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United States Department of Agriculture

Agricultural Research Service

Title: Unfolding and Refolding Studies of Fungal Phytase Using Dynamic Light Scattering

Authors
item Ullah, Abul
item Sethumadhavan, Kandan

Submitted to: Protein Society Symposium
Publication Type: Abstract Only
Publication Acceptance Date: May 14, 2004
Publication Date: May 14, 2004
Citation: Ullah, A.H., Sethumadhavan, K. Unfolding and refolding studies of fungal phytase using dynamic light scattering(abstract). American Society for Biochemistry & Molecular Biology Annual Meeting 2004 FASEB Journal, Enzyme Structure, and Function. 8(18):C24.

Technical Abstract: Aspergillus ficuum phytase encoded by phyA produces a very stable phosphohydrolase (EC 3.1.3.8) capable of degrading phytate at 58ºC. This enzyme is being produced by industry for use in animal feed to degrade phytate. To understand the molecular basis of high stability in fungal phytase, we have studied the unfolding and refolding of the enzyme in the presence and absence of protein denaturant, guanidinium hydrochloride (Gu.HCl). The role of 5 disulfide bridges in refolding mechanism were also probed. We focused our measurements based on two criteria: one, the enzymes shape as determined by hydrodynamic radius (rh) of the molecule by dynamic light scattering (DLS) instrument and two, by enzyme's activity using phytate as substrate. Both ß-mercaptoethanol and Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was used to reduce disulfide bridges under denaturing condition. Phytase's rh increased remarkably concomitant with unfolding from the native state by increasing amounts of Gu.HCl. The unfolded phytase could refold in minutes in assay medium because of the dilution of the denaturant. Both the rh value and the activity restores upon refolding. Incorporating TCEP in the refolding medium could block the refolding process as indicated by a high rh value of the unfolded protein and catalytic demise. These indicate that disulfide bridge formation is a prerequisite to refolding of phytase. The rh values of protein in the presence or absence of a denaturant obtained by DLS instrument could provide valuable information about its folding status. Coupling these data with activity will provide insight into a protein's folding mechanism.

Last Modified: 4/23/2014
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