Search results for: pectinase enzyme
596 STD-NMR Based Protein Engineering of the Unique Arylpropionate-Racemase AMDase G74C
Authors: Sarah Gaßmeyer, Nadine Hülsemann, Raphael Stoll, Kenji Miyamoto, Robert Kourist
Abstract:
Enzymatic racemization allows the smooth interconversion of stereocenters under very mild reaction conditions. Racemases find frequent applications in deracemization and dynamic kinetic resolutions. Arylmalonate decarboxylase (AMDase) from Bordetella Bronchiseptica has high structural similarity to amino acid racemases. These cofactor-free racemases are able to break chemically strong CH-bonds under mild conditions. The racemase-like catalytic machinery of mutant G74C conveys it a unique activity in the racemisation of pharmacologically relevant derivates of 2-phenylpropionic acid (profenes), which makes AMDase G74C an interesting object for the mechanistic investigation of cofactor-independent racemases. Structure-guided protein engineering achieved a variant of this unique racemase with 40-fold increased activity in the racemisation of several arylaliphatic carboxylic acids. By saturation–transfer–difference NMR spectroscopy (STD-NMR), substrate binding during catalysis was investigated. All atoms of the substrate showed interactions with the enzyme. STD-NMR measurements revealed distinct nuclear Overhauser effects in experiments with and without molecular conversion. The spectroscopic analysis led to the identification of several amino acid residues whose variation increased the activity of G74C. While single-amino acid exchanges increased the activity moderately, structure-guided saturation mutagenesis yielded a quadruple mutant with a 40 times higher reaction rate. This study presents STD-NMR as versatile tool for the analysis of enzyme-substrate interactions in catalytically competent systems and for the guidance of protein engineering.Keywords: racemase, rational protein design, STD-NMR, structure guided saturation mutagenesis
Procedia PDF Downloads 299595 Improvement of Activity of β-galactosidase from Kluyveromyces lactis via Immobilization on Polyethylenimine-Chitosan
Authors: Carlos A. C. G. Neto, Natan C. G. e Silva , Thaís de O. Costa, Luciana R. B. Gonçalves, Maria V. P. Rocha
Abstract:
β-galactosidases (E.C. 3.2.1.23) are enzymes that have attracted by catalyzing the hydrolysis of lactose and in producing galacto-oligosaccharides by favoring transgalactosylation reactions. These enzymes, when immobilized, can have some enzymatic characteristics substantially improved, and the coating of supports with multifunctional polymers is a promising alternative to enhance the stability of the biocatalysts, among which polyethylenimine (PEI) stands out. PEI has certain properties, such as being a flexible polymer that suits the structure of the enzyme, giving greater stability, especially for multimeric enzymes such as β-galactosidases. Besides that, protects them from environmental variations. The use of chitosan support coated with PEI could improve the catalytic efficiency of β-galactosidase from Kluyveromyces lactis in the transgalactosylation reaction for the production of prebiotics, such as lactulose since this strain is more effective in the hydrolysis reaction. In this context, the aim of the present work was first to develop biocatalysts of β-galactosidase from K. lactis immobilized on chitosan-coated with PEI, determining the immobilization parameters, its operational and thermal stability, and then to apply it in hydrolysis and transgalactolisation reactions to produce lactulose using whey as a substrate. The immobilization of β-galactosidase in chitosan previously functionalized with 0.8% (v/v) glutaraldehyde and then coated with 10% (w/v) PEI solution was evaluated using an enzymatic load of 10 mg protein per gram support. Subsequently, the hydrolysis and transgalactosylation reactions were conducted at 50 °C, 120 RPM for 20 minutes, using whey supplemented with fructose at a ratio of 1:2 lactose/fructose, totaling 200 g/L. Operational stability studies were performed in the same conditions for 10 cycles. Thermal stabilities of biocatalysts were conducted at 50 ºC in 50 mM phosphate buffer, pH 6.6 with 0.1 mM MnCl2. The biocatalyst whose support was coated was named CHI_GLU_PEI_GAL, and the one that was not coated was named CHI_GLU_GAL. The coating of the support with PEI considerably improved the parameters of immobilization. The immobilization yield increased from 56.53% to 97.45%, biocatalyst activity from 38.93 U/g to 95.26 U/g and the efficiency from 3.51% to 6.0% for uncoated and coated support, respectively. The biocatalyst CHI_GLU_PEI_GAL was better than CHI_GLU_GAL in the hydrolysis of lactose and production of lactulose, converting 97.05% of lactose at 5 min of reaction and producing 7.60 g/L lactulose in the same time interval. QUI_GLU_PEI_GAL biocatalyst was stable in the hydrolysis reactions of lactose during the 10 cycles evaluated, converting 73.45% lactose even after the tenth cycle, and in the lactulose production was stable until the fifth cycle evaluated, producing 10.95 g/L lactulose. However, the thermal stability of CHI_GLU_GAL biocatalyst was superior, with a half-life time 6 times higher, probably because the enzyme was immobilized by covalent bonding, which is stronger than adsorption (CHI_GLU_PEI_GAL). Therefore, the strategy of coating the supports with PEI has proven to be effective for the immobilization of β-galactosidase from K. lactis, considerably improving the immobilization parameters, as well as, the catalytic action of the enzyme. Besides that, this process can be economically viable due to the use of an industrial residue as a substrate.Keywords: β-galactosidase, immobilization, kluyveromyces lactis, lactulose, polyethylenimine, transgalactosylation reaction, whey
Procedia PDF Downloads 108594 Effect of Hormones Priming on Enzyme Activity and Lipid Peroxidation in Wheat Seed under Accelerated Aging
Authors: Amin Abbasi, Fariborz Shekari, Seyed Bahman Mousavi
Abstract:
Seed aging during storage is a complex biochemical and physiological processes that can lead to reduce seed germination. This phenomenon associated with increasing of total antioxidant activity during aging. To study the effects of hormones on seed aging, aged wheat seeds (control, 90 and 80% viabilities) were treated with GA3, Salicylic Acid, and paclobutrazol and antioxidant system were investigated as molecular biomarkers for seed vigor. The results showed that, seed priming treatment significantly affected germination percentage, normality seedling percentage, H2O2, MDA, CAT, APX, and GPX activates. Maximum germination percentage achieve in GA3 priming in control treatment. Germination percentage and normal seedling percentage increased in other GA3 priming treatment compared with other hormones. Also aging increased MDA, H2O2 content. MDA is considered sensitive marker commonly used for assessing membrane lipid peroxidation and H2O2result in toxicity to cellular membrane system and damages to plant cells. Amount of H2O2 and MDA declined in GA3 treatment. CAT, GPX and APX activities were reduced by increasing the aging time and at different levels of priming. The highest APX activity was observed in Salicylic Acid control treatment and the highest GPX and CAT activity was obtained in GA3 control treatment. The lowest MDA and H2O2 showed in GA3 control treatment, too. Hormone priming increased Antioxidant enzyme activity and decreased amount of reactive oxygen space and malondialdehyde (MDA) under aging treatment. Also, GA3 priming treatments have a significant effect on germination percentage and number of normal seedling. Generally aging seed, increase ROS and lipid peroxidation. Antioxidant enzymes activity of aged seeds increased after hormone priming.Keywords: hormones priming, wheat, aging seed, antioxidant, lipid peroxidation
Procedia PDF Downloads 487593 Variation in Carboxylesterase Activity in Spodoptera litura Fabricious (Noctuidae: Lepidoptera) Populations from India
Authors: V. Karuppaiah, J. C. Padaria, C. Srivastava
Abstract:
