Subject(s)
Mutagenesis/genetics , Proteins/genetics , Amino Acid Sequence , Amino Acids/genetics , Codon/genetics , DNA Primers , Escherichia coli/genetics , Genetic Techniques , Hydrolases/chemistry , Hydrolases/genetics , Models, Molecular , Molecular Sequence Data , Protein Conformation , Restriction Mapping/methods , Rhodococcus/enzymology , Rhodococcus/geneticsABSTRACT
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.
Subject(s)
6-Phytase/genetics , 6-Phytase/metabolism , Acid Phosphatase/genetics , Acid Phosphatase/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Gastric Juice/enzymology , Hot Temperature , 6-Phytase/chemistry , Acid Phosphatase/chemistry , Animal Feed , Animals , Dietary Supplements , Enzyme Stability , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Hydrogen-Ion Concentration , Models, Molecular , Mutagenesis, Site-Directed , Phosphates/metabolism , Point MutationABSTRACT
Directed evolution technologies were used to selectively improve the stability of an enzyme without compromising its catalytic activity. In particular, this article describes the tandem use of two evolution strategies to evolve a xylanase, rendering it tolerant to temperatures in excess of 90 degrees C. A library of all possible 19 amino acid substitutions at each residue position was generated and screened for activity after a temperature challenge. Nine single amino acid residue changes were identified that enhanced thermostability. All 512 possible combinatorial variants of the nine mutations were then generated and screened for improved thermal tolerance under stringent conditions. The screen yielded eleven variants with substantially improved thermal tolerance. Denaturation temperature transition midpoints were increased from 61 degrees C to as high as 96 degrees C. The use of two evolution strategies in combination enabled the rapid discovery of the enzyme variant with the highest degree of fitness (greater thermal tolerance and activity relative to the wild-type parent).
