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Applied and Environmental Microbiology, May 2007, p. 3069-3076, Vol. 73, No. 9
0099-2240/07/$08.00+0     doi:10.1128/AEM.02970-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Wanming Zhang,1 Edward J. Mullaney,2 and Xin Gen Lei1*

Department of Animal Science, Cornell University, Ithaca, New York 14853,1 Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, Louisiana 701242

Received 21 December 2006/ Accepted 26 February 2007

Although it has been widely used as a feed supplement to reduce manure phosphorus pollution of swine and poultry, Aspergillus niger PhyA phytase is unable to withstand heat inactivation during feed pelleting. Crystal structure comparisons with its close homolog, the thermostable Aspergillus fumigatus phytase (Afp), suggest associations of thermostability with several key residues (E35, S42, R168, and R248) that form a hydrogen bond network in the E35-to-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 seven mutants of Afp. All seven 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 nine 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), which retained 20% greater (P < 0.05) activity after being heated at 80°C for 10 min and had a 7°C higher melting temperature than that of wild-type PhyA. This study demonstrates the functional importance of the hydrogen bond network and ionic interaction in supporting the high thermostability of Afp and the feasibility of adopting these structural units to improve the thermostability of a homologous PhyA phytase.

* Corresponding author. Mailing address: Department of Animal Science, 252 Morrison Hall, Cornell University, Ithaca, NY 14853. Phone: (607) 254-4703. Fax: (607) 255-9829. E-mail: XL20{at}cornell.edu

{triangledown} Published ahead of print on 9 March 2007.

Applied and Environmental Microbiology, May 2007, p. 3069-3076, Vol. 73, No. 9
0099-2240/07/$08.00+0     doi:10.1128/AEM.02970-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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