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Applied and Environmental Microbiology, May 2004, p. 3041-3046, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.3041-3046.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Enhancing the Thermal Tolerance and Gastric Performance of a Microbial Phytase for Use as a Phosphate-Mobilizing Monogastric-Feed Supplement

James B. Garrett,* Keith A. Kretz, Eileen O'Donoghue, Janne Kerovuo, William Kim, Nelson R. Barton, Geoffrey P. Hazlewood, Jay M. Short, Dan E. Robertson, and Kevin A. Gray

Diversa Corporation, San Diego, California 92121

Received 22 August 2003/ Accepted 17 January 2004

The inclusion of phytase in monogastric animal feed has the benefit of hydrolyzing indigestible plant phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) to provide poultry and swine with dietary phosphorus. An ideal phytase supplement should have a high temperature tolerance, allowing it to survive the feed pelleting process, a high specific activity at low pHs, and adequate gastric performance. For this study, the performance of a bacterial phytase was optimized by the use of gene site saturation mutagenesis technology. Beginning with the appA gene from Escherichia coli, a library of clones incorporating all 19 possible amino acid changes and 32 possible codon variations in 431 residues of the sequence was generated and screened for mutants exhibiting improved thermal tolerance. Fourteen single site variants were discovered that retained as much as 10 times the residual activity of the wild-type enzyme after a heated incubation regimen. The addition of eight individual mutations into a single construct (Phy9X) resulted in a protein of maximal fitness, i.e., a highly active phytase with no loss of activity after heating at 62°C for 1 h and 27% of its initial activity after 10 min at 85°C, which was a significant improvement over the appA parental phytase. Phy9X also showed a 3.5-fold enhancement in gastric stability.

* Corresponding author. Mailing address: Diversa Corporation, 4955 Directors Pl., San Diego, CA 92121. Phone: (858) 526-5262. Fax: (858) 526-5762. E-mail: jgarrett{at}diversa.com.

Applied and Environmental Microbiology, May 2004, p. 3041-3046, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.3041-3046.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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