Sudhakar Poda

Optimization of culture conditions by response surface methodology and unstructured kinetic modeling for bioactive metabolite production by Nocardiopsis litoralis VSM-8

Response surface methodology-based central composite design on five variables incubation time, pH, temperature, sucrose concentration, and soya peptone concentration was employed for optimization of the production of bioactive compounds by Nocardiopsis litoralis strain VSM 8. The main quadratic effects and interactions of the five variables on the production of bioactive metabolites were investigated. A second-order polynomial model produced a satisfied fit for experimental data with regard to the production of the bioactive metabolites. Regression analysis showed that high R2 values of all the five responses are significant and adjusted R2 values showed good agreement with the experimental and predicted values. The present model was used to evaluate the direct interaction and quadratic effects to optimize the physico-chemical parameters for the production of bioactive metabolites that inhibit the pathogenic microorganisms measured in terms of zones of inhibition (responses). Mathematical kinetic model development and estimation of kinetic parameters also showed good approximation in terms of model fitting and regression analysis.

Optimization of process parameters for improved production of bioactive metabolites by Streptomyces tritolerans DAS 165T.

Aims: To optimize the process parameters for enhanced production of bioactive metabolites by Streptomyces tritolerans DAS 165. Place and Duration of Study: Department of Botany and Microbiology, April 2012 to August 2012. Methodology: Agar well diffusion assay was employed to study the effect of environmental parameters such as incubation period, pH, temperature and salt concentration and influence of various nutrients such as carbon and nitrogen sources and minerals on the bioactive metabolite production by Streptomyces tritolerans DAS 165.  Results: The production of antimicrobial metabolite was high when the strain was cultured for six days at 35oC in medium (pH 7.5) with sucrose at the concentration of 2% (carbon source), soya peptone at the concentration of 1% (nitrogen source) and sodium chloride at the concentration of 5%.  Conclusion: This is the first report on the optimization of bioactive metabolite production Original Research Article British Microbiology Research Journal, 4(4): 428-442, 2014 429 by Streptomyces tritolerans DAS 165. As the strain exhibited potent antimicrobial activity, it may be explored for biotechnological purposes. 

Molecular characterization of Mtb-OMP decarboxylase by modeling, docking and dynamic studies

Tuberculosis (TB), the second most deadly disease in the world is caused by Mycobacterium tuberculosis (Mtb). In the present work a unique enzyme of Mtb orotidine 5′ monophosphate decarboxylase (Mtb-OMP Decase) is selected as drug target due to its indispensible role in biosynthesis of pyrimidines. The present work is focused on understanding the structural and functional aspects of Mtb-OMP Decase at molecular level. Due to absence of crystal structure, the 3D structure of Mtb-OMP Decase was predicted by MODELLER9V7 using a known structural template 3L52. Energy minimization and refinement of the developed 3D model was carried out with Gromacs 3.2.1 and the optimized homology model was validated by PROCHECK,WHAT-IF and PROSA2003. Further, the surface active site amino acids were quantified by WHAT-IF pocket. The exact binding interactions of the ligands, 6-idiouridine 5′ monophosphate and its designed analogues with the receptor Mtb-OMP Decase were predicted by docking analysis with AUTODOCK 4.0. This would be helpful in understanding the blockade mechanism of OMP Decase and provide a candidate lead for the discovery of Mtb-OMP Decase inhibitors, which may bring insights into outcome new therapy to treat drug resistant Mtb.

Bioactive natural products from Pseudonocardia endophytica VUK-10

Two proline containing cyclic dipeptides (CDPs), cyclo (L-Pro-L-Tyr) (1) and cyclo (L-Pro-L-Phe) (2) were isolated from the fermentation broth of Pseudonocardia endophytica VUK-10 originating from the Nizampatnam mangrove ecosystem on the south coast of Andhra Pradesh, India. The structures of the compounds were established by 1H NMR and 13C NMR spectroscopy, FTIR and EIMS. The antimicrobial and cytotoxic activities of the compounds were tested against a variety of medicinally and agriculturally important bacteria and fungi as well as on the MDA-MB-231, OAW-42, HeLa and MCF-7 human cell lines. Xanthomonas malvacearum was most sensitive toward 1 (MIC 4 μg/ml), whereas compound 2 had good antibacterial activity against Xanthomonas campestris (MIC 8 μg/ml). Fusarium solani was highly sensitive toward 1 (MIC 16 μg/ml). The compounds were cytotoxic against the human cell lines at micro molar concentrations; the highest activity (IC50 < 10 μM) of 1 was recorded against the MDA-MB-231 cancer cell line.

