2008 Annual Report 1a.Objectives (from AD-416) First, develop phytases with significantly higher specific activity and broad substrate utilization by employing molecular biology techniques. Second, engineer higher heat stability in phytase and combine this with increased specific activity to produce a more cost effective enzyme for the animal feed industry. This will be followed by optimizing the enzymatic and nutritional properties of phytase for specific applications. 1b.Approach (from AD-416) Analyze the sequence and molecular structural data on phytase molecules to achieve higher specific activity for phytic acid and then mutate the substrate specificity site and neighboring amino acid residues in A. niger NRRL 3135 phyA to effect these changes. These same techniques will also be employed to widen the variety of substrates that can be utilized. Increase heat tolerance in phytase will be effected by replacement of specific amino acid residues in the phyA molecule with amino acids occurring at higher frequency in stable proteins; this will result in a mutant phyA with increased heat tolerance. Once this goal is achieved, a combination of increased thermostability with the mutations conferring the higher specific activity will be undertaken. Determination of whether phytate binds to and affects the solubility of chromium and molybdenum, and whether phytate and oxalate interact via cross-linking by calcium and/or magnesium are also planned. 3.Progress Report A patent was granted entitled “Using mutations to improve Aspergillus.” This technology was employed to create a phytase (an enzyme that helps animals digest food)for animal feed with higher activity. This resulted in faster growth and lower phosphorus levels in their manure. A study was published that details the different effects that common salt has on the activity of fungal and bacterial phytase. Calcium chloride extended the pH range of fungal phytase to 8.0 and enhanced its activity. In E. coli phytase, it shifted the optimum pH from 5.5 to 2.0 with no enhancement of activity. Since the two phytases share the same catalytic mechanism, other structural components can account for the observed catalytic and salt effect differences. We broadened the pH range of a second fungal phytase, PhyB to a more characteristic pH range in the stomach of animals through site-directed mutagenesis. Amino acids of the substrate specificity site of PhyA were changed and the resulting mutants characterized. A shift in the optimum pH of mutant E272K from 2.5 to 3.2 meant it more closely matched the gastric environment. This change in pH range of PhyB makes it better suited for development as an animal feed additive. NP 306, Component: 2, Problem Area: c. 4.Accomplishments 1. Adapting phytase to the gastric environment. By altering the optimum pH of fungal phytase to match the pH of the stomach of an animal more of the phytin phosphorus is made available to the animal. This promotes faster animal growth and reduces phosphorus levels in the manure. A low phosphorus level in manure is important because it protects our environment and helps conserves the world’s phosphorus reserves. Application of this technology to a second phytase (Appl. Microbiol. Biotechnol., 76:117-122) demonstrates it has the potential for a wide application. NP 306, Component: 2, Problem Area: c. 2. Increasing thermostability of phytase. Animal feed is sometimes heated briefly during processing. In order to retain the high level of activity, a heat tolerant phytase is desirable. Earlier studies have focused on finding a phytase with thermostability and attempting to incorporate several desirable features to make it commercially viable. We successfully took a different approach to this problem by starting with a widely marketed phytase and increased its thermostability (Appl. Environ. Microbiol., 73:3069-3076). This technique provides the animal feed producers with an improved phytase with an already proven track record. NP 306, Component: 2, Problem Area: c. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of the New MTAs (providing only) 1 Number of Invention Disclosures Submitted 2 Number of New Patent Applications Filed 1 Review Publications Kadan, R.S., Phillippy, B.Q. 2007. Effects of yeast and bran on phytate degradation and minerals in rice bread. Journal of Food Science and Technology. 72(4):C208-C211. Weaver, J.D., Mullaney, E.J., Lei, X.G. 2007. Altering the substrate specificity site of aspergillus niger PhyB shifts the pH optimum to pH 3.2. Applied Microbiology and Biotechnology. (2007)76:117-122. Ullah, A.H., Sethumadhavan, K., Mullaney, E.J. 2008. Salt effect on the pH profile and kinetic parameters of microbial phytases. Journal of Agricultural and Food Chemistry. 56:3398-3402. Lei, X. G., Porres, J. M., Mullaney, E. J. and Brinch-Pedersen, H. 2007. Phytase: source, structure and application. In: Industrial Enzymes, structure, function and applications. Editied by Polina, J. and MacCabe, A. P. Springer, Dordrecht, The Netherlands, p. 505-529. Weaver, J.D., Ullah, A.H., Sethumadhavan, K., Mullaney, E.J., Lei, X.G. 2007. Enzymatic comparisons of Aspergillus Niger PhyA and Escherichia Coli AppA2 Phytases (abstract). Journal of Animal Science. 85(Supplement 1):647.