PURIFICATION AND CHARACTERIZATION OF TWO INTRACELLULAR PHYTASES FROM THE TEMPEH FUNGUS RHIZOPUS OLIGOSPORUS

The fungus commonly used in tempeh production, Rhizopus oligosporus (DSMZ 1964), was grown in rice flour suspension and two intracellular phytases (RO1 and RO2) were isolated from the fungus, purified to near homogeneity in a five-step process and characterized. Enzyme recoveries after purification were 1.3% (RO1) and 1.6% (RO2) in relation to the phytase activity in the crude extract. Their estimated molecular mass was 45.0 +/- 5.0 kDa. The purified proteins belong to the acid phytases and they both exhibit two distinct pH optima at 3.0 and 4.5 for RO1 and 3.0 and 5.0 for RO2. Optimal temperature for the degradation of sodium phytate was determined to be 60C for RO1 and 55C for RO2. Kinetic parameters for the hydrolysis of sodium phytate were K-m = 1.6 mM (RO1) and 0.13 mM (RO2) and V-max = 0.044 U for RO1 and 0.155 U for RO2. The phytases from R. oligosporus exhibit a broad affinity for various phosphorylated compounds. PRACTICAL APPLICATIONS This work reports the isolation, purification and characterization of two intracellular phytases from the tempeh fungus Rhizopus oligosporus. Interest in these enzymes has been stimulated by the fact that phytase supplements increase the availability of phosphorus in pig and poultry feed, and thereby reduce environmental pollution due to excess phosphate excretion in areas where there is intensive livestock production. One of the purified phytase fractions RO2 showed promising enzymatic properties such as low K-m for phytate. This could be exploited for further biotechnological applications especially in the feed industry.