The tobacco caterpillar, Spodoptera litura Fab (Lepidoptera: Noctuidae) is a polyphagous pest various field and horticulture crops in India. Pest had virtually developed resistance to all commonly used insecticides. Enhanced detoxification is the prime mechanism that is dictated by detoxification different enzymes and carboxylesterase is one of the major enzyme responsible development of resistance. In India, insecticide resistance studies on S. litura are mainly deployed on detoxification enzymes activity and investigation at gene level alteration i.e. at nucleotide level is very merger. In the present study, we collected the S. litura larvae from three different cauliflower growing belt viz., IARI, New Delhi (Delhi), Palari, Sonepat (Haryana) and Varanasi (Uttar Pradesh) to study the role of carboxylesterase activity and its gene level variation The CarE activity was measured using UV-VIS spectrophotometer with 3rd instar larvae of S. litura. The elevated activity of CarE was observed in Sonepat strain (28.09 ± 0.09 µmol/min/mg of protein) followed by Delhi (26.72 ± 0.04 µmol/min/mg of protein) and Varanasi strain (10.00 ± 0.44 µmol/min/mg of protein) of S. litura. The genomic DNA was isolated from 3rd instar larvae and CarE gene was amplified using a primer sequence, F:5’tccagagttccttgtcaggcac3’; R:5’ctgcatcaagcatgtctc3. CarE gene, about 500bp was partially amplified, sequenced and submitted to NCBI (Accession No. KF835886, KF835887 and KF835888). The sequence data revealed polymorphism at nucleotide level in all the three strains and gene found to have 88 to 97% similarity with previous available nucleotide sequences of S. litura, S. littoralis and S. exiqua. The polymorphism at the nucleotide level could be a reason for differential activity of carboxylesterase enzymes among the strains. However, investigation at gene expression level would be useful to analyze the overproduction of carboxylesterase enzyme.Keywords: carboxylesterase, CarE gene, nucleotide polymorphism, insecticide resistance, spodoptera litura
Procedia PDF Downloads 916592 Enzyme Producing Psyhrophilic Pseudomonas app. Isolated from Poultry Meats
Authors: Ali Aydin, Mert Sudagidan, Aysen Coban, Alparslan Kadir Devrim
Abstract:
Pseudomonas spp. (specifically, P. fluorescens and P. fragi) are considered the principal spoilage microorganisms of refrigerated poultry meats. The higher the level psychrophilic spoilage Pseudomonas spp. on carcasses at the end of processing lead to decrease the shelf life of the refrigerated product. The aim of the study was the identification of psychrophilic Pseudomonas spp. having proteolytic and lipolytic activities from poultry meats by 16S rRNA and rpoB gene sequencing, investigation of protease and lipase related genes and determination of proteolytic activity of Pseudomonas spp. In the of isolation procedure, collected chicken meat samples from local markets and slaughterhouses were homogenized and the lysates were incubated on Standard method agar and Skim Milk agar for selection of proteolytic bacteria and tributyrin agar for selection of lipolytic bacteria at +4 °C for 7 days. After detection of proteolytic and lipolytic colonies, the isolates were firstly analyzed by biochemical tests such as Gram staining, catalase and oxidase tests. DNA gene sequencing analysis and comparison with GenBank revealed that 126 strong enzyme Pseudomonas spp. were identified as predominantly P. fluorescens (n=55), P. fragi (n=42), Pseudomonas spp. (n=24), P. cedrina (n=2), P. poae (n=1), P. koreensis (n=1), and P. gessardi (n=1). Additionally, protease related aprX gene was screened in the strains and it was detected in 69/126 strains, whereas, lipase related lipA gene was found in 9 Pseudomonas strains. Protease activity was determined using commercially available protease assay kit and 5 strains showed high protease activity. The results showed that psychrophilic Pseudomonas strains were present in chicken meat samples and they can produce important levels of proteases and lipases for food spoilage to decrease food quality and safety.Keywords: Pseudomonas, chicken meat, protease, lipase
Procedia PDF Downloads 383591 Optimization and Kinetic Analysis of the Enzymatic Hydrolysis of Oil Palm Empty Fruit Bunch To Xylose Using Crude Xylanase from Trichoderma Viride ITB CC L.67
Authors: Efri Mardawati, Ronny Purwadi, Made Tri Ari Penia Kresnowati, Tjandra Setiadi
Abstract:
EFB are mainly composed of cellulose (≈ 43%), hemicellulose (≈ 23%) and lignin (≈20%). The palm oil empty fruit bunches (EFB) is the lignosellulosic waste from crude palm oil industries mainly compose of (≈ 43%), hemicellulose (≈ 23%) and lignin (≈20%). Xylan, a polymer made of pentose sugar xylose and the most abundant component of hemicellulose in plant cell wall. Further xylose can be used as a raw material for production of a wide variety of chemicals such as xylitol, which is extensively used in food, pharmaceutical and thin coating applications. Currently, xylose is mostly produced from xylan via chemical hydrolysis processes. However, these processes are normally conducted at a high temperature and pressure, which is costly, and the required downstream processes are relatively complex. As an alternative method, enzymatic hydrolysis of xylan to xylose offers an environmentally friendly biotechnological process, which is performed at ambient temperature and pressure with high specificity and at low cost. This process is catalysed by xylanolytic enzymes that can be produced by some fungal species such as Aspergillus niger, Penicillium crysogenum, Tricoderma reseei, etc. Fungal that will be used to produce crude xylanase enzyme in this study is T. Viride ITB CC L.67. It is the purposes of this research to study the influence of pretreatment of EFB for the enzymatic hydrolysis process, optimation of temperature and pH of the hydrolysis process, the influence of substrate and enzyme concentration to the enzymatic hydrolysis process, the dynamics of hydrolysis process and followingly to study the kinetics of this process. Xylose as the product of enzymatic hydrolysis process analyzed by HPLC. The results show that the thermal pretreatment of EFB enhance the enzymatic hydrolysis process. The enzymatic hydrolysis can be well approached by the Michaelis Menten kinetic model, and kinetic parameters are obtained from experimental data.Keywords: oil palm empty fruit bunches (EFB), xylose, enzymatic hydrolysis, kinetic modelling
Procedia PDF Downloads 383590 Optimization of Biomass Components from Rice Husk Treated with Trichophyton Soudanense and Trichophyton Mentagrophyte and Effect of Yeast on the Bio-Ethanol Yield
Authors: Chukwuma S. Ezeonu, Ikechukwu N. E. Onwurah, Uchechukwu U. Nwodo, Chibuike S. Ubani, Chigozie M. Ejikeme
Abstract:
Trichophyton soudanense and Trichophyton mentagrophyte were isolated from the rice mill environment, cultured and used singly and as di-culture in the treatment of measure quantities of preheated rice husk. Optimized conditions studied showed that carboxymethylcellulase (CMCellulase) activity of 57.61 µg/ml/min was optimum for Trichophyton mentagrophyte heat pretreated rice husk crude enzymes at 50oC and 80oC respectively. Duration of 120 hours (5 days) gave the highest CMcellulase activity of 75.84 µg/ml/min for crude enzyme of Trichophyton mentagrophyte heat pretreated rice husk. However, 96 hours (4 days) duration gave maximum activity of 58.21 µg/ml/min for crude enzyme of Trichophyton soudanense heat pretreated rice husk. Highest CMCellulase activities of 67.02 µg/ml/min and 69.02 µg/ml/min at pH of 5 were recorded for crude enzymes of monocultures of Trichophyton soudanense (TS) and Trichophyton mentagrophyte (TM) heat pretreated rice husk respectively. Biomass components showed that rice husk cooled after heating followed by treatment with Trichophyton mentagrophyte gave 44.50 ± 10.90 (% ± Standard Error of Mean) cellulose as the highest yield. Maximum total lignin value of 28.90 ± 1.80 (% ± SEM) was obtained from pre-heated rice husk treated with di-culture of Trichophyton soudanense and Trichophyton mentagrophyte (TS+TM). The hemicellulose content of 30.50 ± 2.12 (% ± SEM) from pre-heated rice husk treated with Trichophyton soudanense (TS); lignin value of 28.90 ± 1.80 from pre-heated rice husk treated with di-culture of Trichophyton soudanense and Trichophyton mentagrophyte (TS+TM); also carbohydrate content of 16.79 ± 9.14 (% ± SEM) , reducing and non-reducing sugar values of 2.66 ± 0.45 and 14.13 ± 8.69 (% ± SEM) were all obtained from for pre- heated rice husk treated with Trichophyton mentagrophyte (TM). All the values listed above were the highest values obtained from each rice husk treatment. The pre-heated rice husk treated with Trichophyton mentagrophyte (TM) fermented with palmwine yeast gave bio-ethanol value of 11.11 ± 0.21 (% ± Standard Deviation) as the highest yield.Keywords: Trichophyton soudanense, Trichophyton mentagrophyte, biomass, bioethanol, rice husk
Procedia PDF Downloads 671589 Potentialities of Onopordum Tauricum (Willd.) as Milk Clotting Agent
Authors: Massimo Mozzon, Nadia Raffaelli
Abstract:
