Faculty Publications
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Item Production and characterization of biosurfactant produced by a novel Pseudomonas sp. 2B(2012) Aparna, A.; Srinikethan, G.; Smitha, H.Biosurfactant-producing bacteria were isolated from terrestrial samples collected in areas contaminated with petroleum compounds. Isolates were screened for biosurfactant production using Cetyl Tri Ammonium Bromide (CTAB)-Methylene blue agar selection medium and the qualitative drop-collapse test. An efficient bacterial strain was selected based on rapid drop collapse activity and highest biosurfactant production. The biochemical characteristics and partial sequenced 16S rRNA gene of isolate, 2B, identified the bacterium as Pseudomonas sp. Five different low cost carbon substrates were evaluated for their effect on biosurfactant production. The maximum biosurfactant synthesis (4.97g/L) occurred at 96h when the cells were grown on modified PPGAS medium containing 1% (v/v) molasses at 30°C and 150rpm. The cell free broth containing the biosurfactant could reduce the surface tension to 30.14mN/m. The surface active compound showed emulsifying activity against a variety of hydrocarbons and achieved a maximum emulsion index of 84% for sunflower oil. Compositional analysis of the biosurfactant reveals that the extracted biosurfactant was a glycolipid type, which was composed of high percentages of lipid (~65%, w/w) and carbohydrate (~32%, w/w). Fourier transform infrared (FT-IR) spectrum of extracted biosurfactant indicates the presence of carboxyl, hydroxyl and methoxyl functional groups. The mass spectra (MS) shows that dirhamnolipid (l-rhamnopyranosyl-l-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoate, Rha-Rha-C 10-C 10) was detected in abundance with the predominant congener monorhamnolipid (l-rhamnopyranosyl-?-hydroxydecanoyl-?-hydroxydecanoate, Rha-C 10-C 10). The crude oil recovery studies using the biosurfactant produced by Pseudomonas sp. 2B suggested its potential application in microbial enhanced oil recovery and bioremediation. © 2012 Elsevier B.V..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 Enhancement of a Novel Extracellular Uricase Production by Media Optimization and Partial Purification by Aqueous Three-Phase System(Taylor and Francis Inc. 325 Chestnut St, Suite 800 Philadelphia PA 19106, 2015) Ram, S.K.; Raval, K.; JagadeeshBabu, P.E.Uricase (urate: oxygen oxidoreductase, EC 1.7.3.3), an enzyme belonging to the class of oxidoreductases, catalyzes the enzymatic oxidation of uric acid to allantoin and finds a wide variety of application as therapeutic and clinical reagent. In this study, uricase production ability of the bacterial strains isolated from deep litter poultry soil is investigated. The strain with maximum extracellular uricase production capability was identified as Xanthomonas fuscans subsp. aurantifolii based on 16S rRNA sequencing. Effect of various carbon and nitrogen sources on uricase productivity was investigated. The uricase production for this strain was optimized using statistically based experimental designs and resulted in uricase activity of 306 U/L, which is 2 times higher than initial uricase activity. Two-step purification, such as ammonium sulfate precipitation and aqueous two-phase system, was carried out and a twofold increase in yield and specific activity was observed. © 2015 Taylor and Francis Group, LLC.Item Partial Purification and Characterization of Chromate Reductase of a Novel Ochrobactrum sp. Strain Cr-B4(Taylor and Francis Inc. 325 Chestnut St, Suite 800 Philadelphia PA 19106, 2015) Hora, A.; Shetty K, V.K.Hexavalent chromium contamination is a serious problem due to its high toxicity and carcinogenic effects on the biological systems. The enzymatic reduction of toxic Cr(VI) to the less toxic Cr(III) is an efficient technology for detoxification of Cr(VI)-contaminated industrial effluents. In this regard, a chromate reductase enzyme from a novel Ochrobactrum sp. strain Cr-B4, having the ability to detoxify Cr(VI) contaminated sites, has been partially purified and characterized. The molecular mass of this chromate reductase was found to be 31.53 kD, with a specific activity 14.26 U/mg without any addition of electron donors. The