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This article in SSSAJ

  1. Vol. 75 No. 3, p. 880-889
     
    Received: Sept 15, 2010
    Accepted: Apr 19, 2011


    * Corresponding author(s): duberry@vt.edu
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doi:10.2136/sssaj2010.0353

Assessing Phytase Activity in Forested and Agricultural Soils using TInsP5 as a Substrate Analog

  1. Duane F. Berry *a,
  2. Hubert L. Walkera,
  3. Kim Harichb and
  4. Chao Shanga
  1. a Dep. of Crop and Soil Environmental Sciences, Virginia Polytechnic Institute & State Univ., Blacksburg, VA 24061
    b Dep. of Biochemistry, Virginia Polytechnic Institute & State Univ., Blacksburg, VA 24061

Abstract

The usefulness of the chromophoric substrate analog of phytic acid, 5-O-[6-(benzoylamino)hexyl]-d-myo-inositol-1,2,3,4,6-pentakisphosphate (TInsP5) for measuring phytase activity (PA) was recently demonstrated for a limited number of soils using a macroassay approach. In this investigation, we assessed PA in several previously untested forested and agricultural soils. The TInsP5 molecular probe (wavelength of most intense light absorption, λmax = 226 nm) permits direct measurement of the phytase-catalyzed dephosphorylation reaction by quantifying production of intermediate probe species using reversed-phase high-performance liquid chromatography with ultraviolet detection (RP-HPLC/UV). Soil-generated phosphorylated probe intermediate species were characterized or identified with RP-HPLC with diode array detector and RP-HPLC/UV/mass spectrometry (MS). Results of the MS analysis showed that the RP-HPLC system is capable of distinguishing between phosphorylated probe intermediate species at the regioisomeric level. Chromatographic profiles of soil-generated intermediates were established and a streamlined microassay approach for measuring PA was used to examine the influence of assay pH on soil PA. The chromatographic profiles of probe intermediate species produced in the soils tested were comparable to those previously observed. Recognizable chromatographic profiles of the intermediates will undoubtedly facilitate data interpretation among soils. Experimental data gathered in this investigation indicated that microbial populations tend to produce phytate-degrading enzymes exhibiting pH optima corresponding to the soil pH. The PA assay was responsive to environmental conditions.

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