Objective: To study the enzyme kinetic characteristics of recombinant Uricase and PEGylated Uricase. Methods: Two methods for the determination of recombinant uricase activity, substrate depletion method and product generation method were established; the results from both assays were consistent. The urine substrate depletion method was chosen to compare the activity and enzyme kinetic parameters of uricase with or without polyglycol modification. Results: Within the optimal pH (9.0) at 37 ℃, the related Vmax, Km and Kcat of free recombinant Uricase were 0.013 μmol·L-1·min-1, 33.77 μmol·L-1 and 889.0 min-1, respectively; while the optimal pH for recombinant Uricase with polyglycol modification was 9.5 at 37 ℃ with Vmax 0.009 μmol·L-1·min-1, Km 31.52 μmol·L-1 and Kcat 615.5 min-1 correspondingly. Under the optimum pH conditions, the specific enzyme activity of Uricase with and without PEG modification,were both constant at 12.5 U·mg-1. Conclusions: Under the optimum pH conditions, the enzyme activity as well as the Km value of the recombinant uricase with or without PEG modification were consistent, indicating that the ratio activity of the recombinant Uricase was not affected by PEG modification, niether the affinity to substrates. Moreover, compared with the free recombinant Uricase, the Vmax of PEG modified enzyme shows about 30% decrease, indicating that the PEG modification may cause the change of tetramer conformation, which could affect the maximum reaction rate of Uricase.
FAN Neng-quan, ZHANG Pu, JIANG Bo, LIU Ri-yong, PENG Lan
. Comparative study on enzymatic kinetics of recombinant Uricase and PEGylated recombinant Uricase*[J]. Chinese Journal of Pharmaceutical Analysis, 2021
, 41(11)
: 1947
-1953
.
DOI: 10.16155/j.0254-1793.2021.11.12
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