Proteases from herbs, woody plants, and trees are exploited for cheesemaking in several countries, especially in South Europe and West Africa. Particularly, “thistles” belonging to several genera within the Asteraceae family (Cynara, Silybum, Centaurea, Carlina, Cirsium, Onopordum) are traditionally used in Mediterranean countries for clotting raw ewe’s and goat’s milk. For the first time, the clotting performance of an aqueous extract from flowers of Onopordum tauricum Willd. (Taurian thistle, bull cottonthistle) were tested in milk of different origin (cow, goat, ewe). The vegetable material was collected in the Central Apennines range, between the Marche and Umbria regions. A response surface methodology (RSM) approach was used to study the effect of the curdling variables (temperature, pH, amount of enzymatic extract) on the technological performance of the thistle extract. A three-step procedure for the purification of the enzyme (ammonium sulphate precipitation, gel filtration and ion-exchange chromatography) was also carried out. The milk clotting activity (MCA) of O. tauricum crude extracts was strongly affected by temperature, pH and by the interaction between these two variables, according to a second-order response surface model, while the milk/coagulant ratio did not affect in a significant way the clotting properties. Experimental data showed that the addition of 10 mM CaCl2 reduced the clotting time of ewe’s, goat’s, and cow’s milk by about 3-fold, 8-fold, and 14-fold, respectively, at 35°C and pH 6.7-6.8. After purification, an enzymatic preparation very close to homogeneity was obtained, which showed a major band at about 30 kDa when analyzed by SDS-PAGE. The identity of the enzyme as an aspartic protease was confirmed by inhibition studies. Cheese-making trials were carried out to check the scale-up (1 to 5 L of milk; 37 °C; 10 mM CaCl2 fortification) and set the recipe: 35-45% of curd yields were recorded, according to curd cutting and pressing.Keywords: milk clotting activity, Onopordum tauricum, plant proteases, vegetable rennet
Procedia PDF Downloads 154588 Anaerobic Fermentation Process for Production of Biohydrogen from Pretreated Fruit Wastes
Authors: A. K. R. Gobinath, He Jianzhong, Kun-Lin Yang
Abstract:
Fruit waste was used as a feedstock to produce biohydrogen in this study. Fruit waste used in this study was collected from several fruit juice stalls in Singapore. Based on our observation, the fruit waste contained 35-40% orange, 10-20% watermelon, 10-15% apple, 10-15% pineapple, 1-5% mango. They were mixed with water (1:1 ratio based on wet biomass) and blended to attain homogenous mixtures. Later, fruit waste was subjected to one of the following pretreatments: autoclave (121 °C for 20min), microwave (20min) or both. After pretreatment, the total sugar concentration in the hydrolysate was high (>12g/l) when both autoclave and microwave were applied. In contrast, samples without pretreatment measured only less than 2g/l of sugar. While using these hydrolysates as carbon sources, Clostridium strain BOH3 produces 2526-3126 ml/l of hydrogen after 72h of anaerobic fermentation. The hydrogen yield was 295-300 ml/g of sugar which is close to the hydrogen yields from glucose (338 ml/gm) and xylose (330 ml/gm). Our HPLC analysis showed that fruit waste hydrolysate contained oligosugars (25-27%), sucrose (18-23%), fructose (25-30%), glucose (10-15%) and mannose (2-5%). Additionally, pretreatment led to the release of free amino acids (160-512 mg/l), calcium (7.8-12.9 ppm), magnesium (4.32-6.55 ppm), potassium (5.4-65.1 ppm) and sodium (0.4-0.5 ppm) into the hydrolysate. These nutrients were able to support strain-BOH3 to grow and produce high level of hydrogen. Notably, unlike other pretreatment methods (with strong acids and bases), these pretreatment techniques did not generate any inhibitors (e.g. furfural and phenolic acids) to suppress the hydrogen production. Interestingly, strain BOH3 can also ferment pretreated fruit waste slurry and produce hydrogen with a high yield (156-343 ml/gm fruit waste). While fermenting pretreated fruit waste slurry, strain-BOH3 excreted several saccharolytic enzymes majorly xylanase (1.84U/ml), amylase (1.10U/ml), pectinase (0.36U/ml) and cellulase (0.43U/ml). Due to expressions of these enzymes, strain BOH3 was able to directly utilize pretreated fruit waste hydrolysate and produces high-level of hydrogen.Keywords: autoclave pretreatment, biohydrogen production, clostridial fermentation, fruit waste, and microwave pretreatment
Procedia PDF Downloads 527587 Ascidian Styela rustica Proteins’ Structural Domains Predicted to Participate in the Tunic Formation
Authors: M. I. Tyletc, O. I. Podgornya, T. G. Shaposhnikova, S. V. Shabelnikov, A. G. Mittenberg, M. A. Daugavet
Abstract:
Ascidiacea is the most numerous class of the Tunicata subtype. These chordates' distinctive feature of the anatomical structure is a tunic consisting of cellulose fibrils, protein molecules, and single cells. The mechanisms of the tunic formation are not known in detail; tunic formation could be used as the model system for studying the interaction of cells with the extracellular matrix. Our model species is the ascidian Styela rustica, which is prevalent in benthic communities of the White Sea. As previously shown, the tunic formation involves morula blood cells, which contain the major 48 kDa protein p48. P48 participation in the tunic formation was proved using antibodies against the protein. The nature of the protein and its function remains unknown. The current research aims to determine the amino acid sequence of p48, as well as to clarify its role in the tunic formation. The peptides that make up the p48 amino acid sequence were determined by mass spectrometry. A search for peptides in protein sequence databases identified sequences homologous to p48 in Styela clava, Styela plicata, and Styela canopus. Based on sequence alignment, their level of similarity was determined as 81-87%. The correspondent sequence of ascidian Styela canopus was used for further analysis. The Styela rustica p48 sequence begins with a signal peptide, which could indicate that the protein is secretory. This is consistent with experimentally obtained data: the contents of morula cells secreted in the tunic matrix. The isoelectric point of p48 is 9.77, which is consistent with the experimental results of acid electrophoresis of morula cell proteins. However, the molecular weight of the amino acid sequence of ascidian Styela canopus is 103 kDa, so p48 of Styela rustica is a shorter homolog. The search for conservative functional domains revealed the presence of two Ca-binding EGF-like domains, thrombospondin (TSP1) and tyrosinase domains. The p48 peptides determined by mass spectrometry fall into the region of the sequence corresponding to the last two domains and have amino acid substitutions as compared to Styela canopus homolog. The tyrosinase domain (pfam00264) is known to be part of the phenoloxidase enzyme, which participates in melanization processes and the immune response. The thrombospondin domain (smart00209) interacts with a wide range of proteins, and is involved in several biological processes, including coagulation, cell adhesion, modulation of intercellular and cell-matrix interactions, angiogenesis, wound healing and tissue remodeling. It can be assumed that the tyrosinase domain in p48 plays the role of the phenoloxidase enzyme, and TSP1 provides a link between the extracellular matrix and cell surface receptors, and may also be responsible for the repair of the tunic. The results obtained are consistent with experimental data on p48. The domain organization of protein suggests that p48 is an enzyme involved in the tunic tunning and is an important regulator of the organization of the extracellular matrix.Keywords: ascidian, p48, thrombospondin, tyrosinase, tunic, tunning
Procedia PDF Downloads 105586 Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β-Lactamase Inhibitor
Authors: Mohammad Faheem, M. Tabish Rehman, Mohd Danishuddin, Asad U. Khan
Abstract:
The worldwide dissemination of CTX-M type β-lactamases is a threat to human health. Previously, we have reported the spread of blaCTX-M-15 gene in different clinical strains of Enterobacteriaceae from the hospital settings of Aligarh in north India. In view of the varying resistance pattern against cephalosporins and other β-lactam antibiotics, we intended to understand the correlation between MICs and catalytic activity of CTX-M-15. In this study, steady-state kinetic parameters and MICs were determined on E. coli DH5α transformed with blaCTX-M-15 gene that was cloned from Enterobacter cloacae (EC-15) strain of clinical background. The effect of conventional β-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) on CTX-M-15 was also studied. We have found that tazobactam is the best among these inhibitors against CTX-M-15. The inhibition characteristic of tazobactam is defined by its very low IC50 value (6 nM), high affinity (Ki = 0.017 µM) and better acylation efficiency (k+2/K9 = 0.44 µM-1s-1). It forms an acyl-enzyme covalent complex, which is quite stable (k+3 = 0.0057 s-1). Since increasing resistance has been reported against conventional b-lactam antibiotic-inhibitor combinations, we aspire to design a non-b-lactam core containing b-lactamase inhibitor. For this, we screened ZINC database and performed molecular docking to identify a potential non-β-lactam based inhibitor (ZINC03787097). The MICs of cephalosporin antibiotics in combination with this inhibitor gave promising results. Steady-state kinetics and molecular docking studies showed that ZINC03787097 is a reversible inhibitor which binds non-covalently to the active site of the enzyme through hydrogen bonds and hydrophobic interactions. Though, it’s IC50 (180 nM) is much higher than tazobactam, it has good affinity for CTX-M-15 (Ki = 0.388 µM). This study concludes that ZINC03787097 compound can be used as seed molecule to design more efficient non-b-lactam containing b-lactamase inhibitor that could evade pre-existing bacterial resistance mechanisms.Keywords: ESBL, non-b-lactam-b-lactamase inhibitor, bioinformatics, biomedicine
