Current Biotechnology - Volume 7, Issue 5, 2018

Background: Quercetin and apigenin are a new class of drug targets for Helicobacter pylori D-alanine- D-alanine ligase (HpDdl). However, their clinical use is limited by its oral bioavailability and hence requires its molecular modification. The present study is an attempt to investigate the insights of competitive inhibition of these compounds. Methods: In silico ligand-protein interaction was carried out, predicting molecular recognition, explicating the binding modes and its binding affinity. Molecular Dynamic (MD) simulations were also performed and their free binding energies were evaluated. Additionally, the Density Functional Theory (DFT) analysis was carried out. Result: The docking simulation showed quercetin having a rerank score of -67.97 compared to -57.75 of apigenin and -45.04 of ATP molecule respectively. The interaction energy analysis revealed quercetin having a favorable total interaction energy of -101.45 kJ mol-1 compared to -91.67 kJ mol-1 and -70.23 kJ mol-1 for apigenin and ATP molecule, respectively. The Root Mean Squared Deviation (RMSD) plot from the MD simulation clearly explains the variations of the Hpddl enzyme and protein-ligand binding complexes. Additionally, the Density Functional Theory (DFT) analysis revealed the HOMO and LUMO favourable energies of quercetin and apigenin. Conclusion: The study reveals the insights on quercetin and apigenin showing competitive inhibition with ATP. The free binding energy calculation confirms the stable binding energy. The MD simulation revealed the conformation changes and the stability of the docked complex. The molecular orbital’s analysis on HOMO and LUMO energies depicts the regions which might favour electrophilic attack and nucleophilic attack.

Objectives: Extraction and manipulation of bacterial nucleic acid directly from soil has emerged as indispensable technique to explore the genomic potential of uncultured microbes. Major difficulty in separating humic acid from soil DNA is attributed to its strong interactions with the humic acid. Previously several methods were used to purify DNA, however the kits and material used during purification is costly and lack efficieny also. This study reports a cost effective, reliable and time saving method for the purification of metagenomic DNA based on novel Nanocomposite. Methods: Nanocomposite TiO2-PANI-SbSnP (Titanium dioxide-polyaniline Tin (1V) antimonophosphate) was prepared by introducing the SnSbP gel into the green gel of PANI followed by addition of 30 mg of TiO2 nanoparticles. TEM, XRD and Zeta potential studies were carried out to gain further insights about the mechanisms underlying the purification, we carried out the following studies. Results: Nanocomposite showed size ~5nm as validated from TEM images. It was positively charged as evinced from its zeta potential value (+9.2mv) and binds strongly with humic acid.It is capable of removing ~98% of the humic acid. Time dependent restriction digestion of the purified and non-purified DNA revealed that nanocomposite purified DNA was easily digestible with restriction enzyme. Purified DNA also led to PCR amplification of the 16S rRNA employing universal set of primer 27F and 1642 R. In contrary, the non-purified DNA was neither digested nor got amplified. Thus, the synthesized nanocomposite demonstrated its potential application in removing impurities from soil DNA.

Background: Tweaking CHO cell metabolism in fed-batch processes to provide sufficient cellular energy for the synthesis of recombinant proteins at high specific rates forms the basis for achieving superior process yields. Despite the growing understanding of the backgrounds of cellular phenotypes, like the Warburg effect, there is still an unmet need to translate theory into practice. Objective: This study presents a new concept for pool selection, which uses directed metabolic evolution to generate high producer CHO cells with a “low lactate” phenotype. Results: At first, a newly developed indicator called the metabolic rating was used to choose suitable CHO host cell clones for metabolic pool selection. Selected candidates were taken to be tested in distinct metabolic pool selection protocols, which made use of alternating sequences of rationally designed medium (supplemented with, e.g., lactate) and standard medium. The addition of feed medium throughout the pool selection phase was included to increase the robustness of the resulting cellular phenotype. The metabolic selection protocol resulted in a cell pool with improved metabolic efficiency (ratio of lactate produced to glucose consumed was decreased by 1.8x) and increased productivity (1.7x titer increase). The analysis of product quality attributes showed a decreased level of galactosylation but a normal level of high mannose structures and an approximately 7x lower content of IgG1 dimer in the metabolically selected cell pool. After single cell cloning of the metabolically selected cell pool with the best performance and an assessment of resulting clones in a 96 deep well plate, a top clone was identified and tested in a fed-batch process in shake flasks. Titers of 3-4 g/L were achieved using media that were optimized towards copper and iron concentrations. Conclusion: It was shown that the Warburg effect, an effect to be avoided during cell culture-based manufacturing, can be successfully used to drive metabolic selection within the cell line engineering work stream.

