Faculty Publications
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Item Optimization of culture medium for novel cell-associated tannase production from bacillus massiliensis using response surface methodology(2012) Belur, P.D.; Goud, R.; Goudar, D.C.Naturally immobilized tannase (tannin acyl hydrolase, E.C. 3.1.1.20) has many advantages, as it avoids the expensive and laborious operation of isolation, purification, and immobilization, plus it is highly stable in adverse pH and temperature. However, in the case of cell-associated enzymes, since the enzyme is associated with the biomass, separation of the pure biomass is necessary. However, tannic acid, a known inducer of tannase, forms insoluble complexes with media proteins, making it difficult to separate pure biomass. Therefore, this study optimizes the production of cell-associated tannase using a "protein-tannin complex" free media. An exploratory study was first conducted in shake-flasks to select the inducer, carbon source, and nitrogen sources. As a result it was found that gallic acid induces tannase synthesis, a tryptose broth gives higher biomass, and lactose supplementation is beneficial. The medium was then optimized using response surface methodology based on the full factorial central composite design in a 3 l bioreactor. A 2 3 factorial design augmented by 7 axial points (? = 1.682) and 2 replicates at the center point was implemented in 17 experiments. A mathematical model was also developed to show the effect of each medium component and their interactions on the production of cell-associated tannase. The validity of the proposed model was verified, and the optimized medium was shown to produce maximum cell-associated tannase activity of 9.65 U/l, which is 93.8% higher than the activity in the basal medium, after 12 h at pH 5.0, 30°C. The optimum medium consists of 38 g/l lactose, 50 g/l tryptose, and 2.8 g/l gallic acid. © The Korean Society for Microbiology and Biotexhnology.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 Production of propyl gallate in nonaqueous medium using cell-associated tannase of Bacillus massiliensis: Effect of various parameters and statistical optimization(2013) Aithal, M.; Belur, P.D.Enzymatic synthesis of propyl gallate in an organic solvent was studied using cell-associated tannase (E.C. 3.1.1.20) of Bacillus massiliensis. Lyophilized biomass showing tannase activity was used as a biocatalyst. The influence of buffer pH and strength, water activity, temperature, biocatalyst loading, gallic acid concentration, and 1-propanol concentration was studied by the one-factor-at-a-time method. Subsequently, response surface methodology was applied based on a central composite design to determine the effects of three independent variables (biocatalyst loading, gallic acid concentration, and 1-propanol concentration) and their mutual interactions. A total of 20 experiments were conducted, and a statistical model was developed, which predicted the maximum propyl gallate yield of 20.28 ?g/mL in the reaction mixture comprising 40.4 mg biocatalyst, 0.4 mM gallic acid, and 6.52 % (v/v) 1-propanol in 9.5 mL benzene at 30°C. The subsequent verification experiments established the validity of the model. Under optimal conditions, 25% conversion of gallic acid to propyl gallate was achieved on a molar basis. The absence of the need for enzyme purification and subsequent immobilization steps and good conversion efficiency makes this enzyme system an interesting one. Reports on the applications of bacterial whole cell systems for synthetic reactions in organic solvents are scarce, and perhaps this is the first report on bacterial cell-associated tannase-mediated esterification in a nonaqueous medium. © 2013 International Union of Biochemistry and Molecular Biology, Inc.Item Enhancement of propyl gallate yield in nonaqueous medium using novel cell-associated tannase of bacillus massiliensis(2013) Aithal, M.; Belur, P.D.Enzymatic synthesis of propyl gallate in organic solvent was studied using cell-associated tannase (EC 3.1.1.20) of Bacillus massiliensis. Lyophilized biomass showing tannase activity was used as the biocatalyst. The effects of solvent, surfactant treatment, and bioimprinting on the propyl gallate synthesis were studied and subsequently optimized. Among various solvents, benzene followed by hexane was found to be the most favorable. Treatment of the biocatalyst with Triton X-100 at a lower concentration (0.2% w/v), before lyophilization, increased the propyl gallate yield by 24.5% compared to the untreated biocatalyst. The biocatalyst was imprinted with various concentrations of gallic acid and tannic acid. Biocatalyst imprinted with tannic acid showed 50% enhancement in the propyl gallate yield compared to the non-imprinted biocatalyst. © 2013 Taylor & Francis Group, LLC.Item Production of naringinase from a new soil isolate, Bacillus methylotrophicus: Isolation, optimization and scale-up studies(2014) Mukund, P.; Belur, P.D.; Saidutta, M.B.Five strains of naringin-degrading bacteria were isolated and found to be positive for extracellular naringinase activity. The one that showed highest activity in the selective medium was identified by 16S rRNA analysis as Bacillus methylotrophicus. The best combination of carbon-nitrogen source was determined by employing two-level full factorial analyses, comprising 24 experiments in shake flasks. Sucrose-yeast extract showed significant increase in naringinase activity (7.46 U/L) compared to the basal medium. Naringinase production was found to be inducible and naringin was found to be the best inducer among naringin, naringenin, hesperidin, and L-rhamnose. Inoculum size of 2% (v/v) and age of 48 hr favored naringinase and biomass production. Highest naringinase activity of 8 U/L was observed at the initial medium pH of 6. Response surface modeling was applied based on central composite design to determine the effects of three independent variables (sucrose, yeast extract, and naringin) and their mutual interactions. In total, 20 experiments were conducted and a statistical