Purification and Characterization of Two Intracellular Phytases From the Tempeh Fungus Rhizopus Oligosporus

Abstract

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 Ϯ 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 APPLICATIONSThis work reports the isolation, purification and characterization of two intracellular phytases from the tempeh fungus Rhizopus oligosporus. Interest 4 Corresponding 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.

Zygomycetes-based biorefinery: Present status and future prospects

“…(Ramachandran et al, 2005). Two intracellular phytases have recently been isolated from R. oligosporus grown in rice flour suspension, and were shown to have broad affinity for various phosphorylated compounds (Azeke et al, 2011).…”

The Impact of Phytases on the Release of Bioactive Inositols, the Profile of Inositol Phosphates, and the Release of Selected Minerals in the Technology of Buckwheat Beer Production

“…This bond, however, can be hydrolyzed by phytase B, i.e., the nonspecific acid phosphomonoesterase [13]. Phytases are known to be synthetized in plants tissues (i.e., endogenic enzymes that are active during soaking and sprouting of seeds), bacteria, and filamentous fungi [14,15]. Phytate dephosphorylation that results from plant endogenous phytase action in barley, wheat, rye, and particularly, in buckwheat grain tissues during mashing is limited, mainly due to process conditions which are out of the optima for pH (4.5-5.5) and temperature values (45-65 • C) [13].…”

Characterization of a Thermotolerant Phytase Produced by Rhizopus microsporus var. microsporus Biofilm on an Inert Support Using Sugarcane Bagasse as Carbon Source

“…The phytase RMPhy1 was purified 4.18-fold with a 4.78 % yield (Table 4). This yield was better as compared to the values obtained for the purified phytase from A. flavus ITCC 6720 [11] and two purified isoforms of intracellular phytase from R. oligosporus after five steps of purification with a recovery of 1.3-1.6 % [10].…”

“…In this context, filamentous fungi can be highlighted as important producers of phytases with interesting biotechnological characteristics such as thermal stability. Some fungal phytases have been characterized as those obtained from Rhodotorula mucilaginosa [4], Aspergillus niger [5], Paecilomyces variotii [8], Aspergillus caespitosus [9], Rhizopus oligosporus [10], and Aspergillus flavus [11].…”