Engineering of Phytase for Improved Activity at Low pH

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Abstract
For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles ofAspergillus fumigatusand consensus phytases. Decreasing the negative surface charge of theA. fumigatusQ27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH ofA. nigerNRRL 3135 phytase,A. nigerpH 2.5 acid phosphatase, andPeniophora lyciiphytase. Site-directed mutagenesis confirmed that, inA. fumigatuswild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues ofA. nigerphytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in bothA. fumigatusand consensus phytase backbones), as well as the S140Y D141G double mutation (inA. fumigatusphytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.

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