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Item Bio-conjugation of Bacillus Fastidiosus-Uricase with methoxy polyethylene glycol derivative and study of physiochemical properties(2012) Nanda, P.; JagadeeshBabu, P.E.; Tekalkote, S.; Kunnummal, B.M.; Kaleekkal, N.Uricase (EC 1.7.3.3, UC) is an enzyme belonging to the class of oxidoreductases and catalyses the oxidation of uric acid to allantoin, carbon dioxide and hydrogen peroxide. In this present work, Uricase from Bacillus fastidisous was conjugated with methoxypolyethyleneglycol p-nitrophenyl carbonate (mPEG-np) a polyethylene glycol derivative, in order to improve the pharmaceutical properties of therapeutic enzyme uricase. The PEGylated conjugates (uricase-mPEG-np) were synthesized using various ratios of uricase and mPEG-np to get maximum residual activity. The PEGylated uricase showed maximum residual uricolytic activity of 90.9% compared to the unmodified uricase, which was achieved at a ratio of 1:17 of uricase to mPEG-np. PEGylated uricase was further characterized using SDS-PAGE to determine its final molecular weight and approximate number of mPEG molecules attached. The result showed that the molecular weight was increased to 79.4 KDa and the number of mPEG molecules bound per subunit of uricase was approximately 9. Stability of the PEGylated uricase at various temperature and pH was studied and found to be 32°C and pH of 9.0. Further the mechanism of binding and possible sites of binding were studied using molecular modeling and docking software tool ArgusLab 4.0.1 and the two-dimensional image of docked uricase were generated.Item Isolation, screening and production studies of uricase producing bacteria from poultry sources(Taylor and Francis Inc. 325 Chestnut St, Suite 800 Philadelphia PA 19106, 2014) Nanda, P.; JagadeeshBabu, P.E.Uricase (urate oxidase EC 1.7.3.3) is a therapeutic enzyme that is widely used to catalyze the enzymatic oxidation of uric acid in the treatment of hyperuricemia and gout diseases. In this study, three bacterial species capable of producing extracellular uricase were isolated from a poultry source and screened based on the size of the clear zone using a uric acid agar plate. The bacterial species capable of producing uricase with the highest uricolytic activity was identified as Bacillus cereus strain DL3 using a 16SrRNA gene sequencing approach. The time-course study of uricase production was performed and the medium was optimized. Carboxymethylcellulose and asparagine were found to be the best carbon and nitrogen sources. Maximum uricolytic activity was observed at pH 7.0 with an inducer concentration of 2.0 g/L. Inoculum size of 5% gave maximum uricolytic activity. The maximum uricolytic activity of 15.43 U/mL was achieved at optimized conditions, which is 1.61 times more than the initial activity. Further, enzymatic stability was determined at different pH and temperature. © 2014 Taylor and Francis Group, LLC.Item Studies on the Site-specific PEGylation Induced Interferences Instigated in Uricase Quantification Using the Bradford Method(Springer Netherlands, 2016) Nanda, P.; JagadeeshBabu, P.E.Uricase from Bacillus fastidiosus was site-specifically PEGylated using methoxypolyethyleneglycol-maleimide (mPEG-mal) of different molecular weights (750 Da, 5 kDa, 10 kDa) via Thiol PEGylation strategy. The obtained monoPEGylated uricase conjugates were characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the molecular weight of single subunit of the conjugate was found to be 42.6, 48.1 and 56.3 kDa with respect to different molecular weights of m-PEG-mal. The influence of PEGylation on the quantification of uricase using protein quantification techniques like Bradford assay, RP-HPLC detection and Dumbroff method has been evaluated. A gradual decline in the absorbance value was observed with the advancement of the PEGylation reaction, indicating an interferences in the protein quantification due to PEGylation. The extent of interference highly dependence on mPEG-mal concentration and its chain length. The present study indicates that the quantification of PEGylation induced interferences caused in protein measured ought to be prudently measured at every discrete step of the PEGylation process. © 2016, Springer Science+Business Media New York.Item Development of a Spectrophotometric Biphasic Assay for the Estimation of mPEG-maleimide in Thiol PEGylation Reaction Mixtures(Taylor and Francis Ltd. michael.wagreich@univie.ac.at, 2016) Nanda, P.; JagadeeshBabu, P.E.; Gupta, P.; Prasad, A.G.Methoxy(polyethylene glycol)-maleimide (mPEG-mal) is a PEG derivative used for thiol PEGylation of protein molecules and finds application in drug delivery studies. The maleimide group undergoes degradation in aqueous media, resulting in the difficult quantitative analysis of mPEG-mal. Routinely employed methods for separation and estimation of mPEG-mal include tedious chromatographic methods like ion exchange, high-performance liquid chromatography with refractive index detector and techniques like mass spectrometry and proton nuclear magnetic resonance. We present a direct and reproducible spectrophotometric method to quantify free and protein bound mPEG-mal in thiol PEGylation reaction mixtures. This method is based on the partitioning of a PEG bound chromophore between an aqueous ammonium isoferrothiocyanate phase to a chloroform phase in the presence of mPEG-mal. Several important parameters influencing the partitioning and stability of the chromophore, volume ratios of liquid phases, ethylenediaminetetraacetic acid concentration in the reaction mixture, mixing time, and chlorinated solvents used for partitioning have been studied. © 2016, Copyright © Taylor & Francis Group, LLC.Item Production and Optimization of Site-Specific monoPEGylated Uricase Conjugates Using mPEG-Maleimide Through RP–HPLC Methodology(Springer New York LLC barbara.b.bertram@gsk.com, 2016) Nanda, P.; JagadeeshBabu, P.E.; Raju, J.R.Purpose: Uricase (Uc), a therapeutic enzyme, is widely used in its PEGylated form to treat hyperuricemia and is largely manufactured by means of random/first generation PEGylation approach. Currently available randomly PEGylated uricase conjugates exhibit inadequacies like reduced uricolytic activity, risk of inducing immunogenic reactions, lack of selectivity, and molecular heterogeneity. In the present study, site-specific/second generation PEGylation strategy involving modification of specific and rare amino acids by means of terminally functionalized PEG polymers was applied. Methods: Uricase was conjugated with methoxypolyethyelenglycol-maleimide (mPEG-mal) by means of thiol PEGylation to synthesize monoPEGylated uricase conjugates. For enhancing the yield of monoPEGylated uricase conjugates, response surface methodology was employed to determine the yield of monoPEGylated conjugates using reverse phase high performance liquid chromatography. Using the optimized conditions, the developed method was validated for the production of monoPEGylated uricase conjugates which were further purified by size exclusion fast protein liquid chromatography (SE-FPLC). The molecular weights of the purified conjugates were determined by sodium dodecyl sulfide polyacrylamide gel electrophoresis (SDS-PAGE). Results: The optimum values of reaction conditions were determined as 1:12 concentration ratio of Uc to mPEG-mal, 2.76 kDa as mPEG-mal molecular weight and 3.55 mM EDTA concentration which resulted in a very high conjugate yield of 95.16 %. The conjugate synthesized using the optimized method retained a residual uricolytic activity of 84 % and a thiol group modification extent of 68.3 %. Conclusion: The PEGylation reaction was optimized using OVAT and statistical methods. Using the optimized conditions very high yield of conjugates were obtained and RP–HPLC method was used to quantify the PEGylated uricase. © 2016, Springer Science+Business Media New York.