Produção, caracterização bioquímica e purificação de fitase produzida por Aspergillus niger var. phoenicis URM 4924

Abstract

During ripening, vegetable seeds and grain accumulated substantial amount of phytic acid, representing over 60% of total phosphorus in them. Phytate acts as an energy source for seed germination and is rarely available for non-ruminants, since they do not synthesize the enzyme. Due to the unavailability of biological organic phosphorus in many vegetable foods, research has sought alternative sources of phosphorus in order to better use. Incorporated in this context are the phytases, which form a group of enzymes that catalyze reactions gradual dephosphorylation of phytate. For enzyme purification the aqueous two-phase systems (ATPS) have been widely used for protein separation due to its low cost compared to other purification processes. The aim of this study was to evaluate the variables that influence production and purification of phytase by aqueous two-phase systems, as well as the biochemical characteristics of phytase produced by Aspergillus niger var. phoenicis URM 4924. The culture medium for the production of phytase were studied using factorial design and response surface methodology. The best condition for the production of phytase (8.80 U/mL) was found matching 1.25% of rice bran and 3.0% corn steep liquor. The optimum pH was 5.0, and remains at over 80% of residual activity at pH 5.0 to 9.0 for 15 hours. The phytase showed affinity constant of 0.12 mM and maximum velocity of 7.9 ηmol.s-1. The best conditions of extraction in aqueous two-phase systems were obtained with 26% (w/w) PEG 8000 (g/mol), pH 8.0 and 12% (w/w) citrate thus promoting purification factor of 7.58, partition coefficient of 3.62 and yield of 113.4%. The extraction using ATPS PEG/citrate proved to be promising for purification of phytase produced by A. niger var. phoenicis URM 4924 and may be applied in the composition of animal feed non-ruminants.