Salam Pradeep Singh

Assessment of five soil DNA extraction methods and a rapid laboratory-developed method for quality soil DNA extraction for 16S rDNA-based amplification and library construction

Extraction of DNA from soil samples using standard methods often results in low yield and poor quality making them unsuitable for community analysis through polymerase chain reaction (PCR) due to the formation of chimeric products with smaller template DNAs and the presence of humic substances. The present study focused on the assessment of five different methods for metagenomic DNA isolation from soil samples on the basis of processing time, purity, DNA yield, suitability for PCR, restriction digestion and mDNA library construction. A simple and rapid alkali lysis based on indirect DNA extraction from soil was developed which could remove 90% of humic substances without shearing the DNA and permits the rapid and efficient isolation of high quality DNA without the requirement of hexadecyltrimethylammonium bromide and phenol cleanup. The size of DNA fragment in the crude extracts was >23 kb and yield 0.5-5 μg/g of soil. mDNA purification using Sephadex G-50 resin yielded high concentration of DNA from soil samples, which has been successfully used for 16S rDNA based amplification of a 1500 bp DNA fragment with 27F and 1492R universal primers followed by restriction digestion and mDNA library construction.

Optimization of Nutrient Requirements and Culture Conditions for the Production of Rhamnolipid from Pseudomonas aeruginosa (MTCC 7815) using Mesua ferrea Seed Oil

Environmental awareness has led to a serious consideration for biological surfactants and hence non-edible vegetable oils may serve as a substitute carbon source for bio-surfactant production (rhamnolipid) which might be an alternative to complex synthetic surfactants. There are reports of rhamnolipid production from plant based oil giving higher production than that of glucose because of their hydrophobicity and high carbon content. Therefore the contribution of non-edible oil such as Mesua ferrea seed oil could serve as a good carbon source for rhamnolipid production. Moreover the use of rhamnolipid production from non-edible plant based seed oil has not been reported elsewhere. The present work focus on the optimal production of rhamnolipid by considering both micro and macro nutrients and culture conditions using response surface methodology. The study observes that micronutrients play a significant role in rhamnolipid production from Pseudomonas aeruginosa (MTCC 7815). The investigation results with the statistically optimize parameters able to produce a higher rhamnolipid production and this methodology could be used to optimize the nutrients requirements and culture conditions. The present findings would assist in bioremediation of crude oil contaminated ecosystems.

Metagenomics: An Application Based Perspective

Metagenomics deals with the isolation of genetic material directly recovered from environmental samples. Metagenomics as an approach has emerged over the past two decades to elucidate a host of microbial communities inhabiting a specific niche with the goal of understanding their genetic diversity, population structure, and ecological role played by them. A number of new and novel molecules with significant functionalities and applications have been identified through this approach. In fact, many investigators are engaged in this field to unlock the untapped genetic resources with funding from governments sector. The sustainable economic future of modern industrialized societies requires the development of novel molecules, enzymes, processes, products, and applications. Metagenomics can also be applied to solve practical challenges in the field of medicine, agriculture, sustainability, and ecology. Metagenomics promises to provide new molecules and novel enzymes with diverse functions and enhanced features compared to the enzymes from the culturable microorganisms. Besides the application of metagenomics for unlocking novel biocatalysts from nature, it also has found applications in fields as diverse as bioremediation, personalized medicine, xenobiotic metabolism, and so forth.

Molecular docking studies of quercetin and its analogues against human inducible nitric oxide synthase.

Nitric oxide synthases (NOS) catalyze to produce nitric oxide (NO) from L-arginine. The isoform of NOS i.e. inducible nitric oxide synthases (iNOS) expression is observed in various human malignant tumors such as breast, lung, prostate and bladder, colorectal cancer, and malignant melanoma. Also an increased level of iNOS expression and activity has been found in the tumor cells of gynecological malignancies, stroma of breast cancer and tumor cells of head and neck cancer. Because of its importance in causing tumors and cancer, iNOS enzyme has become a new target in finding novel inhibitors as anti cancer agents. The present work focuses on the molecular docking analysis of quercetin and its analogues against iNOS enzyme. Earlier there are reports of quercetin inhibiting iNOS enzyme in certain experiments as anti cancer agent. But the clinical use of quercetin is limited by its low oral bioavailability and therefore needed its molecular modification to improve its pharmacological properties. In the present study ten analogues of quercetin were found to be docked at the active site cavity with favorable ligand-protein molecular interaction and interestingly from the ADME-Toxicity analysis these analogues have enhanced pharmacological properties than quercetin.

Valorization and Miscellaneous Prospects of Waste Musa balbisiana Colla Pseudostem

Resourceful utilization of the enormous quantum of agrowastes generated via agricultural practices can be supportive in waste management, environmental upgradation, and subsequent material and energy recovery. In this regard, the present study aimed at highlighting waste banana (Musa balbisiana Colla) pseudostem (an agrowaste) as a potential bio-based feedstock with miscellaneous applications. The pseudostem was characterized by carbon, nitrogen, and hydrogen (CHN) analysis, thermogravimetric-differential thermal analysis (TGDTA), and Fourier transform infrared (FTIR) spectroscopy. Cellulose, hemicellulose, and lignin were estimated as a part of biochemical characterization. Total phenolic content, total flavonoid content, 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, and ferric reducing antioxidant power (FRAP) were carried out as a part of antioxidant characterization. The waste banana pseudostem biomass (WBPB) was also tried successfully as a natural filler in polyvinyl chloride (PVC) polymer composite. Thermal properties and water uptake test of the WBPB polymer composite were accessed as a part of composite characterization. The pseudostem had calorific value (15.22 MJ/kg), high holocellulose (58.67%), high free radical scavenging potential (69.9%), and a low ash content (6.8%). Additionally, the WBPB polymer composite showed improved water resistance and thermostability. The study suggests feasibility of WBPB as a prospective bioenergy feedstock, primary antioxidant source, and reinforcing agent in polymer composites.

