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Engineering of phytase for improved activity at low pH
Andrea Tomschy1, Roland Brugger, Martin Lehmann
1Biotechnology Department, Roche Vitamins, Ltd., 4070 Basel, Switzerland.
Applied and Environmental Microbiology
|March 28, 2002
Summary
Researchers engineered phytase enzymes for animal feed by modifying amino acids. These changes successfully lowered the pH optimum, enhancing enzyme activity in the digestive tract and providing new tools for enzyme design.
Area of Science:
- Enzymology
- Protein Engineering
- Biotechnology
Background:
- Phytase enzymes are crucial for animal feed applications, requiring optimal activity within the digestive tract's pH range.
- Engineering phytase pH activity profiles is essential for improving industrial applications.
Purpose of the Study:
- To rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases.
- To identify and modify active-site amino acids to achieve desired pH optima.
Main Methods:
- Site-directed mutagenesis was employed to alter specific amino acid residues in phytase enzymes.
- Glycinamidylation was used to decrease the negative surface charge of a phytase mutant.
- Amino acid sequence alignments and structural analysis guided the identification of key residues.
Main Results:
- Decreasing negative surface charge via glycinamidylation lowered the pH optimum but caused significant inactivation.
- Mutating Gly-277 and Tyr-282 to Lys and His, respectively, in A. fumigatus phytase created a second pH optimum at 2.8–3.4.
- Specific mutations (K68A, S140Y D141G) decreased pH optima by 0.5–1.0 unit with maintained or increased specific activity.
Conclusions:
- Rational engineering of phytase active sites can effectively modulate pH activity profiles.
- Mutations altering surface charge and active-site residues provide tools for optimizing phytases for specific industrial needs.
- These findings offer significant advancements in designing tailored phytase enzymes.