Research For Nonexponential Thermoinactivation Process Of Candida Uricase

Uricase is a therapeutic enzyme to treat refractory gout and a tool enzyme for serum uric acid assay in clinical laboratories, but require both thermostability and activity as high as possible. A new fungal uricase from a strain of Candida was found to have a non-exponential decrease of activity; its apparent t0.5 was much longer at pH 9.2 or in the presence of oxonate. Kinetic analysis of the non-exponential decrease of activity of the fungal uricase during thermoinactivation at 37 0C with a model to mine structural factors of thermostability will guide the design of a mutant of uricase for stronger thermostability.1. Characterization of thermoinactivation process of Candida uricase.After expanding culture the strain of Candida utilis(C.G.M.C.C. 2.1008), uricase was expressed by uric acid-induced. Crude enzyme solution was collected by centrifuging fungal cells, cell sonication, centrifuging and discarding cell debris. After purification by DEAE-cellulose twice and characterization by SDS-PAGE, purified uricase was obtained to have specific activity of 10.0 kU g-1.Sample analysis gave following results: 1) protein sample had purity of 93.26%. 2) the relative molecular mass was 17.35 KD. 3) MALDI- TOF / TOF showed sequence similar to FAD-dependent pyridine nucleotide-disulfide oxidoreductase(gi | 154253900). 4) No amino acid residues were released after ten Edman degradation, indicating the blocked N-terminusTo monitor thermoinactivation process, all solution samples were diluted to 0.03 g L-1 proteins, with 20 mM sodium borate at pH 7.4 or 9.2 plus antibiotics, 2.0 mM p-aminobenzamidine, 0.20 mM EDTA and 0.20 mM PMSF. Samples of the fungal uricase were incubated at 37 0C after the tubes were sealed with parafilm. At an indicated time, aliquots of samples were taken under sterile conditions to measure residual activity. The prepared fungal uricase had much stronger thermostability at 37 0C and pH 7.4 or 9.2, but a non-exponential decrease of residual activities, with a plateau period prior to the exponential decrease of activity of 4 days at pH 7.4 and 12 days pH 9.2, the binding of oxonate greatly prolonged the plateau period to 24 days at both pH values.By SDS-PAGE and acidic PAGE, it was found thermoinactivation at 37 0C caused the dissociation of the homotetratmer into homodimers and then monomers of smaller weight and higher mobility; there was negligible degradation of polypeptides during thermoinactivation of the uricase. 2. Kinetic model for thermoinactivation processKinetic model for thermoinactivation process of the prepared fungal uricase was proposed based on the some hypotheses: There are surely multiple noncovalent interactions between homodimers in a homotetramer and thus multiple conformational intermediates of the homotetramer of the fungal uricase before its dissociation into two nondenatured homodimers. With a set of preset parameters for the group of rate equations, a simulated thermoinactivation curve was calculated by iterative numerical integration. By fitting to the experimental curve, the simulated thermoinactivation curve for least-square-fitting(LSF) gave parameters for the kinetic model.Fitting by software Matlab,Clearly, m, rather than k-2 and k-1, primarily determined the plateau period prior to the exponential decrease. With no ligand induced, m7.4 =24,m9.2 = 64; after the addition of the ligand Oxonate, m7.4 =24, m9.2 = 64. The binding of oxonate may cause conformation changes to stabilize the nondenatured homodimer as a complex and retard its reassociation into the homotetramer, with much smaller k-2, k-1 and k2 for the fungal uricase. 3. Rational design of thermostable mutants of a bacterial uricaseBased on the kinetic model for thermoinactivation of the fungal uricase, a mutant of Bacillus fastidiosus uricase was designed and purified successfully. Without the ligand, there were no plateau period before the exponential decrease of activity, and the thermoinactivation of the nondenatured homodimers is the rate-determinant. But when induced by oxonate, it can be observed a plateau period for 7-9 days. Adding of ligand can increase the reversible conformation changes, and the dissociation of the homotetramer into the homodimers became the rate-determinant, thus the plateau period showed up. The increase in the number of diverse noncovalent interactions helpful to thermostability of this structure form, or the increase of the strengths of noncovalent interactions crucial for its thermostability, may have large enough m to produce a plateau period prior to the exponential decrease of activity. Kinetic analysis of thermoinactivation process was helpful to design thermostable mutants...