Procedia PDF Downloads 233585 Biodegradation of 2,4-Dichlorophenol by Pseudomonas chlororaphis Strain Isolated from Activated Sludge Sample from a Wastewater Treatment Plant in Durban, South Africa
Authors: Boitumelo Setlhare, Mduduzi P. Mokoena, Ademola O. Olaniran
Abstract:
Agricultural and industrial activities have led to increasing production of xenobiotics such as 2,4-dichlorophenol (2,4-DCP), a derivative of 2,4-dichlorophenoxyacetic acid (2,4-D), which is a widely used herbicide. Bioremediation offers an efficient, cost-effective and environmentally friendly method for degradation of the compound through the activities of the various microbial enzymes involved in the catabolic pathway. The aim of this study was to isolate and characterize bacterial isolate indigenous to contaminated sites in Durban, South Africa for 2,4-DCP degradation. One bacterium capable of utilizing 2,4-DCP as sole carbon source was isolated using culture enrichment technique and identified as Pseudomonas chlororaphis strain UFB2 via PCR amplification and analysis of 16S rRNA gene sequence. This isolate was able to degrade up to 75.11% of 2,4-DCP in batch cultures within 10 days, with the degradation rate constant of 0.14 mg/l/d. Phylogenetic analysis revealed the relatedness of this bacterial isolate to other Pseudomonas sp. previously characterized for chlorophenol degradation. PCR amplification of the catabolic genes involved in 2,4-DCP degradation revealed the presence of the correct amplicons for phenol hydroxylase (600 bp), catechol 1,2-dioxygenase (214 bp), muconate isomerase (851 bp), cis-dienelactone hydrolase (577 bp), and trans-dienelactone hydrolase (491 bp) genes. Enzyme assays revealed activity as high as 21840 mU/mg, 15630 mU/mg, 2340 mU/mg and 1490 mU/mg obtained for phenol hydroxylase, catechol 1,2-dioxygenase, cis-dienelactone hydroxylase and trans-dienelactone hydroxylase, respectively. The absence of catechol 2,3-dioxygenase gene and the corresponding enzyme in this isolate suggests that the organism followed ortho-pathway for 2,4-DCP degradation. Furthermore, the absence of malaycetate reductase genes showed that the bacterium may not be able to completely mineralize 2,4-DCP. Further studies are required to optimize 2,4-DCP degradation by this isolate as well as to elucidate the mechanism of 2,4-DCP degradation.Keywords: biodegradation, catechol 1, 2-dioxygenase, 2, 4-dichlorophenol, phenol hydroxylase, Pseudomonas chlororaphis
Procedia PDF Downloads 244584 Aminopeptidase P (DAP) Expression Pattern in Drosophila Melanogaster
Authors: Suneeta Gireesh Panicker
Abstract:
Aim: Aminopeptidase P (APP) is an enzyme that has specificity for proline, can specifically cleave Xaa-Proline peptides and is a metallo-aminopeptidase. The bonds nearby to the imino acid proline are tough to cleave by many peptidases, but APP can specifically break peptide bonds engaged with proline. Membrane-bound form and a cytosolic form are the two forms in which this enzyme exists. The exact physiological function of APP remains unclear and hence the present work attempts to determine it. Methods: In the present study, the expression pattern of cytosolic Aminopeptidase P (DAP) was determined in all the embryonic stages and larval stages of wild-type Drosophila by using polyclonal monospecific antibodies. To show the presence of DAP RNA in embryonic and larval stages, RNA in situ hybridization was performed. DAP promoter-LacZ fusion reporter gene vector was used to construct transgenic embryos to study the regulation pattern of DAP. To study the DAP expression profile, a transgenic fly consisting of a DAP promoter with β-gal and GFP reporter genes in front of it was constructed. Results: DAP protein expression was observed in neuroectodermal cells, posterior midgut primordium, proctodeum, ventral neuroblast and primordial stomatogastric nervous system. It was observed in the ventral cord and midgut in stage 12. The completely developed embryos showed the intense occurrence of it in the ventral cord and gut region. The eye-antennal disc, wing disc and leg disc also showed the presence of DAP protein. LacZ expression in transgenic embryos also showed the same pattern. Conclusion: Similar to various known multiple-functional proteins, DAP could be one with different functions at different stages and in different cells. Data presented here designates DAP functions in the early embryonic and imaginal dics differentiation and development, suggesting that it may be required for the metabolism of proteins like neuropeptides and tachykinins.Keywords: aminopeptidase P, in situ hybridization, transgenic fly, embryonic stages
Procedia PDF Downloads 77583 Identification of the Key Enzyme of Roseoflavin Biosynthesis
Authors: V. Konjik, J. Schwartz, R. Sandhoff, M. Mack
Abstract:
The rising number of multi-resistant pathogens demands the development of new antibiotics in order to reduce the lethal risk of infections. Here, we investigate roseoflavin, a vitamin B2 analogue which is produced by Streptomyces davawensis and Streptomyces cinnabarinus. We consider roseoflavin to be a 'Trojan horse' compound. Its chemical structure is very similar to riboflavin but in fact it is a toxin. Furthermore, it is a clever strategy with regard to the delivery of an antibiotic to its site of action but also with regard to the production of this chemical: The producer cell has only to convert a vitamin (which is already present in the cytoplasm) into a vitamin analog. Roseoflavin inhibits the activity of Flavin depending proteins, which makes up to 3.5 % of predicted proteins in organisms sequenced so far. We sequentially knocked out gene clusters and later on single genes in order to find the ones which are involved in the roseoflavin biosynthesis. Consequently, we identified the gene rosB, coding for the protein carrying out the first step of roseoflavin biosynthesis, starting form Flavin mononucleotide. Here we show, that the protein RosB has so far unknown features. It is per se an oxidoreductase, a decarboxylase and an aminotransferase, all rolled into one enzyme. A screen of cofactors revealed needs of oxygen, NAD+, thiamine and glutamic acid to carry out its function. Surprisingly, thiamine is not only needed for the decaboxylation step, but also for the oxidation of 8-demethyl-8-formyl Flavin mononucleotide. We had managed to isolate three different Flavin intermediates with different oxidation states, which gave us a mechanistic insight of RosB functionality. Our work points to a so far new function of thiamine in Streptomyces davawensis. Additionally, RosB could be extremely useful for chemical synthesis. Careful engineering of RosB may allow the site-specific replacement of methyl groups by amino groups in polyaromatic compounds of commercial interest. Finally, the complete clarification of the roseoflavin biosynthesis opens the possibility of engineering cost-effective roseoflavin producing strains.Keywords: antibiotic, flavin analogue, roseoflavin biosynthesis, vitamin B2
Procedia PDF Downloads 238582 Synthesis of Carbon Nanotubes from Coconut Oil and Fabrication of a Non Enzymatic Cholesterol Biosensor
Authors: Mitali Saha, Soma Das
Abstract:
The fabrication of nanoscale materials for use in chemical sensing, biosensing and biological analyses has proven a promising avenue in the last few years. Cholesterol has aroused considerable interest in recent years on account of its being an important parameter in clinical diagnosis. There is a strong positive correlation between high serum cholesterol level and arteriosclerosis, hypertension, and myocardial infarction. Enzyme-based electrochemical biosensors have shown high selectivity and excellent sensitivity, but the enzyme is easily denatured during its immobilization procedure and its activity is also affected by temperature, pH, and toxic chemicals. Besides, the reproducibility of enzyme-based sensors is not very good which further restrict the application of cholesterol biosensor. It has been demonstrated that carbon nanotubes could promote electron transfer with various redox active proteins, ranging from cytochrome c to glucose oxidase with a deeply embedded redox center. In continuation of our earlier work on the synthesis and applications of carbon and metal based nanoparticles, we have reported here the synthesis of carbon nanotubes (CCNT) by burning coconut oil under insufficient flow of air using an oil lamp. The soot was collected from the top portion of the flame, where the temperature was around 6500C which was purified, functionalized and then characterized by SEM, p-XRD and Raman spectroscopy. The SEM micrographs showed the formation of tubular structure of CCNT having diameter below 100 nm. The XRD pattern indicated the presence of two predominant peaks at 25.20 and 43.80, which corresponded to (002) and (100) planes of CCNT respectively. The Raman spectrum (514 nm excitation) showed the presence of 1600 cm-1 (G-band) related to the vibration of sp2-bonded carbon and at 1350 cm-1 (D-band) responsible for the vibrations of sp3-bonded carbon. A nonenzymatic cholesterol biosensor was then fabricated on an insulating Teflon material containing three silver wires at the surface, covered by CCNT, obtained from coconut oil. Here, CCNTs worked as working as well as counter electrodes whereas reference electrode and electric contacts were made of silver. The dimensions of the electrode was 3.5 cm×1.0 cm×0.5 cm (length× width × height) and it is ideal for working with 50 µL volume like the standard screen printed electrodes. The voltammetric behavior of cholesterol at CCNT electrode was investigated by cyclic voltammeter and differential pulse voltammeter using 0.001 M H2SO4 as electrolyte. The influence of the experimental parameters on the peak currents of cholesterol like pH, accumulation time, and scan rates were optimized. Under optimum conditions, the peak current was found to be linear in the cholesterol concentration range from 1 µM to 50 µM with a sensitivity of ~15.31 μAμM−1cm−2 with lower detection limit of 0.017 µM and response time of about 6s. The long-term storage stability of the sensor was tested for 30 days and the current response was found to be ~85% of its initial response after 30 days.Keywords: coconut oil, CCNT, cholesterol, biosensor
