Enhancing the thermal tolerance and gastric performance of a microbial phytase for use as a phosphate-mobilizing monogastric-feed supplement.
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 degrees C for 1 h and 27% of its initial activity after 10 min at 85 degrees C, which was a significant improvement over the appA parental phytase. Phy9X also showed a 3.5-fold enhancement in gastric stability.
Garrett
James B
JB
Diversa Corporation, San Diego, California 92121, USA. jgarrett@diversa.com
Kretz
Keith A
KA
O'Donoghue
Eileen
E
Kerovuo
Janne
J
Kim
William
W
Barton
Nelson R
NR
Hazlewood
Geoffrey P
GP
Short
Jay M
JM
Robertson
Dan E
DE
Gray
Kevin A
KA
D010832:6-Phytase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000135:Acid Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000821:Animal Feed; D000818:Animals; D019587:Dietary Supplements; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005750:Gastric Juice / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D017354:Point Mutation
D029968:Escherichia coli Proteins; D010710:Phosphates; D000135:Acid Phosphatase; D010832:6-Phytase; C406788:appA protein, E coli