Two current methods (McKercher and Anderson and Steward and Tate 18 ) for the estimation of inositol polyphosphates in soil extracts were examined for accuracy by subjecting the inositol polyphosphate fraction obtained by each method to oxidation with alkaline hypobromite. Anion-exchange chromatography of the resistant phosphate showed that with both methods a large proportion of the organic phosphorus in the fraction was neither inositol pentakisphosphate nor inositol hexakisphosphate. The results of methods for the estimation of these compounds in soil extracts which do not include an oxidation with alkaline hypobromite must therefore be suspect. 495 Copyright© 1981 by Marcel Dekker, Inc. 496 IRVING AND.COSGROVE

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)....

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