Procedia PDF Downloads 279581 Greening the Blue: Enzymatic Degradation of Commercially Important Biopolymer Dextran Using Dextranase from Bacillus Licheniformis KIBGE-IB25
Authors: Rashida Rahmat Zohra, Afsheen Aman, Shah Ali Ul Qader
Abstract:
Commercially important biopolymer, dextran, is enzymatically degraded into lower molecular weight fractions of vast industrial potential. Various organisms are associated with dextranase production, among which fungal, yeast and bacterial origins are used for commercial production. Dextranases are used to remove contaminating dextran in sugar processing industry and also used in oral care products for efficient removal of dental plaque. Among the hydrolytic products of dextran, isomaltooligosaccharides have prebiotic effect in humans and reduces the cariogenic effect of sucrose in oral cavity. Dextran derivatives produced by hydrolysis of high molecular polymer are also conjugated with other chemical and metallic compounds for usage in pharmaceutical, fine chemical industry, cosmetics, and food industry. Owing to the vast application of dextran and dextranases, current study focused on purification and analysis of kinetic parameters of dextranase from a newly isolated strain of Bacillus licheniformis KIBGE-IB25. Dextranase was purified up to 35.75 folds with specific activity of 1405 U/mg and molecular weight of 158 kDa. Analysis of kinetic parameters revealed that dextranase performs optimum cleavage of low molecular weight dextran (5000 Da, 0.5%) at 35ºC in 15 min at pH 4.5 with a Km and Vmax of 0.3738 mg/ml and 182.0 µmol/min, respectively. Thermal stability profiling of dextranase showed that it retained 80% activity up to 6 hours at 30-35ºC and remains 90% active at pH 4.5. In short, the dextranase reported here performs rapid cleavage of substrate at mild operational conditions which makes it an ideal candidate for dextran removal in sugar processing industry and for commercial production of low molecular weight oligosaccharides.Keywords: Bacillus licheniformis, dextranase, gel permeation chromatograpy, enzyme purification, enzyme kinetics
Procedia PDF Downloads 438580 Short-Term Impact of a Return to Conventional Tillage on Soil Microbial Attributes
Authors: Promil Mehra, Nanthi Bolan, Jack Desbiolles, Risha Gupta
Abstract:
Agricultural practices affect the soil physical and chemical properties, which in turn influence the soil microorganisms as a function of the soil biological environment. On the return to conventional tillage (CT) from continuing no-till (NT) cropping system, a very little information is available from the impact caused by the intermittent tillage on the soil biochemical properties from a short-term (2-year) study period. Therefore, the contribution made by different microorganisms (fungal, bacteria) was also investigated in order to find out the effective changes in the soil microbial activity under a South Australian dryland faring system. This study was conducted to understand the impact of microbial dynamics on the soil organic carbon (SOC) under NT and CT systems when treated with different levels of mulching (0, 2.5 and 5 t/ha). Our results demonstrated that from the incubation experiment the cumulative CO2 emitted from CT system was 34.5% higher than NT system. Relatively, the respiration from surface layer (0-10 cm) was significantly (P<0.05) higher by 8.5% and 15.8 from CT; 8% and 18.9% from NT system w.r.t 10-20 and 20-30 cm respectively. Further, the dehydrogenase enzyme activity (DHA) and microbial biomass carbon (MBC) were both significantly lower (P<0.05) under CT, i.e., 7.4%, 7.2%, 6.0% (DHA) and 19.7%, 15.7%, 4% (MBC) across the different mulching levels (0, 2.5, 5 t/ha) respectively. In general, it was found that from both the tillage system the enzyme activity and MBC decreased with the increase in depth (0-10, 10-20 and 20-30 cm) and with the increase in mulching rate (0, 2.5 and 5 t/ha). From the perspective of microbial stress, there was 28.6% higher stress under CT system compared to NT system. Whereas, the microbial activity of different microorganisms like fungal and bacterial activities were determined by substrate-induced inhibition respiration using antibiotics like cycloheximide (16 mg/gm of soil) and streptomycin sulphate (14 mg/gm of soil), by trapping the CO2 using an alkali (0.5 M NaOH) solution. The microbial activities were confirmed through platting technique, where it was that found bacterial activities were 46.2% and 38.9% higher than fungal activity under CT and NT system. In conclusion, it was expected that changes in the relative abundance and activity of different microorganisms (bacteria and fungi) under different tillage systems could significantly affect the C cycling and storage due to its unique structures and differential interactions with the soil physical properties.Keywords: tillage, soil respiration, MBC, fungal-bacterial activity
Procedia PDF Downloads 256579 Modified Poly (Pyrrole) Film-Based Biosensors for Phenol Detection
Authors: S. Korkut, M. S. Kilic, E. Erhan
Abstract:
In order to detect and quantify the phenolic contents of a wastewater with biosensors, two working electrodes based on modified Poly (Pyrrole) films were fabricated. Enzyme horseradish peroxidase was used as biomolecule of the prepared electrodes. Various phenolics were tested at the biosensor. Phenol detection was realized by electrochemical reduction of quinones produced by enzymatic activity. Analytical parameters were calculated and the results were compared with each other.Keywords: carbon nanotube, phenol biosensor, polypyrrole, poly (glutaraldehyde)
Procedia PDF Downloads 411578 Developing Novel Bacterial Primase (DnaG) Inhibitors
Authors: Shanakr Bhattarai, V. S. Tiwari, Barak Akabayov
Abstract:
The plummeting number of infections and death is due to the development of drug-resistant bacteria. In addition, the number of approved antibiotic drugs by the Food and Drug Administration (FDA) is insufficient. Therefore, developing new drugs and finding novel targets for central metabolic pathways in bacteria is urgently needed. One of the promising targets is DNA replication machinery which consists of many essential proteins and enzymes. DnaG primase is an essential enzyme and a central part of the DNA replication machinery. DnaG primase synthesizes short RNA primers that initiate the Okazaki fragments by the lagging strand DNA polymerase. Therefore, it is reasonable to assume that inhibition of primase activity will stall DNA replication and prevent bacterial proliferation. We did the expression and purification of eight different bacterial DnaGs (Mycobacterium tuberculosis(Mtb), Bacillus anthracis (Ba), Mycobacterium smegmatis (Msmeg), Francisella tularencis (Ft), Vibrio cholerae (Vc) and Yersinia pestis (Yp), Staphylococcus aureus(Saureus), Escherichia coli(Ecoli)) followed by the radioactive activity assay. After obtaining the pure and active protein DnaG, we synthesized the inhibitors for them. The inhibitors were divided into five different groups, each containing five molecules, and the cocktail inhibition assay was performed against each DnaGs. The groups of molecules inhibiting the DnaGs were further tested with individual molecules belonging to inhibiting groups. Each molecule showing inhibition was titrated against the corresponding DnaGs to find IC50. We got a molecule(VS167) that acted as broad inhibitors, inhibiting all eight DnaGs. Molecules VS180 and VS186 inhibited seven DnaGs (except Saureus). Similarly, two molecules(VS 173, VS176) inhibited five DnaGs (Mtb, Ba, Ft, Yp, Ecoli). VS261 inhibited four DnaGs (Mtb, Ba, Ft, Vc). MS50 inhibited Ba and Vc DnaGs. And some of the inhibitors inhibited only one DnaGs. Thus we found the broad and specific inhibitors for different bacterial DnaGs, and their Structure-activity analysis(SAR) was done. Further, We tried to explain the similarities among the enzyme DnaGs from different bacteria based on their inhibition pattern.Keywords: DNA replication, DnaG, okazaki fragments, antibiotic drugs
