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Title: Altering the Substrate Specificity Site of Aspergillus Niger PhyB shifts the pH optimum to pH 3.2

Author
item WEAVER, JEREMY - CORNELL UNIVERSITY
item Mullaney, Edward
item LEI, XIN - CORNELL UNIVERSITY

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/28/2007
Publication Date: 4/21/2007
Citation: 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.

Interpretive Summary: Fungal phytase has been shown to be a very effective means to lower phosphorus levels in manure when used as an animal feed additive. Phytase allows animals with simple digestive systems (poultry and swine) to utilize the phosphorus found in phytic acid, which is abundant in the seeds and grains commonly used in animal feed. Without this enzyme the animal absorbs very little of this phosphorus and instead it ends up in the manure where it can potentially harm the environment by spurring the growth of harmful algae in waterways. These algal blooms have been known to cause large fish kills. Previously we have use molecular technology to alter the fungal gene that produces a commercial phytase so that its product works better at the pH levels found in the stomach of pigs. In this study we applied these same techniques to a second fungal phytase that is not commercially market. The second phytase has many desire features, but its narrow pH range has limited its commercial application. By altering it pH profile of the enzyme to match conditions found in the stomach, this other phytase now becomes a candidate for utilization in animal feed and also provides another means to protect the environment.

Technical Abstract: Phytases are of biotechnological importance as animal feed additives for their ability to catalyze the hydrolysis of phosphate from phytate for absorption by simple-stomached animals, and to reduce their fecal phosphorus excretion. Aspergillus niger PhyB has high catalytic activity at low pHs around 2.5, but has little activity at the commonly observed gastric pH of young animals (3.0-3.5). Our objective was to determine if the pH optima of PhyB could be broadened to a more characteristic pH range in the stomach of young animals through site-directed mutagenesis. We created two mutants, E272K and E272Q, each with a single amino acid substitution of the same residue in the substrate specificity site. Mutants were designed to replace an acidic amino acid, with either a neutral amino acid (E272Q) or basic amino acid (E272K), and were overexpressed in the yeast Pichia pastoris. While the wild-type (WT) pH optimum was 2.5, mutant E272K shifted to a new optimum of pH 3.2. E272K had a concomitant reduction in K (m) of 36-fold at pH 2.5 and 6-fold at pH 3.2 compared to the WT. Our results indicate that the pH optimum of PhyB can be altered to match the stomach pH, along with an improved substrate affinity.