Faculty Publications
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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 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 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.