Procedia PDF Downloads 85577 Evaluation of Microwave-Assisted Pretreatment for Spent Coffee Grounds
Authors: Shady S. Hassan, Brijesh K. Tiwari, Gwilym A. Williams, Amit K. Jaiswal
Abstract:
Waste materials from a wide range of agro-industrial processes may be used as substrates for microbial growth, and subsequently the production of a range of high value products and bioenergy. In addition, utilization of these agro-residues in bioprocesses has the dual advantage of providing alternative substrates, as well as solving their disposal problems. Spent coffee grounds (SCG) are a by-product (45%) of coffee processing. SCG is a lignocellulosic material, which is composed mainly of cellulose, hemicelluloses, and lignin. Thus, a pretreatment process is required to facilitate an efficient enzymatic hydrolysis of such carbohydrates. In this context, microwave pretreatment of lignocellulosic biomass without the addition of harsh chemicals represents a green technology. Moreover, microwave treatment has a high heating efficiency and is easy to implement. Thus, microwave pretreatment of SCG without adding of harsh chemicals investigated as a green technology to enhance enzyme hydrolysis. In the present work, microwave pretreatment experiments were conducted on SCG at varying power levels (100, 250, 440, 600, and 1000 W) for 60 s. By increasing microwave power to a certain level (which vary by varying biomass), reducing sugar increases, then reducing sugar from biomass start to decrease with microwave power increase beyond this level. Microwave pretreatment of SCG at 60s followed by enzymatic hydrolysis resulted in total reducing sugars of 91.6 ± 7.0 mg/g of biomass (at microwave power of 100 w). Fourier transform Infrared Spectroscopy (FTIR) was employed to investigate changes in functional groups of biomass after pretreatment, while high-performance liquid chromatography (HPLC) was employed for determination of glucose. Pretreatment of lignocellulose using microwave was found to be an effective and energy efficient technology to improve saccharification and glucose yield. Energy performance will be evaluated for the microwave pretreatment, and the enzyme hydrolysate will be used as media component substitute for the production of ethanol and other high value products.Keywords: lignocellulose, microwave, pretreatment, spent coffee grounds
Procedia PDF Downloads 409576 A Gold-Based Nanoformulation for Delivery of the CRISPR/Cas9 Ribonucleoprotein for Genome Editing
Authors: Soultana Konstantinidou, Tiziana Schmidt, Elena Landi, Alessandro De Carli, Giovanni Maltinti, Darius Witt, Alicja Dziadosz, Agnieszka Lindstaedt, Michele Lai, Mauro Pistello, Valentina Cappello, Luciana Dente, Chiara Gabellini, Piotr Barski, Vittoria Raffa
Abstract:
CRISPR/Cas9 technology has gained the interest of researchers in the field of biotechnology for genome editing. Since its discovery as a microbial adaptive immune defense, this system has been widely adopted and is acknowledged for having a variety of applications. However, critical barriers related to safety and delivery are persisting. Here, we propose a new concept of genome engineering, which is based on a nano-formulation of Cas9. The Cas9 enzyme was conjugated to a gold nanoparticle (AuNP-Cas9). The AuNP-Cas9 maintained its cleavage efficiency in vitro, to the same extent as the ribonucleoprotein, including non-conjugated Cas9 enzyme, and showed high gene editing efficiency in vivo in zebrafish embryos. Since CRISPR/Cas9 technology is extensively used in cancer research, melanoma was selected as a validation target. Cell studies were performed in A375 human melanoma cells. Particles per se had no impact on cell metabolism and proliferation. Intriguingly, the AuNP-Cas9 internalized spontaneously in cells and localized as a single particle in the cytoplasm and organelles. More importantly, the AuNP-Cas9 showed a high nuclear localization signal. The AuNP-Cas9, overcoming the delivery difficulties of Cas9, could be used in cellular biology and localization studies. Taking advantage of the plasmonic properties of gold nanoparticles, this technology could potentially be a bio-tool for combining gene editing and photothermal therapy in cancer cells. Further work will be focused on intracellular interactions of the nano-formulation and characterization of the optical properties.Keywords: CRISPR/Cas9, gene editing, gold nanoparticles, nanotechnology
Procedia PDF Downloads 93575 Caged Compounds as Light-Dependent Initiators for Enzyme Catalysis Reactions
Authors: Emma Castiglioni, Nigel Scrutton, Derren Heyes, Alistair Fielding
Abstract:
By using light as trigger, it is possible to study many biological processes, such as the activity of genes, proteins, and other molecules, with precise spatiotemporal control. Caged compounds, where biologically active molecules are generated from an inert precursor upon laser photolysis, offer the potential to initiate such biological reactions with high temporal resolution. As light acts as the trigger for cleaving the protecting group, the ‘caging’ technique provides a number of advantages as it can be intracellular, rapid and controlled in a quantitative manner. We are developing caging strategies to study the catalytic cycle of a number of enzyme systems, such as nitric oxide synthase and ethanolamine ammonia lyase. These include the use of caged substrates, caged electrons and the possibility of caging the enzyme itself. In addition, we are developing a novel freeze-quench instrument to study these reactions, which combines rapid mixing and flashing capabilities. Reaction intermediates will be trapped at low temperatures and will be analysed by using electron paramagnetic resonance (EPR) spectroscopy to identify the involvement of any radical species during catalysis. EPR techniques typically require relatively long measurement times and very often, low temperatures to fully characterise these short-lived species. Therefore, common rapid mixing techniques, such as stopped-flow or quench-flow are not directly suitable. However, the combination of rapid freeze-quench (RFQ) followed by EPR analysis provides the ideal approach to kinetically trap and spectroscopically characterise these transient radical species. In a typical RFQ experiment, two reagent solutions are delivered to the mixer via two syringes driven by a pneumatic actuator or stepper motor. The new mixed solution is then sprayed into a cryogenic liquid or surface, and the frozen sample is then collected and packed into an EPR tube for analysis. The earliest RFQ instrument consisted of a hydraulic ram unit as a drive unit with direct spraying of the sample into a cryogenic liquid (nitrogen, isopentane or petroleum). Improvements to the RFQ technique have arisen from the design of new mixers in order to reduce both the volume and the mixing time. In addition, the cryogenic isopentane bath has been coupled to a filtering system or replaced by spraying the solution onto a surface that is frozen via thermal conductivity with a cryogenic liquid. In our work, we are developing a novel RFQ instrument which combines the freeze-quench technology with flashing capabilities to enable the studies of both thermally-activated and light-activated biological reactions. This instrument also uses a new rotating plate design based on magnetic couplings and removes the need for mechanical motorised rotation, which can otherwise be problematic at cryogenic temperatures.Keywords: caged compounds, freeze-quench apparatus, photolysis, radicals
Procedia PDF Downloads 204574 Nutrigenetic and Bioinformatic Analysis of Rice Bran Bioactives for the Treatment of Lifestyle Related Disease Diabetes and Hypertension
Authors: Md. Alauddin, Md. Ruhul Amin, Md. Omar Faruque, Muhammad Ali Siddiquee, Zakir Hossain Howlader, Mohammad Asaduzzaman
Abstract:
Diabetes and hypertension are the major lifestyle related diseases. The α-amylase and angiotensin converting enzymes (ACE) are the key enzymes that regulate diabetes and hypertension. The aim was to develop a drug for the treatment of diabetes and hypertension. The Rice Bran (RB) sample (Oryza sativa; BRRI-Dhan-84) was collected from the Bangladesh Rice Research Institute (BRRI), and rice bran proteins were isolated and hydrolyzed by hydrolyzing enzyme alcalase and trypsin. In vivo experiment suggested that rice bran bioactives has an effect on regulating the expression of several key gluconeogenesis and lipogenesis-regulating genes, such as glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, and fatty acid synthase. The above genes have a connection of regulating the glucose level, lipids profile as well as act as an anti-inflammatory agent. A molecular docking, bioinformatics and in vitro experiments were performed. We found rice bran protein hydrolysates significantly (<0.05) influence the peptide concentration in the case of trypsin, alcalase, and (trypsin + alcalase) digestion. The in vitro analysis found that protein hydrolysate significantly (<0.05) reduced diabetic and hypertension as well as oxidative stress. A molecular docking study showed that the YY and IP peptide have a significantly strong binding affinity to the active site of the ACE enzyme and α-amylase with -7.8Kcal/mol and -6.2Kcal/mol, respectively. The Molecular dynamics (MD) simulation and Swiss ADME data analysis showed that less toxicity risk, good physicochemical properties, pharmacokinetics, and drug-likeness with drug scores 0.45 and 0.55 of YY and IP peptides, respectively. Thus, rice bran bioactive could be a good candidate for the treatment of diabetes and hypertension.Keywords: anti-hypertensive and anti-hyperglycemic, anti-oxidative, bioinformatics, in vitro study, rice bran proteins and peptides
