Faculty of Applied Sciences
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Item Development of an electrochemical immunosensor for the detection of steviol glycosides by experimental and computational methods(2020-04) Hloma, Phathisanani; Bisetty, Krishna; Sabela, M. I.; Kanchi, S.An electrochemical immunosensor employs antibodies as a capture and detection mechanism to produce an electrical charge for the quantitative analysis of target molecules. The current analytical methods for the separation and detection of stevia glycosides can be tedious in terms of sample preparation and the lack of selectivity. However, electrochemical immunosensors provide selective, sensitive and costeffective detection routes for these widely consumed sweeteners. In this study, the author developed an electrochemical immunosensor for the detection and quantification of steviol glycosides, a non-nutritive sweetener widely employed in the food and beverage industries. Most of the artificial sweeteners are low-calorie sweeteners recommended for health-related illnesses. The stability of these sweeteners at even high temperatures has increased their applications in foodstuffs widely. Constant exposure to these sweeteners is somehow associated with health complications, as some are cancer-causing agents. Although there are no reports on stevia glycosides as a health risk sweetener, its widespread use in the food industry needs to be regulated. Herein, the developed immunosensor was achieved by fabricating the platinum electrodes with graphene oxide (GO) assimilated in Zinc Oxide nanoparticles (ZnONPs) with multiwalled carbon nanotubes (MWCNTs) and immobilized with the human sweet receptor subunit T1R2. The electrochemical detection of the natural sweetening compound, Rebaudioside A (Reb A) was evaluated qualitatively and quantitatively using cyclic and differential pulse voltammetry, respectively under optimised conditions in pH 11 borate buffer from -0.4 V to 0.8 V vs Ag/AgCl. The GO/MWCNT/ZnONPs nanocomposite was characterized using High-resolution Transmission Electron microscope (HR-TEM), Thermogravimetric Analysis (TGA), Attenuated Total Reflection Mode Fourier transform infrared (ATR-FTIR) and UV-VIS spectroscopy characterization techniques. Also, asymmetric flow-field-flow fractionation and centrifugal flow-field-flow fractionation equipped with a UV-vis and multi-angle angle light scattering detectors were used to separate and characterize the size distribution of the synthesised ZnO nanostructures. The field flow fractionation (FFF) is one of the efficient separation techniques known, and centrifugal flow fieldflow fractionation separates different particle sized nanoparticles by density, thus determining size variation within the synthesised batch. The results obtained using FFF were compared and validated with the conventional characterisation techniques described above. Computational studies were used to supplement experimental results using docking and adsorption methods. Adsorption studies were carried out to better understand the mechanistic aspects between T1R2, the nanocomposite used to modify the platinum working electrode, and the analyte Reb A. Docking studies between the T1R2 receptor and the steviol glycosides were used to explore the interaction and mechanism of the immunosensor detection. The results of this study may contribute to the development of an immunosensor that can potentially be used to quantify steviol glycosides in the food and beverage industryItem Crystallography, in silico studies, and In vitro antifungal studies of 2,4,5 trisubstituted 1,2,3-triazole analogues(MDPI AG, 2020-06-20) Venugopala, Katharigatta N.; Khedr, Mohammed A.; Girish, Yarabahally R.; Bhandary, Subhrajyoti; Chopra, Deepak; Morsy, Mohamed A.; Aldhubiab, Bandar E.; Deb, Pran Kishore; Attimarad, Mahesh; Nair, Anroop B.; Sreeharsha, Nagaraja; V, Rashmi; Kandeel, Mahmoud; Akrawi, Sabah H.; Reddy M B, Madhusudana; Shashikanth, Sheena; Alwassil, Osama I.; Mohanlall, VireshA series of 2,4,5 trisubstituted-1,2,3-triazole analogues have been screened for their antifungal activity against five fungal strains, Candida parapsilosis, Candida albicans, Candida tropicalis, Aspergillus niger, and Trichophyton rubrum, via a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) microdilution assay. Compounds GKV10, GKV11, and GKV15 emerged as promising antifungal agents against all the fungal strains used in the current study. One of the highly active antifungal compounds, GKV10, was selected for a single-crystal X-ray diffraction analysis to unequivocally establish its molecular structure, conformation, and to understand the presence of different intermolecular