model was developed, which predicted naringinase production of 10.61 U/L. Subsequently, verification experiments were conducted and validity of the model was verified. Bioreactor studies conducted with the optimized medium showed an enzyme production of 12.05 U/L within 34 hr of fermentation. Copyright © Taylor & 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 Scale-up of naringinase production process based on the constant oxygen transfer rate for a novel strain of Bacillus methylotrophicus(Taylor and Francis Inc. 325 Chestnut St, Suite 800 Philadelphia PA 19106, 2017) Raval, K.; Gehlot, K.; Belur, B.D.Naringinase bioprocess based on Bacillus methylotrophicus was successfully scaled up based on constant oxygen transfer rate (OTR) as the scale-up criterion from 5-L bioreactor to 20-L bioreactor. OTR was measured in 5 and 20-L bioreactor under various operating conditions using dynamic method. The operating conditions, where complete dispersion was observed were identified. The highest OTR of 0.035 and 0.04 mMol/L/s was observed in 5 and 20-L bioreactor, respectively. Critical dissolved oxygen concentration of novel isolated strain B. methylotrophicus was found to be 20% of oxygen saturation in optimized medium. The B. methylotrophicus cells grown on sucrose had maximum oxygen uptake rate of 0.14 mMol/L/s in optimized growth medium. The cells produced the maximum naringinase activity of 751 and 778 U/L at 34 hr in 5 and 20-L bioreactors, respectively. The maximum specific growth rate of about 0.178/hr was observed at both the scales of operations. The maximum naringinase yield of 160 and 164 U/g biomass was observed in 5 and 20-L bioreactors, respectively. The growth and production profiles at both scales were similar indicating successful scale-up strategy for B. methylotrophicus culture. © 2017 Taylor & Francis.Item The combined effects of carbon/nitrogen ratio, suspended biomass, hydraulic retention time and dissolved oxygen on nutrient removal in a laboratory-scale anaerobic–anoxic–oxic activated sludge biofilm reactor(IWA Publishing, 2018) Manu, D.S.; Thalla, A.K.The current trend in sustainable development deals mainly with environmental management. There is a need for economically affordable, advanced treatment methods for the proper treatment and management of domestic wastewater containing excess nutrients (such as nitrogen and phosphorus) which can cause eutrophication. The reduction of the excess nutrient content of wastewater by appropriate technology is of much concern to the environmentalist. In the current study, a novel integrated anaerobic–anoxic–oxic activated sludge biofilm (A2O-AS-biofilm) reactor was designed and operated to improve the biological nutrient removal by varying reactor operating conditions such as carbon to nitrogen (C/N) ratio, suspended biomass, hydraulic retention time (HRT) and dissolved oxygen (DO). Based on various trials, it was seen that the A2O-AS-biofilm reactor achieved good removal efficiencies with regard to chemical oxygen demand (95.5%), total phosphorus (93.1%), ammonia nitrogen concentration (NH4þ-N) (98%) and total nitrogen (80%) when the reactor was maintained at C/N ratio of 4, suspended biomass of 3 to 3.5 g/L, HRT of 10 h, and DO of 1.5 to 2.5 mg/L. Scanning electron microscopy (SEM) of suspended and attached biofilm showed a dense structure of coccus and bacillus bacteria with the diameter ranging from 0.3 to 1.2 ?m. The Fourier transform infrared (FTIR) spectroscopy results indicated phosphorylated macromolecules and carbohydrates mix or bind with extracellular proteins in exopolysaccharides. © IWA Publishing 2018.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.Item Cloning, expression, purification and characterization of chitin deacetylase extremozyme from halophilic Bacillus aryabhattai B8W22(Springer Science and Business Media Deutschland GmbH, 2021) Pawaskar, G.M.; Raval, K.; Rohit, P.; Shenoy, R.P.; Raval, R.Chitin deacetylase (CDA) (EC 3.5.1.41) is a hydrolytic enzyme that belongs to carbohydrate esterase family 4 as per the CAZY database. The CDA enzyme deacetylates chitin into chitosan. As the marine ecosystem is a rich source of chitin, it would also hold the unexplored extremophiles. In this study, an organism was isolated from 40 m sea sediment under halophilic condition and identified as Bacillus aryabhattai B8W22 by 16S rRNA sequencing. The CDA gene from the isolate was cloned and overexpressed in E. coli Rosetta pLysS and purified using a Ni–NTA affinity chromatography. The enzyme was found active on both ethylene glycol chitin (EGC) and chitooligosaccharides (COS). The enzyme characterization study revealed, maximum enzyme velocity at one hour, optimum pH at 7 with 50 mM Tris–HCl buffer, optimum reaction temperature of 30 ºC in standard assay conditions. The co-factor screening affirmed enhancement in the enzyme activity by 142.43 ± 7.13% and 146.88 ± 4.09% with substrate EGC and COS, respectively, in the presence of 2 mM Mg2+. This activity was decreased with the inclusion of EDTA and acetate in the assay solutions. The enzyme was found to be halotolerant; the relative activity increased to 116.98 ± 3.87% and 118.70 ± 0.98% with EGC and COS as substrates in the presence of 1 M NaCl. The enzyme also demonstrated thermo-stability, retaining 87.27 ± 2.85% and 94.08 ± 0.92% activity with substrate EGC and COS, respectively, upon treatment at 50 ºC for 24 h. The kinetic parameters Km, Vmax, and Kcat were 3.06E?05 µg mL?1, 3.06E + 01 µM mg?1 min?1 and 3.27E + 04 s?1, respectively, with EGC as the substrate and 7.14E?07 µg mL?1, 7.14E + 01 µM mg?1 min?1 and 1.40E + 06 s?1, respectively, with COS as the substrate. The enzyme was found to be following Michaelis–Menten kinetics with both the polymeric and oligomeric substrates. In recent years, enzymatic conversion of chitosan is gaining importance due to its known pattern of deacetylation and reproducibility. Thus, this BaCDA extremozyme could be used for industrial production of chitosan polymer as well as chitosan oligosaccharides for biomedical application. © 2021, The Author(s).