Inositol Phosphate Phosphatases of Microbiological Origin: the Inositol Pentaphosphate Products of Aspergillus ficuum Phytases

Abstract

The fungus Aspergillus ficuum NRRL 3135 is known to produce an extracellular nonspecific orthophosphoric monoester phosphohydrolase (EC 3.1.3.2) with a pH optimum of 2.0, as well as an extracellular myo-inositol hexaphosphate phosphohydrolase (EC 3.1.3.8; phytase) with pH optima of 2.0 and 5.5. Both these enzymes are also known to hydrolyze myo-inositol hexaphosphate. The pentaphosphates liberated in the first step of this hydrolysis have been isolated and identified by ion-exchange chromatography and optical rotation. The nonspecific orthophosphoric monoester phosphohydrolase produces a single pentaphosphate, D-myo-inositol-1,2,4,5,6-pentaphosphate, whereas the phytase, at both pH 2.0 and 5.5, produces a mixture of two pentaphosphates. The major component of this mixture is D-myo-inositol-1,2,4,5,6-pentaphosphate and the other is D-myo-inositol-1,2,3,4,5-pentaphosphate. Thus the pathways of dephosphorylation of myo-inositol hexaphosphate by these two enzymes differ from that of wheat-bran phytase which forms L-myo-inositol-1,2,3,4, 5-pentaphosphate.Phytases (EC 3.1.3.8) isolated from wheatbran (12) and a soil bacterium (Pseudomonas sp.) (2) have been shown to dephosphorylate myo-inositol hexaphosphate in a stepwise manner. The predominant myo-inositol pentaphosphates formed by these enzymes are, respectively, L-myo-inositol-1,2,3,4,5-pentaphosphate and D-myo-inositol-1,2,4,5,6-pentaphosphate. No similar study of phytases of fungal origin has been published although Theodorou (11), by using electrophoresis, has shown that the phytase of a mycorrhizal fungus, Rhizopogan luteolus, produces a mixture of at least two pentaphosphates. The major component of this mixture corresponds to the major pentaphosphate liberated by Neurospora crassa phytase (7) and by Pseudomonas phytase (2), whereas the minor component corresponds to the pentaphosphate liberated by wheat-bran phytase (12).Shieh, Wodzinski, and Warl (10) recently described the isolation and properties of two fractions with phytase activity from culture filtrates of the fungus Aspergillus ficuum NRRL 3135. One of these fractions had a single pH 434 optimum at 2.0, whereas the other had pH optima at 2.0 and at 5.5.The work reported in this paper examines the pentaphosphates produced by both the extracellular phytases from A. ficuum NRRL 3135 to establish whether the pathways of dephosphorylation of myo-inositol hexaphosphate by these enzymes resemble that of wheat-bran phytase (12) or Pseudomonas phytase (2). MATERIALS AND METHODSAll chemicals were reagent grade unless otherwise specified.Culture of organism. A. ficuum 3135 was maintained on agar slopes consisting of Czapek Dox medium (Difco) in which the inorganic orthophosphate had been replaced by dodecasodium myo-inositol hexaphosphate (1.75 g/liter; Sigma). Cultures of the organism were prepared by using the low-phosphate medium (2 mg of P per 100 ml) and technique of Shieh and Ware (9). Enzyme activity. Phytase activity was assayed at 40 C by determining the rate of release of inorganic orthophosphate (5)....

Phytase, a New Life for an “Old” Enzyme

“…The project was terminated in 1968 when the company failed to identify an organism that produced phytase activity sufficiently high to be commercially viable. However, IMC's pioneering attempt provided a very valuable isolate of Aspergillus (ficuum) niger NRRL 3135 (ATCC 66876), which enabled the later identification of the first marketed phytase in the 1990s after the initial characterization by Irving & Cosgrove (53). Application of recombinant DNA technology in the 1980s led to another milestone in the development of phytase as an animal-feed additive.…”

Molecular and catalytic properties of phytate‐degrading enzymes (phytases)

“…The phytate‐degrading enzymes from rye, barley, spelt, oat, wheat bran, rice and mung bean fit into this general consideration, because the major myo ‐inositol pentakisphosphate generated by these enzymes has been identified as D‐Ins(1,2,3,5,6)P5 [identical with l ‐Ins(1,2,3,4,5)P5] (Maiti et al ., 1974; Hayakawa et al ., 1990; Greiner & Larsson Alminger, 2001). The phytate‐degrading enzymes from S. cerevisiae (Greiner et al ., 2001a), Pseudomonas (Cosgrove, 1970), K. terrigena (Greiner et al ., 1997), and A. niger (Irving & Cosgrove, 1972) generate d ‐Ins(1,2,4,5,6)P5 as the major myo ‐inositol pentakisphosphate. Thus, according to the general consideration these enzymes are 3‐phytases.…”

The Influence of Environmental Temperature and Substrate Initial Moisture Content on Aspergillus Niger Growth and Phytase Production in Solid-State Cultivation

“…Upon hydrolysis of phytic acid, phosphorus is freed and the bioavailability of nutrients increases. Phytase is a phosphomonoesterase and is capable of hydrolyzing phytic acid to inorganic orthophosphate, lower esters of myoinositol, and free inositol (Irvine and Cosgrove, 1972). This enzyme is present in plants and tissues, and it is also produced by many species of fungi and bacteria (Consgrove, 1966).…”