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Appl. Environ. Microbiol. doi:10.1128/AEM.02970-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Adopting Selected Hydrogen Bonding and Ionic Interactions from Aspergillus fumigatus Phytase Structure Improves Thermostability of Aspergillus niger PhyA Phytase

Department of Animal Science, Cornell University, Ithaca, New York 14853; Southern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, New Orleans, Louisiana 70124

^* To whom correspondence should be addressed. Email: XL20{at}cornell.edu.

	Abstract

Although it has been widely used as a feed supplement to reduce manure phosphorus pollution of swine and polutry, Aspergillus niger PhyA phytase is unable to withstand heat inactivation during feed pelleting. Crystal structure comparisons with its close homolog, the thermostable A. fumigatus phytase (Afp), suggest associations of thermostability with several key residues (E35, S42, R168, and R248) that form hydrogen bond network in E35-S42 region, and ionic interactions between R168 and D161, and between R248 and D244. In this study, loss-of-function mutations (E35A, R168A, and R248A) were introduced singularly or in combination into 7 mutants of Afp. All 7 mutants displayed decreases in thermostability, with the highest loss (25%, P < 0.05) in the triple mutant (E35A/R168A/R248A). Subsequently, a set of corresponding substitutions were introduced into 9 mutants of PhyA to strengthen the hydrogen bonding and ionic interactions. While four mutants showed improved thermostability, the best response came from the quadruple mutant (A58E/P65S/Q191R/T271R) that retained 20% greater (P < 0.05) activity after being heated at 80°C for 10 min and had 7°C higher melting temperature than that of wild type PhyA. This study demonstrates the functional importance of hydrogen bond network and ionic interaction in supporting the high thermostability of Afp and the feasibility of adopting these structural units in improving thermostability of another homologous PhyA phytase.

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