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ARS Home » Southeast Area » New Orleans, Louisiana » Southern Regional Research Center » Commodity Utilization Research » Research » Publications at this Location » Publication #172493


item Ullah, Abul
item Sethumadhavan, Kandan
item Mullaney, Edward

Submitted to: Biochemical and Biophysical Research Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/29/2004
Publication Date: 2/25/2005
Citation: Ullah, A.H., Sethumadhavan, K., Mullaney, E.J. 2005. Monitoring of unfolding and refolding in fungal phytase (phyA) by dynamic light scattering. Biochemical and Biophysical Research Communications. 327(4):993-998.

Interpretive Summary: Phytic acid, a natural compound produced in soybean and other legume as the seeds mature, is a known antinutrient. It binds important minerals rendering them unavailable to poultry, hog, and human because an enzyme by the name of phytase that could degrade phytic acid is absent in the gut of these animals. One way to circumvent the problem is to add phytase in the soybean meal that then will be able to breakdown phytic acid and make the meal more nutritious. A fungal strain makes phytase, which has been used to supplement soybean meal for use as poultry and hog meal. However, the fungal phytase has some weak properties one of them being the heat lability. We are trying to understand at molecular level what controls the stability in phytase. Once that is known, then phytase could be improved by knowledge-based protein engineering. In this paper, we show that disulfide bridges, which phytase has 5 of them, play an important role in its folding. Perhaps by incorporating one additional disulfide bridge near the catalytic center of phytase we could improve the stability and heat tolerance of the protein. We also have shown in this paper that a dynamic light scattering instrument can be used to monitor both the unfolding and refolding processes in phytase. These processes are very important for any enzyme and hold the key for their stability. These fundamental processes should be unraveled before one could do a rational design to improve any enzyme's structure leading up to their improvement and impart stability. This would then translate into improving the usefulness any biocatalyst. In our case, these findings will help us design a better phytase for mankind.

Technical Abstract: Role of disulfide bridges in phytase's unfolding-refolding was probed using dynamic light scattering. Phytase was unfolded by Gu.HCl and then refolded by removing the denaturant by rapid dialysis. Thiol active reagents prevented refolding; thus, disulfide bridge formation in phytase is an integral step in folding. Catalytic demise of phytase after unfolding and refolding in presence of TCEP indicates that disulfide bridges are necessary for active site formation. The rh of the active and unfolded phytase is 4 and 14 nm, respectively. Removal of denaturant through dialysis refolds phytase; its rh shifts back to 4 nm. When TCEP remains in the refolding media, the rh remains high. The unfolded phytase when diluted in assay medium refolds as a function of time at 25º C and 37º C, but not higher temperature. Monitoring rh of phytase under denaturing and renaturing condition gives an accurate measure of the folding status of the enzyme.