Journal Articles

Permanent URI for this collectionhttps://idr.nitk.ac.in/handle/123456789/19884

Browse

Author: search.filters.author.Nanda, P.Subject: search.filters.subject.Bacillus

Search Results

Now showing 1 - 2 of 2
  • No Thumbnail Available
    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.
  • No Thumbnail Available
    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.