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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 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.Item Extraction, optimization and characterization of collagen from sole fish skin(Elsevier B.V., 2018) Arumugam, G.K.S.; Sharma, D.; Mohan Balakrishnan, R.M.; JagadeeshBabu, P.E.In this study, collagen was successfully extracted from marine waste i.e. Sole fish skin, which is available in the coastal area of Mangalore, India. The extraction process was optimized using One Variable at a Time (OVAT) and Response Surface Methodology (RSM) with Box-Behnken Design (BBD) was to achieve maximum yield and the extracted collagen was characterized. The optimal conditions to obtain highest collagen yield was determined to be, an acetic acid concentration of 0.54 M, NaCl concentration of 1.90 M, solvent/solid ratio of 8.97 ml/g and time of 32.32 h. The maximum collagen yield of 19.27 ± 0.05 mg/g of fish skin was achieved under the optimal conditions. The analysis of variance and contour plots exhibited a significant interaction of all the selected variables over collagen extraction process. The SDS-PAGE (Sodium dodecyl sulfate - polyacrylamide gel electrophoresis) analysis suggested that the extracted collagen contained three ?-chains i.e. (?1)2, ?2 (M.W. 118, 116 kDa) and one ? chain (M.W. 200 kDa) which was similar to commercially available calfskin Type I collagen. FT-IR (Fourier Transform Infrared Spectroscopy) analysis confirmed the existence of helical arrangements of collagen. SEM (Scanning electron microscopy) observation revealed that the extracted collagen was in the form of fibrils with irregular linkages. © 2018 Elsevier B.V.Item In silico structural and functional analysis of bacillus uricases(Bentham Science Publishers, 2021) Nelapati, A.K.; Meena, S.; Singh, A.K.; Bhakta, N.; JagadeeshBabu, P.E.Background: Excluding humans, the peroxisomal uricase is responsible for the catabolism of uric acid into allantoin in many species like microorganisms, plants, and inverte-brates. Particularly in humans, the synthesis and excretion of uric acid are naturally balanced. When the uric acid concentration crosses 7 mg/dl, it results in conditions such as hyperuricemia and gout. Uricase is one of the potential sources for the reduction of uric acid in humans. Uricase is also widely used as a commercial diagnostic reagent in medical and clinical biochemistry to esti-mate the uric acid concentration in blood and other biological fluids. Computational approaches can be used for screening and investigation of uricase enzyme with desirable characteristics that can be employed in diverse industrial applications. Objectives: The present study deals with computational-based structural, functional, and phylogenetic analyses of uricase enzymes from various Bacillus species. Methods: Seventy uricase protein sequences from Bacillus species were selected for multiple sequence alignment, phylogenetic analysis, motif assessment, domain architecture examination, understanding of basic physicochemical properties and in silico identification of the composition of amino acids in uricase. Further, structural (secondary and tertiary structure prediction), and functional (CYS_REC, MOTIF scan, CD-search, STRING, SOSUI, and PeptideCutter) analyses of uric-ase were performed. Results: Bacillus simplex (WP_063232385.1) was chosen as the representative species of the Bacillus genera. The three-dimensional (3D) structure of B. simplex uricase was predicted and validated using QMEAN, RAMPAGE, ERRAT, Verify 3D and PROQ servers. The analysis revealed that the tertiary structure of the selected uricase has good quality and acceptability. Conclusion: Computational analysis of uricase from various Bacillus sources revealed that all the selected Bacillus uricases are active within acidic to a neutral environment, and thermally stable with a molecular weight ranging from 35.59-59.85kDa. The secondary structure analysis showed that all uricases are rich in alpha-helices and sheets. The CDD tool identified two conserved do-mains, one of which belongs to OHCU decarboxylase and another belongs to Uricase superfamily. The quality estimation of 3D modeled protein gave a high overall quality factor score of 94.64. Al-so, all Bacillus species of uricase enzyme and their corresponding genes showed a strong correlation from the phylogenetic comparison of the selected taxa. The present detailed computational investigation on the uricase protein could help in screening a suitable uricase producing microbe with desirable characteristics for industrial application. © 2021 Bentham Science Publishers.