess for production of a new A. niger FS3 Phytase : studies of its purification and characterization

Abstract

Abstract: Phytases have important applications in human and animal nutrition because they hydrolyze the phytate present in legumes, cereal grains and oil seeds. This results in an increased availability of minerals, trace elements and amino acids as well as phosphate. Fifty potential phytase-producing fungal strains were isolated from a fertile soil obtained from the northern part of Paraná State in Brazil and other alternative sources using a selective media. Heat treatment resulted in an increase of inorganic phosphate concentration, which is well known as a microbial phytase production repressor, and also reduces phytase production. UV exposure of the substrate was shown to reduce microbial contamination without affecting phytase production. A Plackett-Burman screening design was applied to identify significant physicochemical variables in phytase formation. These pre-selected variables were subsequently optimized using a Central Composite Rotational Design (CCRD). The maximum phytase production was achieved with the optimum variables temperature 30ºC, initial moisture 65%, Na-citrate buffer concentration 0.3M, initial pH 5.0 and urea concentration 1.5%. An overall 4.3-fold improvement in phytase production was successfully achieved. Respirometric data of phytase production by citric pulp fermentation in a columntype bioreactor was monitored using a new data acquisition system. Respirometric activity during fermentation and its relation with fungal growth, forced air and enzyme production using a column-type bioreactor were also objectives of this work. Phytase synthesis by a new isolate, A. niger FS3, increased with forced air. The O2 consumption and CO2 production during solid-state fermentation were monitored by controllers, acquired by sensors in the bottom and top of the columns linked to controllers, recorded by the acquisition software and processed by Fersol2 software tool without the need of a gas chromatography. Phytase synthesis was associated to fungal growth, and therefore could be used to estimate biomass formed in citric pulp fermentation. The identification of suitable conditions for the first step of phytase purification was performed in batch. The results showed that parameters established as SPsepharose FF in glycine-HCl pH 2.85 and enzyme diluted in the same buffer in the proportion 1:3 (v/v), resulted in a substantial recovery of enzyme (59.61%) compared to the recovery percentages in CM and DEAE-sepharose (56.61% and 37.64%, respectively) considering the study conducted in tubes instead of column. This batch study is a fast way to define parameters to run ion exchange chromatography. The phytase was purified to electrophoretic homogeneity by cationic-exchange, anionicexchange chromatography and cromatofocusing steps. On SDS–PAGE analysis, the molecular weight of the purified phytase was calculated to be approximately 100 kDa. The phytase has an optimum pH of 5.0-5.5 and an optimum temperature of 60ºC. The phytase displayed high affinity for phytate and the Km was 0.52 mM.