Procedia PDF Downloads 55573 Plasmonic Biosensor for Early Detection of Environmental DNA (eDNA) Combined with Enzyme Amplification
Authors: Monisha Elumalai, Joana Guerreiro, Joana Carvalho, Marta Prado
Abstract:
DNA biosensors popularity has been increasing over the past few years. Traditional analytical techniques tend to require complex steps and expensive equipment however DNA biosensors have the advantage of getting simple, fast and economic. Additionally, the combination of DNA biosensors with nanomaterials offers the opportunity to improve the selectivity, sensitivity and the overall performance of the devices. DNA biosensors are based on oligonucleotides as sensing elements. These oligonucleotides are highly specific to complementary DNA sequences resulting in the hybridization of the strands. DNA biosensors are not only an advantage in the clinical field but also applicable in numerous research areas such as food analysis or environmental control. Zebra Mussels (ZM), Dreissena polymorpha are invasive species responsible for enormous negative impacts on the environment and ecosystems. Generally, the detection of ZM is made when the observation of adult or macroscopic larvae's is made however at this stage is too late to avoid the harmful effects. Therefore, there is a need to develop an analytical tool for the early detection of ZM. Here, we present a portable plasmonic biosensor for the detection of environmental DNA (eDNA) released to the environment from this invasive species. The plasmonic DNA biosensor combines gold nanoparticles, as transducer elements, due to their great optical properties and high sensitivity. The detection strategy is based on the immobilization of a short base pair DNA sequence on the nanoparticles surface followed by specific hybridization in the presence of a complementary target DNA. The hybridization events are tracked by the optical response provided by the nanospheres and their surrounding environment. The identification of the DNA sequences (synthetic target and probes) to detect Zebra mussel were designed by using Geneious software in order to maximize the specificity. Moreover, to increase the optical response enzyme amplification of DNA might be used. The gold nanospheres were synthesized and characterized by UV-visible spectrophotometry and transmission electron microscopy (TEM). The obtained nanospheres present the maximum localized surface plasmon resonance (LSPR) peak position are found to be around 519 nm and a diameter of 17nm. The DNA probes modified with a sulfur group at one end of the sequence were then loaded on the gold nanospheres at different ionic strengths and DNA probe concentrations. The optimal DNA probe loading will be selected based on the stability of the optical signal followed by the hybridization study. Hybridization process leads to either nanoparticle dispersion or aggregation based on the presence or absence of the target DNA. Finally, this detection system will be integrated into an optical sensing platform. Considering that the developed device will be used in the field, it should fulfill the inexpensive and portability requirements. The sensing devices based on specific DNA detection holds great potential and can be exploited for sensing applications in-loco.Keywords: ZM DNA, DNA probes, nicking enzyme, gold nanoparticles
Procedia PDF Downloads 235572 The Pathology of Bovine Rotavirus Infection in Calves That Confirmed by Enzyme Linked Immunosorbant Assay, Reverse Transcription Polymerase Chain Reaction and Real-Time RT-PCR
Authors: Shama Ranjan Barua, Tofazzal M. Rakib, Mohammad Alamgir Hossain, Tania Ferdushy, Sharmin Chowdhury
Abstract:
Rotavirus is one of the main etiologies of neonatal diarrhea in bovine calves that causes significant economic loss in Bangladesh. The present study was carried out to investigate the pathology of neonatal enteritis in calves due to bovine rotavirus infection in south-eastern part of Bangladesh. Rotavirus was identified by using ELISA, RT-PCR (Reverse Transcription Polymerase Chain Reaction), real-time RT-PCR. We examined 12 dead calves with history of diarrhea during necropsy. Among 12 dead calves, in gross examination, 6 were found with pathological changes in intestine, 5 calves had congestion of small intestine and rest one had no distinct pathological changes. Intestinal contents and/or faecal samples of all dead calves were collected and examined to confirm the presence of bovine rotavirus A using Enzyme linked immunosorbant assay (ELISA), RT-PCR and real-time RT-PCR. Out 12 samples, 5 (42%) samples revealed presence of bovine rotavirus A in three diagnostic tests. The histopathological changes were found almost exclusively limited in the small intestine. The lesions of rotaviral enteritis ranged from slight to moderate shortening (atrophy) of villi in the jejunum and ileum with necrotic crypts. The villi were blunt and covered by immature epithelial cells. Infected cells, stained with Haematoxylin and Eosin staining method, showed characteristic syncytia and eosinophilc intracytoplasmic inclusion body. The presence of intracytoplasmic inclusion bodies in enterocytes is the indication of viral etiology. The presence of rotavirus in the affected tissues and/or lesions was confirmed by three different immunological and molecular tests. The findings of histopathological changes will be helpful in future diagnosis of rotaviral infection in dead calves.Keywords: calves, diarrhea, pathology, rotavirus
Procedia PDF Downloads 245571 Elucidating the Defensive Role of Silicon-Induced Biochemical Responses in Wheat Exposed to Drought and Diuraphis noxia Infestation
Authors: Lintle Mohase, Ninikoe Lebusa, Mpho Stephen Mafa
Abstract:
Wheat is an economically important cereal crop. However, the changing climatic conditions that intensify drought in production areas, and additional pest infestation, such as the Russian wheat aphid (RWA, Diuraphis noxia), severely hamper its production. Drought and pest management require an additional water supply through irrigation and applying inorganic nutrients (including silicon) as alternative strategies to mitigate the stress effects. Therefore, other approaches are needed to enhance wheat productivity during drought stress and aphid abundance. Two wheat cultivars were raised under greenhouse conditions, exposed to drought stress, and treated with silicon before infestation with the South African RWA biotype 2 (RWASA2). The morphological evaluations showed that severe drought or a combination of drought and infestation significantly reduced the plant height of wheat cultivars. Silicon treatment did not alleviate the growth reduction. The biochemical responses were measured using spectrophotometric assays with specific substrates. An evaluation of the enzyme activities associated with oxidative stress and defence responses indicated that drought stress increased NADPH oxidase activity, while silicon treatment significantly reduced it in drought-stressed and infested plants. At 48 and 72 hours sampling periods, a combination of silicon, drought and infestation treatment significantly increased peroxidase activity compared to drought and infestation treatment. The treatment also increased β-1,3-glucanase activity 72 hours after infestation. In addition, silicon and drought treatment increased glucose but reduced sucrose accumulation. Furthermore, silicon, drought, and infestation treatment combinations reduced the sucrose content. Finally, silicon significantly increased the trehalose content under severe drought and infestation, evident at 48 and 72-hour sampling periods. Our findings shed light on silicon’s ability to induce protective biochemical responses during drought and aphid infestation.Keywords: drought, enzyme activity, silicon, soluble sugars, Russian wheat aphid, wheat
Procedia PDF Downloads 71570 Canine Visceral Leishmaniasis In Brazil
Authors: Elisangela Sobreira, Denise Teixeira
Abstract:
