Persistence of {221}-Propeller Phytase in Soil and Its Implication in Phosphorus Mobilization

This dissertation, "Persistence of {221}-propeller Phytase in Soil and Its Implication in Phosphorus Mobilization" by Miu-fun, Leung, 梁妙芬, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled PERSISTENCE OF β-PROPELLER PHYTASE IN SOIL AND ITS IMPLICATION IN PHOSPHORUS MOBILIZATION Submitted by Leung Miu Fun for the degree of Master of Philosophy at The University of Hong Kong in August 2004 Plants meet their phosphorus requirement by the uptake of phosphate ions via their roots. In terrestrial systems, organic phosphorus constitutes 20-80% of the total soil phosphorus. A range of organic P compounds are found in soil, with inositol hexakisphosphate (phytate) as the predominant constituent. Phytate is the major phosphorus storage compound in plants. Release of plant phytate during plant decay is the major origin of soil phytate. The preferential accumulation of inositol phosphates in soil is due to their adsorption to soil colloids, which hampers their biodegradation. To be available to plants, soil phytate must first be mineralized to phosphate, a process mediated by phosphatases. To improve phosphorus availability, a neutral β-propeller phytase from Bacillus subtilis was previously transformed into tobacco to enhance its ability to absorb phosphorus from soil phytate. The current study determined how long the phytase derived from transgenic tobacco could persist in soil by simulating field conditions. Transgenic phytase was found to be more resistant to biotic degradation than crude Bacillus phytase in soil, possibly due to glycosylation which might render it less susceptible to protease digestion. Longer persistence of phytase during plant decay might reduce the release of plant phytate to soil, or alternatively, promote the release of inorganic phosphate to soil. Phosphorus from soil phytates are not readily available for enzyme decomposition, as they are tightly complexed with cations or adsorbed to aluminum and iron(III) precipitates in acid soils, or to insoluble calcium salts and clays in alkaline soils. The abilities of phytases from Bacillus subtilis, Aspergillus ficuum and wheat in mobilizing soil phytate were compared in vitro. Their amino acid sequences, 3-dimensional structures and biochemical properties revealed that they belonged to three different classes of phytases: β-propella phytase, histidine acid phosphatase, and purple acid phosphatase, respectively. When phytate was complexed with cations, all three classes of phytases were able to utilize phytate complexed with calcium, magnesium and manganese ions. However, they exhibited no activity towards phytate salts complexed with copper(II), zinc(II), iron(II), iron(III) and aluminum(III) ions. On the other hand, when phytate was adsorbed to aluminum and iron(III) precipitates, wheat phytase was the most effective enzyme in releasing inorganic phosphates from phytate. Plant roots also excreted organic acids in response to P deficiency. Interference by organic acids on the activities of all three classes of phytases was also examined. Oxalate and citrate were inhibitory to the phytases with wheat phytase being the most resistant enzyme to increasing concentrations of citrate. Based on the above results, wheat phytase might be a better candidate than histidine acid phosphatase and β-propella phytase for mobilizing soil phytate in transgenic studies. DOI: 10.5353/th_b3073651 Subjects: Phytases Soils - Phosphorus content