Production and statistical optimization of biodiesel from kitchen chimney dump lard

BackgroundThe global upsurge in world energy demand, sharp decline in petrofuels reserves, and greater concerns about the environment has constrained mankind to investigate newer and renewable feedstocks for liquid transportation fuels. Existent bioenergy feedstocks cannot suffice for the petro crude reserves, new feedstock for the same will serve as an incremental step for addressing the long recognized problem of energy supply and simultaneously also support further research endeavors related to green chemistry, feed stock biology and bio-energy research. In this regard, the present investigation aims to highlight kitchen chimney dump lard (a bio-waste), as a prospective feedstock for biodiesel production.ResultsStatistical implication (response surface methodology, RSM) can be helpful in design of experiments for biodiesel production and allied approaches. Biodiesel was produced from KCDL which contains high amount of free fatty acids (acid value 28 mg KOH/gm) by a two stage transesterification process. The biodiesel yield was optimized using RSM for optimal production and the yield was found to be 82% compared to 80% predicted using RSM. The biodiesel was characterized by CHN, GC-MS and FTIR spectroscopy. The fuel properties namely density, viscosity, cloud point, pour point, flash point and calorific value were analyzed to access the quality of the fuel. The results suggest the feasibility of biodiesel production from KCDL.ConclusionProduction of biodiesel from KCDL is feasible and warrants necessary research.

Molecular docking studies on analogues of quercetin with d-alanine:d-alanine ligase of Helicobacter pylori

Helicobacter pylori (Hp) is a human pathogen associated with myriad of diseases such as gastritis, peptic ulceration, piles and gastric cancer. The resistance of Hp against antimicrobial agents has increased just as that of other pathogens worldwide, thus emphasizing an urgent need for developing new antibacterial agents. The d-alanine:d-alanine ligase (Ddl, EC 6.3.2.4) has been considered as a putative antimicrobial drug target and a lot of inhibitor screening efforts have been made. Quercetin, a member of the flavonoids, characterized by a flavone nucleus composed of two benzene rings linked through a heterocyclic pyrone ring is reported to possess antibacterial activity against H. pylori Ddl (HpDdl) enzyme. In this milieu, we have performed molecular docking analysis of quercetin and its analogues at the active site of HpDdl. Some of the screened compounds showed better affinity and interaction with HpDdl enzyme. The docking analysis and absorption, distribution, metabolism and toxicity study forward few of them as plausible lead molecule or a novel class of drugs with enhanced pharmacological properties.

Molecular docking simulation analysis of the interaction of dietary flavonols with heat shock protein 90

Hsp90 is a major protein involved in the stabilization of various proteins in cancer cells. The present investigation focused on the molecular docking simulation studies of flavanols as inhibitors of Hsp90 at the high affinity adenosine triphosphate (ATP) binding site and analyzed absorption, distribution, metabolism, excretion and toxicity (ADME-toxicity). The molecular docking analysis revealed that the flavanols showed competitive inhibition with ATP molecule at the active site and enhanced pharmacological parameters.

Prediction of the three-dimensional structure of serine/threonine protein kinase pto of Solanum lycopersicum by homology modelling.

The resistant gene Pto of Solanum lycopersicum interacts with the avr Pto gene product of the bacterial pathogen Pseudomonas syringae pv tomato to launch a cascade of molecular events that triggers the hypersensitive disease-resistance response in tamato. The paper describes attempts to predict the structure of Pto encoding a serine/threonine protein kinase to understand the mechanism and function. A three-dimensional model based on the crystal structure of effect protein Avr ptob complexed with Kinase Pto and bacterial effector protein Avrpto was generated using Modeller9v7. We adopted different modelling approaches for our study, Intialy, we generated a model based on a single template protein and then a model based on multiple templates. The models generated through these approaches were further assessed with ANOLEA energy assessment, Ram Page server and PROCHECK for stereochemistry and geometry check. Comparative analysis suggested that the model generated was better than the templates. This study paves the way for generating computer molecular models for proteins whose crystal structures are not available and which would aid in studying protein-protein interactions.

MD Simulation Studies of Fumarase Reveal Thermo Dynamical Stability

Fumarase enzyme is known to catalyse the stereo specific inter conversion of fumarate to L-malate which is a part of the Krebs cycle. Despite the biological significance and importance of this enzyme, the reaction mechanism of fumarase is not completely understood or known. In this context an experiment on molecular dynamics simulation was carried out for at least 10 nanoseconds molecular dynamics simulation run using Nano Scale Molecular Dynamics program implemented in Discovery Studio 4.0. The trajectory analysis of various energy parameters revealed the thermo dynamical stability of the enzyme. The present findings may aid in understanding the biological significance of this enzyme.