temperature and pH optima for chromate reductase activity were 40°C and 8.0, respectively. The activation energy (Ea) for the chromate reductase was found to be 34.7 kJ/mol up to 40°C and the activation energy for its deactivation (Ed) was found to be 79.6 kJ/mol over a temperature range of 50-80°C. The frequency factor for activation of chromate reductase was found to be 566.79 s-1, and for deactivation of chromate reductase it was found to be 265.66 × 103 s-1. The reductase activity of this enzyme was affected by the presence of various heavy metals and complexing agents, some of which (ethylenediamine tetraacetic acid [EDTA], mercaptoethanol, NaN3, Pb2+, Ni2+, Zn2+, and Cd2+) inhibited the enzyme activity, while metals like Cu2+ and Fe3+ significantly enhanced the reductase activity. The enzyme followed Michaelis-Menten kinetics with Km of 104.29 ?M and a Vmax of 4.64 ?M/min/mg. © 2015 Taylor and Francis Group, LLC.Item Isolation and screening of endophytes from the rhizomes of some Zingiberaceae plants for L-asparaginase production(Taylor and Francis Inc. 325 Chestnut St, Suite 800 Philadelphia PA 19106, 2016) Krishnapura, P.R.; Belur, P.D.Endophytes are described as microorganisms that colonize the internal tissues of healthy plants without causing any disease. Endophytes isolated from medicinal plants have been attracting considerable attention due to their high biodiversity and their predicted potential to produce a plethora of novel compounds. In this study, an attempt was made to isolate endophytes from rhizomes of five medicinal plants of Zingiberaceae family, and to screen the endophytes for L-asparaginase activity. In total, 50 endophytes (14 bacteria, 22 actinomycetes, and 14 fungi) were isolated from Alpinia galanga, Curcuma amada, Curcuma longa, Hedychium coronarium, and Zingiber officinale; of these, 31 endophytes evidenced positive for L-asparaginase production. All the L-asparaginase-positive isolates showed L-asparaginase activity in the range of 54.17–155.93 U/mL in unoptimized medium. An endophytic fungus isolated from Curcuma amada, identified as Talaromyces pinophilus, was used for further experiments involving studies on the effect of certain nutritional and nonnutritional factors on L-asparaginase production in submerged fermentation. Talaromyces pinophilus initially gave an enzyme activity of 108.95 U/mL, but gradually reduced to 80 U/mL due to strain degeneration. Perhaps this is the first report ever on the production of L-asparaginase from endophytes isolated from medicinal plants of Zingiberaceae family. © 2016, Taylor & Francis Group, LLC.Item Optimised microwave-assisted biosynthesis of silver nanoparticles from Nothapodytes foetida leaf extracts and its anti-microbial activities(Taylor and Francis Ltd. michael.wagreich@univie.ac.at, 2016) Roopesh, R.; Geedhika, G.; D'Souza, J.; Anandhan, S.; Bhat, K.U.; Jaya M, J.; Fathima B, S.; Mohan Balakrishnan, R.M.The present investigation reports the biogenesis of silver nanoparticles (Ag NPs) using extracts of a medicinal plant Nothapodytes foetida. Total phenolic content (TPC) and ferric reducing antioxidant power (FRAP) assay were carried out for the microwave-assisted extract (MAE) of N. foetida using methanol as solvent and the conditions for extraction were optimised by response surface methodology (RSM). The effects of operating variables such as extraction time, temperature and ratio of sample to solvent were studied using central composite design (CCD). A mathematical model with a high determination coefficient (R2) for TPC (0.991) and FRAP (0.995) was obtained. The optimal conditions of extraction for TPC were 48.6 ºC, 23.15 min and 2.04:30 (g/mL) and for FRAP 52.31ºC, 12.32 min and 1.67: 30 (g/mL). Under these conditions, the experimental yields of TPC and FRAP were 2.426 mg gallic acid equivalents (GAE)/g dry powder and 14.985mg of FeSO4·7H2O/g of dry powder, respectively. Ag NPs were characterised using UV–Vis spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. The synthesised Ag NPs have also shown potent activity against the human pathogenic Staphylococcus aureus. © 2016 Informa UK Limited, trading as Taylor & Francis Group.Item New extracellular thermostable oxalate oxidase produced from endophytic Ochrobactrum intermedium CL6: Purification and biochemical characterization(Taylor and Francis Inc. 325 Chestnut St, Suite 800 Philadelphia PA 19106, 2016) Kumar, K.; Belur, P.D.Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide with the reduction of molecular oxygen to hydrogen peroxide. Oxalate oxidase found its application in clinical assay for oxalate in blood and urine. This study describes the purification and biochemical characterization of an oxalate oxidase produced from an endophytic bacterium, Ochrobactrum intermedium CL6. The cell-free fermentation broth was subjected to two-step enzyme purification, which resulted in a 58.74-fold purification with 83% recovery. Specific activity of the final purified enzyme was 26.78 U mg?1 protein. The enzyme displayed an optimum pH and temperature of 3.8 and 80°C, respectively, and high stability at 4–80°C for 6 h. The enzymatic activity was not influenced by metal ions and chemical agents (K+, Na+, Zn2+, Fe3+, Mn2+, Mg2+, glucose, urea, lactate) commonly found in serum and urine, with Cu2+ being the exception. The enzyme appears to be a metalloprotein stimulated by Ca2+ and Fe2+. Its Km and Kcat for oxalate were found to be 0.45 mM and 85 s?1, respectively. This enzyme is the only known oxalate oxidase which did not show substrate inhibition up to a substrate concentration of 50 mM. Thermostability, kinetic properties, and the absence of substrate inhibition make this enzyme an ideal candidate for clinical applications. © 2016, Copyright © Taylor & Francis Group, LLC.Item Artificial neural network based modeling to evaluate methane yield from biogas in a laboratory-scale anaerobic bioreactor(Elsevier Ltd, 2016) Nair, V.V.; Dhar, H.; Kumar, S.; Thalla, A.K.; Mukherjee, S.; Wong, J.W.C.The performance of a laboratory-scale anaerobic bioreactor was investigated in the present study to determine methane (CH4) content in biogas yield from digestion of organic fraction of municipal solid waste (OFMSW). OFMSW consists of food waste, vegetable waste and yard trimming. An organic loading between 40 and 120 kg VS/m3 was applied in different runs of the bioreactor. The study was aimed to focus on the effects of various factors, such as pH, moisture content (MC), total volatile solids (TVS), volatile fatty acids (VFAs), and CH4 fraction on biogas production. OFMSW witnessed high CH4 yield as 346.65 L CH4/kg VS added. A target of 60–70% of CH4 fraction in biogas was set as an optimized condition. The experimental results were statistically optimized by application of ANN model using free forward back propagation in MATLAB environment. © 2016 Elsevier LtdItem 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 Exploring the fungal protein cadre in the biosynthesis of PbSe quantum dots(Elsevier B.V., 2017) Jacob, J.M.; Sharma, S.; Mohan Balakrishnan, R.M.While a large number of microbial sources have recently emerged as potent sources for biosynthesis of chalcogenide quantum dots (QDs), studies regarding their biomimetic strategies that initiate QD biosynthesis are scarce. The present study describes several mechanistic aspects of PbSe QD biosynthesis using marine Aspergillus terreus. Scanning electron microscopic (SEM) studies indicated distinctive morphological features such as abrasion and agglomeration on the fungal biomass after the biosynthesis reaction. Further, the biomass subsequent to the heavy metal/metalloid precursor was characterized with spectral signatures typical to primary and secondary stress factors such as thiol compounds and oxalic acid using Fourier Transform Infra-Red Spectroscopic (FTIR) analysis. An increase in the total protein content in the reaction mixture after biosynthesis was another noteworthy observation. Further, metal-phytochelatins were identified as the prominent metal-ion trafficking components in the reaction mixture using Liquid Chromatography Mass Spectroscopic analysis (LCMS). Subsequent assays confirmed the involvement of metal binding peptides namely metallothioneins and other anti-oxidant enzymes that might have played a prominent role in the microbial metal detoxification system for the biosynthesis of PbSe QDs. Based on these findings a possible mechanism for the biosynthesis of PbSe QDs by marine A. terreus has been elucidated. © 2016 Elsevier B.V.