Development of a FRET-based fluorescence aptasensor for the detection of aflatoxin B1 in contaminated food grain samples

The present study aimed to develop an aptamer-based FRET detection strategy for the specific and sensitive detection of AFB1 in contaminated food grains. The study comprises generation of ssDNA aptamers against AFB1 by whole-cell SELEX and their application in a FRET-based platform utilizing graphene oxide (GO) and quantum dots (QDs). The generated aptamers were characterized to determine their specificity and sensitivity using indirect ELISA where AFB1–OVA was used as a coating antigen. Among the aptamers generated, the ATB1 aptamer showed good reactivity and selectivity against AFB1. This aptamer was further characterized to determine its secondary structure and KD value, which was found to be 5.9 kcal mol−1. The characterized aptamers were conjugated onto Cd/Se quantum dots to develop a fluorimetric system for the detection of aflatoxin B1 using a graphene oxide platform. The presence of graphene oxide quenches the fluorescence ability of the quantum dots due to π–π stacking interactions between the aptamer and GO. Upon target addition, the aptamer forms a complex with aflatoxin B1 thereby restoring the fluorescence intensity. The developed assay shows a linear response from 0.002 μg μl−1 to 0.2 μg μl−1 with a detection limit of 0.004 μg μl−1 for the AFB1 standard toxin and showed no cross-reactivity with other closely related mycotoxins. To validate the reliability of the developed method, several field samples spiked with AFB1 were included in this study and the results obtained were cross verified using a standard commercial AFB1 kit. In conclusion, the developed method may find good utility in routine food testing laboratories for risk assessment of AFB1.

Optimization of culture conditions by Response Surface Methodology and Unstructured kinetic modeling for L-Asparaginase production by Pseudonocardia endophytica VUK-10

L-asparaginase is an anti-tumor enzyme and widely accepted as chemotherapeutic agent which has activity against acute lymphoblastic leukemia. The current study targets the production of L-asparaginase by Pseudonocardia endophytica VUK-10 by a statistically designed model. Experiments were performed according to central composite design of RSM with five independent variables such as time, pH, temperature, concentrations of maltose and L-asparagine concentration for optimization. All the five conditions had significant interaction with other variables for the maximum response (L-asparaginase production). Maximum L-asparaginase production was recorded as 7.42 IU/ml slightly higher than the model predicted value of 6.8 IU/ml, from statistical optimization studies. An unstructured kinetic model was proposed to depict the profiles of biomass, substrate utilization and L-asparaginase production in optimized medium under shake flask level. The logistic and Leudeking-Piret expressions were modified to predict the kinetic model parameters (µmax, X0, Xmax, α, β, γ and η) and we found that L-asparaginase production was growth-associated. High significant correlation (R2) values of 0.86, 0.96 and 0.94 were observed with the experimental and predicted results for Pseudonocardia endophytica VUK-10 growth, L-asparaginase activity and Maltose utilization, respectively. The results obtained from medium optimization using RSM and unstructured mathematical models describe the L-asparaginase fermentation kinetics more effectively.

Optimization of Operating Conditions for the Production of Enhanced Antifungal Metabolites from Streptomonospora arabica VSM 25 by Full Factorial Design

Objectives: To execute the influence of the physico-chemical variables on the production of the bioactive metabolites by Streptomonospra arabica VSM-25 using Response Surface Methodology. Materials and Methods: An actinobacterium strain isolated from the deep sea marine sediment samples was identified as Streptomonospra arabica VSM-25 by conventional and molecular approaches. RSM was employed to study the impact of five variables, viz. incubation time, pH, temperature, galactose and peptone concentrations on the production of antifungal metabolites by VSM-25. Growth related production formation kinetics and substrate utilization in batch system was analysed using mathematical and unstructured kinetic models. Results: Statistical study showed that the incubation time, pH, Temperature, Concentration of galactose and peptone has a significant effect (p Conclusion: To the best of our knowledge this is the first report on the production of anti fungal metabolites from S. arabica using RSM and kinetic modelling studies which firmly support the application of RSM and kinetic modelling for optimization. The study may find potential application in rapid screening and production of novel drug molecules from unexploited natural sources. Key words: Streptomonospora arabica , Antimycotic Activity, Optimization, Kinetic Modelling, Response Surface Methodology, Bioactive Metabolites.

Pullulan-Stabilized Silver Nanoparticles -Their Synthesis, Characterization and Application as Bactericidal Agents.

A rapid method for Pullulan-stabilized silver nanoparticles (PuAgNPs) synthesis has been developed. Different concentrations of Pullulan and Silver nitrate and effects of reaction time, pH was used to investigate the synthesis of silver nanoparticles. The synthesized Pu-AgNPs were first screened and identified using surface plasmon peaks of UV–VIS spectroscopy.The research results indicated that the surface plasmon resonance peaks were observed between 410–460 nm wavelengths in UV-VIS spectroscopy studies.The morphology of the synthesized AgNPs proved a variation in spherical shape and polydispersed with an averagesize of 10-55 nm, using TEM. Further, five characteristic peaksconfirmed the presence of elemental silver and the crystalline structure of silver nanoparticles from XRD analysis. From FTIR spectra, stretching vibrations of hydroxyl (O-H), carbonyl (C=O) and C=C stretches exhibits the reduction and stabilization of AgNPs. Further, clear zones of inhibition (about 10-25 mm) against four bacterial pathogens obtained in the antibacterial studies for the synthesized PuAgNPs.The experimental results demonstrated that pullulan could be used as reducing and stabilizing agentfor formation of AgNPs and can be used as redoubtablebactericidal agents.

Optimization of the Cultural Parameters for Improved Production of Antimicrobial Metabolites by Streptomyces gulbargensis DAS 131

Aims: To investigate the influence of appropriate culture medium by optimizing the cultural conditions affecting the growth and bioactive metabolite production by Streptomyces gulbargensisDAS 131 under submerged culture conditions in order to reduce the cost of fermentation process to improve the formation of antimicrobial compounds.