Visceral leishmaniasis is a public health problem in Brazil, it is the main reservoir dog. In the period 2012-2016 78 diagnoses were performed in dogs suspected. Blood samples were collected from the cephalic vein obtaining serum used for the indirect immunofluorescence test and enzyme-linked immunosorbent assay, while it collected a drop of blood for the rapid chromatographic immunoassay. Obtained in 32 dogs positive. The test is important for the control of this disease and is used routinely in the Zoonoses Control Center.Keywords: Brazil, dogs, Leismaniasis, Zoonoses center
Procedia PDF Downloads 252569 Characterising Rates of Renal Dysfunction and Sarcoidosis in Patients with Elevated Serum Angiotensin-Converting Enzyme
Authors: Fergal Fouhy, Alan O’Keeffe, Sean Costelloe, Michael Clarkson
Abstract:
Background: Sarcoidosis is a systemic, non-infectious disease of unknown aetiology, characterized by non-caseating granulomatous inflammation. The lung is most often affected (90%); however, the condition can affect all organs, including the kidneys. There is limited evidence describing the incidence and characteristics of renal involvement in sarcoidosis. Serum angiotensin-converting enzyme (ACE) is a recognised biomarker used in the diagnosis and monitoring of sarcoidosis. Methods: A single-centre, retrospective cohort study of patients presenting to Cork University Hospital (CUH) in 2015 with first-time elevations of serum ACE was performed. This included an initial database review of ACE and other biochemistry results, followed by a medical chart review to confirm the presence or absence of sarcoidosis and management thereof. Acute kidney injury (AKI) was staged using the AKIN criteria, and chronic kidney disease (CKD) was staged using the KDIGO criteria. Follow-up was assessed over five years tracking serum creatinine, serum calcium, and estimated glomerular filtration rates (eGFR). Results: 119 patients were identified as having a first raised serum ACE in 2015. Seventy-nine male patients and forty female patients were identified. The mean age of patients identified was 47 years old. 11% had CKD at baseline. 18% developed an AKI at least once within the next five years. A further 6% developed CKD during this time period. 13% developed hypercalcemia. The patients within the lowest quartile of serums ACE had an incidence of sarcoidosis of 5%. None of this group developed hypercalcemia, 23% developed AKI, and 7% developed CKD. Of the patients with a serum ACE in the highest quartile, almost all had documented diagnoses of sarcoidosis with an incidence of 96%. 3% of this group developed hypercalcemia, 13% AKI and 3% developed CKD. Conclusions: There was an unexpectedly high incidence of AKI in patients who had a raised serum ACE. Not all patients with a raised serum ACE had a confirmed diagnosis of sarcoidosis. There does not appear to be a relationship between increased serum ACE levels and increased incidence of hypercalcaemia, AKI, and CKD. Ideally, all patients should have biopsy-proven sarcoidosis. This is an initial study that should be replicated with larger numbers and including multiple centres.Keywords: sarcoidosis, acute kidney injury, chronic kidney disease, hypercalcemia
Procedia PDF Downloads 94568 Application of Low Frequency Ac Magnetic Field for Controlled Delivery of Drugs by Magnetic Nanoparticles
Authors: K. Yu Vlasova, M. A. Abakumov, H. Wishwarsao, M. Sokolsky, N. V. Nukolova, A. G. Majouga, Y. I. Golovin, N. L. Klyachko, A. V. Kabanov
Abstract:
Introduction:Nowadays pharmaceutical medicine is aimed to create systems for combined therapy, diagnostic, drug delivery and controlled release of active molecules to target cells. Magnetic nanoparticles (MNPs) are used to achieve this aim. MNPs can be applied in molecular diagnostics, magnetic resonance imaging (T1/T2 contrast agents), drug delivery, hyperthermia and could improve therapeutic effect of drugs. The most common drug containers, containing MNPs, are liposomes, micelles and polymeric molecules bonded to the MNPs surface. Usually superparamagnetic nanoparticles are used (the general diameter is about 5-6 nm) and all effects of high frequency magnetic field (MF) application are based on Neel relaxation resulting in heating of surrounded media. In this work we try to develop a new method to improve drug release from MNPs under super low frequency MF. We suppose that under low frequency MF exposures the Brown’s relaxation dominates and MNPs rotation could occur leading to conformation changes and release of bioactive molecules immobilized on MNPs surface.The aim of this work was to synthesize different systems with active drug (biopolymers coated MNPs nanoclusters with immobilized enzymes and doxorubicin (Dox) loaded magnetic liposomes/micelles) and investigate the effect of super low frequency MF on these drug containers. Methods: We have synthesized MNPs of magnetite with magnetic core diameter 7-12 nm . The MNPs were coated with block-copolymer of polylysine and polyethylene glycol. Superoxide dismutase 1 (SOD1) was electrostatically adsorbed on the surface of the clusters. Liposomes were prepared as follow: MNPs, phosphatidylcholine and cholesterol were dispersed in chloroform, dried to get film and then dispersed in distillated water, sonicated. Dox was added to the solution, pH was adjusted to 7.4 and excess of drug was removed by centrifugation through 3 kDa filters. Results: Polylysine coated MNPs formed nanosized clusters (as observed by TEM) with intensity average diameter of 112±5 nm and zeta potential 12±3 mV. After low frequency AC MF exposure we observed change of immobilized enzyme activity and hydrodynamic size of clusters. We suppose that the biomolecules (enzymes) are released from the MNPs surface followed with additional aggregation of complexes at the MF in medium. Centrifugation of the nanosuspension after AC MF exposures resulted in increase of positive charge of clusters and change in enzyme concentration in comparison with control sample without MF, thus confirming desorption of negatively charged enzyme from the positively charged surface of MNPs. Dox loaded magnetic liposomes had average diameter of 160±8 nm and polydispersity index (PDI) 0.25±0.07. Liposomes were stable in DW and PBS at pH=7.4 at 370C during a week. After MF application (10 min of exposure, 50 Hz, 230 mT) diameter of liposomes raised to 190±10 nm and PDI was 0.38±0.05. We explain this by destroying and/or reorganization of lipid bilayer, that leads to changes in release of drug in comparison with control without MF exposure. Conclusion: A new application of low frequency AC MF for drug delivery and controlled drug release was shown. Investigation was supported by RSF-14-13-00731 grant, K1-2014-022 grant.Keywords: magnetic nanoparticles, low frequency magnetic field, drug delivery, controlled drug release
Procedia PDF Downloads 479567 Ability of Bentonite-lactobacillus Rhamnosus GAF06 Mixture to Mitigate Aflatoxin M1 Damages in Balb/C Mice
Authors: Amina Aloui, Jalila Ben Salah-Abbès, Abdellah Zinedine, Amar Riba, Noel Durand, Catherine Brabet, Didier Montet, Samir Abbès
Abstract:
Mycotoxin contamination of food and feed-isa globaconcern, both economically and for public health. Aflatoxin M1 (AFM1) is the principal hydroxylated metabolite of aflatoxin B1. It is frequently found in milk and other dairy products. It is responsible for the development of hepatocellular carcinoma and immunotoxic in humans and animals. The reduction of its bioavailabilitybecomesa great demand in order to protect human and animal health. The use of probiotic bacteria and clay are demonstrated to be able to bind AFM1 in vitro. This study aimed to investigate, in vivo, the activity of two-component mixture: L. rhamnosusGAF06 (LR) and bentonite for reducing the oxidative stress and the histological alterationsinduced by AFM1 in the liver andkidneys. For the experiment, male mice were divided into 7 groups (6 mice/group) and treated, orally, by AFM1, alone or in combination with LR and/or bentonite, for 10 days as follows: group 1 control, group 2 treated with LR alone (2.108 CFU/ml), group 3 treated with bentonite alone (1g/kg), group 4 treated with AFM1 alone (100μg/kg), group 5 co-treated with LR+AFM1, group 6 co-treated with bentonite+AFM1, group 7 co-treated with bentonite+LR+AFM1. At the end of the treatment, the mice were sacrificed, and the livers and kidneys were collected for histological assays. Intracellular antioxidant activities and lipid peroxidation were also studied. The results showed that AFM1causeddamage in liver and kidney tissues, being evidence of hepatotoxicity and nephrotoxicity marked by necrotic cells. It increased the MDA level and decreased the antioxidant enzyme activities (SOD) in both organs. In contrast, the co-treatment with AFM1 plus LR and/or bentonitesignificantly improved the hepatic and renal tissues, regulated kidney, and liver antioxidant enzyme activities. This improvement was more remarkable with the administration of LR-bentonite mixture with AFM1.LR and bentonite alone showed to be safe during the treatment. This mixture can be a promising candidate for future applications in biotechnological processes that aimed to detoxify AFM1in food and feed.Keywords: aflatoxin M1, bentonite, L. rhamnosus GAF06, oxidative stress, prevention
Procedia PDF Downloads 182