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Applied and Environmental Microbiology, June 2006, p. 4397-4403, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02612-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Shifting the pH Profile of Aspergillus niger PhyA Phytase To Match the Stomach pH Enhances Its Effectiveness as an Animal Feed Additive

Taewan Kim,1 Edward J. Mullaney,2 Jesus M. Porres,1 Karl R. Roneker,1 Sarah Crowe,1 Sarah Rice,1 Taegu Ko,1 Abul H. J. Ullah,2 Catherine B. Daly,2 Ross Welch,3 and Xin Gen Lei1*

Department of Animal Science, Cornell University, Ithaca, New York 14853,1 Southern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, New Orleans, Louisiana 70124,2 Soil and Nutrition Laboratory, United States Department of Agriculture, Ithaca, New York 148533

Received 5 November 2005/ Accepted 4 April 2006

Environmental pollution by phosphorus from animal waste is a major problem in agriculture because simple-stomached animals, such as swine, poultry, and fish, cannot digest phosphorus (as phytate) present in plant feeds. To alleviate this problem, a phytase from Aspergillus niger PhyA is widely used as a feed additive to hydrolyze phytate-phosphorus. However, it has the lowest relative activity at the pH of the stomach (3.5), where the hydrolysis occurs. Our objective was to shift the pH optima of PhyA to match the stomach condition by substituting amino acids in the substrate-binding site with different charges and polarities. Based on the crystal structure of PhyA, we prepared 21 single or multiple mutants at Q50, K91, K94, E228, D262, K300, and K301 and expressed them in Pichia pastoris yeast. The wild-type (WT) PhyA showed the unique bihump, two-pH-optima profile, whereas 17 mutants lost one pH optimum or shifted the pH optimum from pH 5.5 to the more acidic side. The mutant E228K exhibited the best overall changes, with a shift of pH optimum to 3.8 and 266% greater (P < 0.05) hydrolysis of soy phytate at pH 3.5 than the WT enzyme. The improved efficacy of the enzyme was confirmed in an animal feed trial and was characterized by biochemical analysis of the purified mutant enzymes. In conclusion, it is feasible to improve the function of PhyA phytase under stomach pH conditions by rational protein engineering.

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

Applied and Environmental Microbiology, June 2006, p. 4397-4403, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02612-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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