Location: Commodity Utilization Research
Title: A single mutation in the hepta-peptide active site of Aspergillus niger PhyA phytase leads to myriad of biochemical changes Authors
Submitted to: Advances in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 13, 2012
Publication Date: September 1, 2012
Citation: Ullah, A.H.J., Sethumadhavan, K., Boone, S., Mullaney, E.J. 2012. A single mutation in the hepta-peptide active site of Aspergillus niger PhyA phytase leads to myriad of biochemical changes. Advances in Microbiology. 2(3):388-394. Interpretive Summary: Phytic acid is a plant metabolite rich in phosphate. Soybean and other legume produce and store phytic acid in the seeds for future use during germination. Meals made from beans and legumes for poultry, swine, and humans when consumed in large amounts cause problems to the animals because phytic acid acts as antinutrient because it binds minerals; thereby robbing the body of essential nutrients such as calcium, manganese, copper, iron, etc. One way to make soybean meals and other legume-based meal nutritious is to enzymatically breakdown phytic acid prior to feeding the meals to simple-stomached animals. Of all studied phytic acid-degrading enzyme, phytase, one produced by the fungus Aspergillus niger drew the most attention of researchers worldwide. Thus, phytase produced by this fungus was commercialized in early 1990s and the enzyme is marketed as a feed supplement to commercial feed producers. There is a quest for making an improved version of the enzyme to make the biocatalyst more effective and stable. We have taken a knowledge-based route to improve the function of the fungal phytase and have employed various biochemical and bioinformatics-based approach to improve the structure of phytase so that genetically modified phytase would perform optimally when fed to the animals as a feed supplement. In this paper we provide scientific evidences for a improved phytase, which we generated by changing only one amino acid from alanine to glutamic acid at residue number 69. We characterized the mutant protein and compared its functionality against the unmodified phytase, which is termed the native and wild type. The mutant phytase has not only altered pH and temperature optima but also favorable enzymatic properties. Furthermore, the chemical stability of the mutant phytase was also enhanced by the knowledge-based single mutation. This mutation could be incorporated into existing mutants of phytase to come up with a superior phosphate degrading enzyme for animal feed industries.
Technical Abstract: The active site motif of proteins belonging to ‘Histidine Acid Phosphatase’ (HAP) contains a hepta-peptide region, RHGXRXP. A close comparison among fungal and yeast HAPs has revealed the fourth residue of the hepta-peptide to be E instead of A, which is the case with A. niger phyA phytase. However, another phytase, phyB, from the same microorganism has a higher turnover number and it shows E in this position. We have mutated A69 residue to E in the fungal phyA phytase. The mutant phytase shows a myriad of new kinetic properties. The pH profile shifted 0.5 pH unit in both 5.0 and 2.5 bi-hump peaks that are characteristics of the fungal phytase. The optimum temperature shifted from 58º C to 55º C. However, the greatest difference was observed in the mutant protein’s reaction to GuCl at a concentration of 0.1 to 0.2 M. The activity of the mutant phytase jumped 100% while the wild type protein showed no activity enhancement in the same concentration range of GuCl. The kinetics performed at higher concentration of Gu.Cl also contrasted the difference between the wild type and mutant phytase. While Km was least affected, the Vmax increased for the mutant and decreased for the wild type. The sensitivity towards myo-inositol hexasulfate, a potent inhibitor, was decreased by the mutation. All in all, A69E mutation has affected a multitude of enzymatic properties of the protein even though the residue was thought to be non-critical for phytase’s catalytic function notwithstanding its location in the conserved hepta peptide region of the biocatalyst.