interactions in its crystal lattice. A cooperative synergy of the C-H···O, C-H···N, C-H···S, C-H···π, and π···π intermolecular interactions was present in the crystal structure, which contributed towards the overall stabilization of the lattice. A molecular docking study was conducted for all the test compounds against Candida albicans lanosterol-14α-demethylase (pdb = 5 tzl). The binding stability of the highly promising antifungal test compound, GKV15, from the series was then evaluated by molecular dynamics studies.Item Design, synthesis, anticancer, antimicrobial activities and molecular docking studies of novel quinoline bearing dihydropyridines(Elsevier, 2016) Nkosi, S'busiso Mfan'vele; Anand, Krishnan; Anandakumar, S.; Singh, Sanil; Chuturgoon, Anil A.; Gengan, Robert MoonsamyA new series of eight quinoline bearing dihydropyridine derivatives (A1–A8) were synthesized in high yield and in short reaction time by a four component reaction of 2-chloro-3-fomyl quinoline, malononitrile, arylamines and dimethyl acetylenedicarboxylate in the presence of a catalytic amount of triethylamine. The compounds were fully characterized by IR, NMR and GC–MS. These compounds were screened for potential biological activity in an A549 lung cancer cell line and were also evaluated for their antibacterial activities against Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 whilst their molecular docking properties in an enzymatic system were also determined. Compounds A2, A3, A4 and A8 showed anti-proliferative activity; with A4 having the highest toxicity at 250 μg/mL and A8 has high toxicity at 125, 250 and 500 μg/mL, respectively. Antibacterial results indicated that A4 have significant activity against tested microorganisms at the minimum inhibitory concentration (MIC) values of 32 μg/mL against Pseudomonas aeruginosa and Escherichia coli,and 16μg/mL against Staphylococcus aureus. Docking of A1 with human mdm2 indicated the lowest binding energy (−6.111 Kcal/mol) thereby showing strong affinity of the ligand molecule with the receptor which has been stabilized by strong hydrogen bond interactions in the binding pocket. This confirms that A1 is a better inhibitor for E3 ubiquitin-protein ligase mdm2.Item Molecular dynamics simulation of chitinase I from Thermomyces lanuginosus SSBP to ensure optimal activity(Taylor and Fancis Online, 2016-09-22) Khan, Faez Iqbal; Bisetty, Krishna; Gu, Ke-Ren; Singh, Suren; Permaul, Kugen; Hassan, Md. Imtaiyaz; Wei, Dong-QingThe fungal chitinase I obtained from Thermomyces lanuginosus SSBP, a thermophilic deuteromycete, has an optimum growth temperature and pH of 323.15 K and 6.5, respectively. This enzyme plays an important task in the defence mechanism of organisms against chitin-containing parasites by hydrolysing β-1, 4-linkages in chitin. It acts as both anti-fungal and biofouling agents, with some being thermostable and suitable for the industrial applications. Three-dimensional model of chitinase I enzyme was predicted and analysed using various bioinformatics tools. The structure of chitinase I exhibited a well-defined TIM barrel topology with an eight-stranded α/β domain. Structural analysis and folding studies at temperatures ranging from 300 to 375 K using 10 ns molecular dynamics simulations clearly showed the stability of the protein was evenly distributed even at higher temperatures, in accordance with the experimental results. We also carried out a number of 20 ns constant pH molecular dynamics simulations of chitinase I at a pH range 2–6 in a solvent. This work was aimed at establishing the optimum activity and stability profiles of chitinase I. We observed a strong conformational pH dependence of chitinase I and the enzyme retained their characteristic TIM barrel topology at low pH.Item Thermostable chitinase II from Thermomyces lanuginosus SSBP: Cloning, structure prediction and molecular dynamics simulations(Elsevier, 2015) Khan, Faez Iqbal; Govender, Algasan; Permaul, Kugen; Singh, Suren; Bisetty, KrishnaThermomyces lanuginosus is a thermophilic fungus that produces large number of industrially-significant enzymes owing to their inherent stability at high temperatures and wide range of pH optima, including thermostable chitinases that have not been fully characterized. Here, we report cloning, characterization and structure prediction of a gene encoding thermostable chitinase II. Sequence analysis revealed that chitinase II gene encodes a 343 amino acid protein of molecular weight 36.65 kDa. Our study reports thatchitinase II exhibits a well-defined TIM-barrel topology with an eight-stranded α/β domain. Structural analysis and molecular docking studies suggested that Glu176 is essential for enzyme