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ARS Home » Southeast Area » New Orleans, Louisiana » Southern Regional Research Center » Commodity Utilization Research » Research » Publications at this Location » Publication #194717
Title: Unfolding and Refolding of Aspergillus Niger PhyB Phytase: Role of Disulfide Bridges

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

Submitted to: Journal of Agriculture and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/15/2008
Publication Date: 9/10/2008
Citation: Ullah, A.H., Sethumadhavan, K., Mullaney, E.J. 2008. Unfolding and Refolding of Aspergillus Niger PhyB Phytase: Role of Disulfide Bridges. Journal of Agriculture and Food Chemistry. 56(17):8179-8183.

Interpretive Summary: Phytic acid, a phosphorus containing sugar is 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 to soybean meal, fed to animals, it 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 elucidate at molecular level what controls the stability in phytase. Once that is known, only then could phytase be improved by genetic engineering. We have discovered a second phytase that works in lower pH. This enzyme is somewhat more heat stable than the other phytase that work in less acidic environment. In this paper, we show that disulfide bridge, an internal component of the enzyme, plays 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. We also show in this paper that higher salts inhibit the activity in this class of phytase. All the data presented in this communication would help to re-design this important feed additive.

Technical Abstract: Role of disulfide bridges in folding of Aspergillus niger phytase pH 2.5-optimum (PhyB) was investigated using dynamic light scattering (DLS). Guanidinium chloride (GuCl) at 1.0 M unfolded phytase; however, its removal by dialysis refolded the protein. Thiol reagent, tris (2-carboxyethyl) phosphine (TCEP) reduces the refolding kinetics by 68%. Unlike PhyA, hydrodynamic radius (rh) value of PhyB phytase decreased from 5.5 to 4.14 nm. The active homo-dimer, 183-kDa, was reduced to 92-kDa monomer. The DLS data taken together with activity measurements could indicate whether refolding took place or not in PhyB protein. The correlation between molecular mass and the state of unfolding and refolding is a very strong one in fungal phytase belonging to Histidine Acid Phosphatase.