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Title: Site-directed mutagenesis of disulfide bridges in Aspergillus niger NRRL 3135 phytase (PhyA), their expression in Pichia pastoris and catalytic characterization

item Mullaney, Edward
item Locovare, Heather
item Sethumadhavan, Kandan
item Boone, Stephanie
item Ullah, Abul

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/4/2010
Publication Date: 7/1/2010
Citation: Mullaney, E.J., Locovare, H., Sethumadhavan, K., Boone, S., Lei, X.G., Ullah, A.H.J. 2010. Site-directed mutagenesis of disulfide bridges in Aspergillus niger NRRL 3135 phytase (PhyA), their expression in Pichia pastoris and catalytic characterization. Applied Microbiology and Biotechnology. 87(4):1367-1372.

Interpretive Summary: When used as an animal feed additive, phytase has been shown to be a very successful in lowering phosphorus levels in manure. This enzyme allows the animals with simple digestive systems (poultry and swine) to utilize the phosphorus found in phytic acid, which is abundant in seeds and grains. Without this enzyme the animal absorbs very little of this nutrient and it ends up in the manure were it can potentially harm the environment by prompting the growth of harmful algae in waterways. A phytase that is currently widely marketed has certain structural features (disulfide bridges) that allow the enzyme to function properly. ARS scientists have now shown how important the structures are to enzymes activity. Bio-molecular techniques were employed to change the fungal gene that codes for this phytase so a series of mutant phytase were produced. This allowed the contribution of each of these five bonds to be quantified. One disulfide bond is essential for activity, remove it and the enzyme is dead. Remove any of the other four disulfide bridges and the characteristics of the enzyme changes notably. One potential important feature observed in these mutants is the shift in the optimum temperature at which the enzyme performs to match the body temperature of swine and poultry. The optimum temperature for the phytase marketed currently is 580C. This mean the enzyme does not perform at its best at body temperature. This discovery of a means to produce a phytase with a lower optimum temperature may have practical significance in the market that has already accepted phytase as an animal feed additive.

Technical Abstract: Earlier studies have established the importance of disulfide bridges in Aspergillus niger NRRL 3135 phytase (phyA). A. niger NRRL 3135 phytase contains five disulfide bridges. In surveying other less characterized fungal phytases the conservation of 8 of the cysteines necessary for the formation of 4 disulfide bridges in A. niger phytase, suggests that they are also utilized for disulfide bridge formation in their respective enzyme. While these bonds are not structural components of the enzyme’s activity site, it is generally accepted that collectively they do contribute to the proper folding of the molecule, which is necessary for activity. In this study the relative importance of each individual disulfide bridges is determined by its removal by site-directed mutagenesis of specific cysteines in the cloned A. niger phyA gene. Individually, these mutant phytases were expressed in a Pichia expression system and their product purified and characterized. The removal of disulfide bridge 2 yielded a mutant phytase with a complete loss of catalytic activity. The other disulfide mutants displayed a broad array of altered catalytic properties including a lower optimum temperature and a modified pH profile. This information now furthers our ability to apply a knowledge-based approach to enhance the effectiveness of phytase as an animal feed additive and other applications.