activity. Folding studies of chitinase II using molecular dynamics simulations clearly demonstrated that the stability of the protein was evenly distributed at 350 K.Item Thermostable chitinase II from Thermomyces lanuginosus SSBP : Cloning, structure prediction and molecular dynamics simulations(Elsevier, 2015-04-08) Khan, Faez Iqbal; Govender, Algasan; Permaul, Kugen; Singh, Suren; Bisetty, KrishnaThermomyces lanuginosus is a thermophilic fungus that produces large number of industrially-significant enzymes owing to their inherent stability at high temperatures and wide range of pH optima, including thermostable chitinases that have not been fully characterized. Here, we report cloning, characterization and structure prediction of a gene encoding thermostable chitinase II. Sequence analysis revealed that chitinase II gene encodes a 343 amino acid protein of molecular weight 36.65 kDa. Our study reports that chitinase II exhibits a well-defined TIM-barrel topology with an eight-stranded α/β domain. Structural analysis and molecular docking studies suggested that Glu176 is essential for enzyme activity. Folding studies of chitinase II using molecular dynamics simulations clearly demonstrated that the stability of the protein was evenly distributed at 350 K.Item Studies on bacterial proteins corona interaction with saponin imprinted ZnO nano-honeycombs and their toxic responses(ACS Publications, 2015-10-07) Sharma, Deepali; Ashaduzzaman, Md.; Golabi, Mohsen; Shriwastav, Amritanshu; Bisetty, Krishna; Tiwari, AshutoshMolecular imprinting generates robust, efficient and highly mesoporous surfaces for bio-interactions. Mechanistic interfacial interaction between the surface of core substrate and protein corona is crucial to understanding the substantial microbial toxic responses at a nanoscale. In this study, we have focused on the mechanistic interactions between synthesised saponin imprinted zinc oxide nano-honeycombs (SIZnO NHs), average size 80-125 nm, surface area 20.27 m2/g, average pore density 0.23 pore/nm and number average pore size 3.74 nm and proteins corona of bacteria. The produced SIZnO NHs as potential anti-fungal and anti-bacterial agents have been studied on Sclerotium rolfsii (S. rolfsii), Pythium debarynum (P. debarynum) and Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), respectively. SIZnO NHs exhibited the highest antibacterial (~50%) and antifungal (~40%) activity against gram-negative bacteria (E. coli) and fungus (P. debarynum) respectively at concentration of 0.1 mol. Scanning electron spectroscopy (SEM) observation showed that the ZnO NHs ruptured the cell wall of bacteria and internalised into the cell. The molecular docking studies have been carried out using lipopolysaccharide and lipocalin Blc as binding proteins. It was envisaged that the proteins present in the bacterial cell wall were found to interact and adsorb on the surface of SIZnO NHs thereby blocking the active sites of the proteins used for cell wall synthesis. The binding affinity and interaction energies for lipopolysaccharide were higher than those of the lipocalin Blc. In addition, a kinetic mathematical model (KMM) was developed in MATLAB to predict the internalisation in the bacterial cellular uptake of the ZnO NHs for better understanding of their controlled toxicity. The results obtained from KMM exhibited a good agreement with the experimental data. Exploration of mechanistic interactions, as well as the formation of bioconjugate of proteins and ZnO NHs would play a key role to interpret more complex biological systems in nature.Item Evaluation of enantioresolution of (±)-catechin using electrokinetic chromatography and molecular docking(SRP, 2012) Sabela, Myalowenkosi Innocent; Singh, Parvesh; Gumede, Njabulo Joyfull; Bisetty, Krishna; Sagrado, SagradoThis study involves the enantioresolution of (±) catechin with the highly sulphated beta cyclodextrin (HS-β-CD) as a chiral selector using capillary electrophoresis (CE). The purpose of this study was to be tter understand enantioresolution amongst host-guest interactions. Furthermore, molecular docking was carried out to elucidate the mechanism of the enantioselective separations of (±) catechin enantiomers obtained in Electrokinetic chroma tography (EKC). A large difference in the interaction energies observed between the two enantiomers represents significant enantiodifferentiation. Our results also suggest that the host-guest interactions between the phenyl ring of the ligand and the open cavity of the HS-β-CD are due mainly to hydrophobic interactions. Interestingly, the stronger interactions observed with (+)-catechin is consistent with the elution order observed in the CE experiments.