Objectives: The main objective of this study was to investigate the molecular diversity of α- subunit of bacterial Ring Hydroxylating Dioxygenases (RHD) in agriculture soil. These enzymes catalyze break down of polyaromatic hydrocarbon to non-toxic form without forming harmful products like 3-PBA (3-Phenoxybenzoic acid). To meet our objectives, we carried out PCR amplification, cloning and analysis of genes encoding partial catalytic domain of α-subunit of phenanthrene (phn) RHD genes from metagenome sample of agricultural soil. Methods: Partial sequences of RHDs were amplified from soil metagenome using degenerate set of primers. A library of ~25 clones was generated by cloning PCR amplified product in pGEM-T easy vector using E. coli Dh5 α cell as host. Plasmid DNA was extracted from recombinant clones using plasmid DNA extraction kit followed by sequencing of positive clones. Phylogeny analysis of the recovered clones was carried out using Mega programs version 7.0. Several bioinformatics tools were used to predict the active site cleft and active site complementarities of RHDs towards various aromatic hydrocarbons. Results: Selective PCR amplification of alpha subunit of bacterial RHDs and its cloning thereafter resulted in a library consisting of 25 clones. Notably, sequencing of these clones demonstrated the presence of four sequences, encoding RHDs. These sequences were grouped into three main clusters. Among four sequences encoding RHDs, one sequence shared ~ 76 % similarity to reported RHD protein (PDB ID: 2GBW). In silico molecular modelling and analysis revealed that active site residues in this sequence were conversed except Gly205Ala mutation in the conserved domain (Glu209-His217). Comparative analysis of the predicted model displayed a significant level of variations in the binding site (at the entrance of loop regions Ala259-Val268). In silico characterization of the substrate binding site with chemical probes demonstrated binding preference of active site towards various polyaromatic hydrocarbons.

Background: Dextran is a bacterial polysaccharide composed of chains of D-glucose units. Most of the commercial strains for dextran production belong to Leuconostoc Spp. Dextran has many uses in the pharmaceutical, food and textile industries. Objectives: The objective of this study was to elucidate the characteristics and structural aspect of the dextran synthesized by purified novel dextransucrase of Acetobacter tropicalis. Methods: The solubility of the dextran was checked in various solvents and viscosity was determined by jacketed Ostwald Viscometer. Optical rotation and molecular weight were evaluated by using polarimeter and gel permeation chromatography, respectively. Finally, the structural characteristics were studied by FTIR, NMR and SEM techniques. Results: From the results, it has been concluded that the water-soluble dextran was dextrorotatory. The FTIR, 1H NMR, 13C observations confirmed the presence of D-glucose residues arranged in a linear chain with consecutive α (1-6) linkages without any branching. The surface morphology of vacuum dried, powdered dextran studied by using Scanning electron microscopy revealed its porous nature. The molecular weight determined by gel exclusion chromatography was found to be in the range from 10-40 kDa (~ 25 kDa). Conclusion: Unique dextran was synthesized by purified dextransucrase from newly isolated A. tropicalis. It is unique because of its linear structure, highly porous nature and small molecular weight. Native dextran with such combination has not been reported by dextransucrase of indigenous bacterial strains. This kind of dextran has high value in food industries, clinical use and therapeutics.

Objective: The objective of this study was to analyze microbial quality of raw milk and some milk products from Nanded region and to determine their antibiotic susceptibility pattern. Method: A total of 80 samples was collected and screened for the presence of some bacterial pathogens. These bacterial pathogens were identified based on morphological characters and biochemical tests and were further subjected for antibiotic susceptibility tests. Results: Among all the samples, the raw milk samples showed the highest total viable count, total coliform count and the total Staphylococcus count. The highest contamination in curd was found to be with S. aureus (40%) followed by S. epidermis (10%), E. coli (10%) and P. aeruginosa (03%). The paneer samples were found to be contaminated with E. coli (90%) followed by S. aureus (50%), S. epidermis (50%), P. aeruginosa (50%), Micrococcus sp. (25%) and Shigella sp. (10%). Similarly, peda samples were found to be contaminated with S. aureus (95%), E. coli (75%), P. aeruginosa (60%), S. epidermis (50%) and Shigella sp. (25%). All the raw milk samples were found to be contaminated with S. aureus (100%) followed by E. coli (90%), P. aeruginosa (80%), S. epidermis (50%) and Shigella sp. (20%). The antibiotic study suggests that out of 92 isolates, the highest resistance was found for penicillin (38.04%) as compared to other antibiotics. Similarly, the multidrug antibiotic resistance pattern (MAR) was detected in 36% (9/25) of S. aureus. Conclusion: The finding of this study revealed that the consumption of raw milk and traditional milk product may be a potential risk for foodborne infection in this region.

Introduction: Scientists and the public are interested in rapid and continuous monitoring of pesticide residues present in or on food using sensitive and low-cost tools. The present study developed and optimized a sensitive visual screening tool using a bio-based paper for the detection of cholinesteraseinhibiting insecticides. Method: The technique based on the change of the intensity of a purple color formed from diazonium (azo) dye that appears when α- naphthyl acetate (α-NA) is hydrolyzed by Acetylcholinesterase (AChE) in the presence of Fast blue-B as a chromogenic agent. Results: The color intensity is proportional to the concentration of ester-containing Organophosphorus (OP) and carbamate compounds. The experiment used Plackett-Burman Design (PBD) protocol in Minitab software to estimate the influence of different variables on the in vitro activity of AChE determinations under immobilization conditions. I50 values of six OP and five carbamate insecticides were calculated. Conclusion: Limit of Detection (LOD, ug/mL) of the rapid visual screening for the tested compounds were also determined, being as low as 4 μM for carbaryl and 13 μM for fenamiphos.