Search results for: enzyme hydrolysis

Authors: Alok Kumar, Hari Ram, Lebin Thomas, Ved Pal Singh

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Organic solvent tolerant amylases and proteases of microbial origin are in great demand for their application in transglycosylation of water-insoluble flavanoids and in peptide synthesizing reaction in organic media. Most of the amylases and proteases are unstable in presence of organic solvent. In the present work two different bacterial strains M-11 and VP-07 were isolated from the soil sample of a dairy farm in Delhi, India, for the efficient production of extracellular amylase and protease through their screening on starch agar (SA) and skimmed milk agar (SMA) plates, respectively. Both the strains (M-11 and VP-07) were identified based on morphological, biochemical and 16S rRNA gene sequencing methods. After analysis through Ez-Taxon software, the strains M-11 and VP-07 were found to have maximum pairwise similarity of 98.63% and 100% with Bacillus subtilis subsp. inaquosorum BGSC 3A28 and Bacillus anthracis ATCC 14578 and were therefore identified as Bacillus sp. UKS1 and Bacillus sp. UKS2, respectively. Time course study of enzyme activity and bacterial growth has shown that both strains exhibited typical sigmoid growth behavior and maximum production of amylase (180 U/ml) and protease (78 U/ml) by these strains (UKS1 and UKS2) was commenced during stationary phase of growth at 24 and 20 h, respectively. Thereafter, both amylase and protease were tested for their tolerance towards organic solvents and were found to be active as well stable in p-xylene (130% and 115%), chloroform (110% and 112%), isooctane (119% and 107%), benzene (121% and 104%), n-hexane (116% and 103%) and toluene (112% and 101%, respectively). Owing to such properties, these enzymes can be exploited for their potential application in industries for organic synthesis.

Keywords: amylase, enzyme activity, industrial applications, organic solvent tolerant, protease

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Authors: John Carlo Combista, Jimbert Tan

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The orchid, Vanda tessellata was chosen by the researchers because of the presence of the constituents in the family Orchidaceae such as alkaloids, flavonoids and glycosides that might give an inhibition activity of the carbonic anhydrase enzyme. This study aimed to determine the in vitro inhibition of carbonic anhydrase of Vanda tessellata flower extract. With the use of modified colorimetric Maren T.H. (1960) method, the time in seconds each test solution changed its color after the rate of CO2 hydration were recorded. Two solvents were used: the semi-polar, 95% ethanol and the non-polar, dichloromethane solvents. The percent inhibition activity of carbonic anhydrase of the different concentrations of solvents ethanol (1%, 25% and 50%) and dichloromethane (1% and 10%) test solutions were determined. Results showed that the ethanol-based extract of Vanda tessellata in different concentrations showed an inhibitory effect while the dichloromethane-based extract of Vanda tessellata showed no inhibitory effect of carbonic anhydrase activity. For ethanol extract, the concentration with the highest activity was 50% followed by 25% which changed its color from red to yellow with an average time of 13.11 seconds and 11.57 seconds but 1% with an average time of 7.56 seconds did not exhibit an effect. The researchers recommend the isolation of the specific active constituents of Vanda tessellata that is responsible for the inhibitory effect of carbonic anhydrase enzyme. It is also recommended to utilize different blood types to observe different reactions to the inhibition of the carbonic anhydrase.

Keywords: carbonic anhydrase, inhibition, modified colorimetric Maren TH method, Vanda orchid

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Authors: Jignasha Derasari, Patel Krishna M, Modi Jignasa G.

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Chemical stability of pharmaceutical molecules is a matter of great concern as it affects the safety and efficacy of the drug product.Stability testing data provides the basis to understand how the quality of a drug substance and drug product changes with time under the influence of various environmental factors. Besides this, it also helps in selecting proper formulation and package as well as providing proper storage conditions and shelf life, which is essential for regulatory documentation. The ICH guideline states that stress testing is intended to identify the likely degradation products which further help in determination of the intrinsic stability of the molecule and establishing degradation pathways, and to validate the stability indicating procedures. A simple, accurate and precise stability indicating RP- HPLC method was developed and validated for simultaneous estimation of Amiloride Hydrochloride and Furosemide in tablet dosage form. Separation was achieved on an Phenomenexluna ODS C18 (250 mm × 4.6 mm i.d., 5 µm particle size) by using a mobile phase consisting of Ortho phosphoric acid: Acetonitrile (50:50 %v/v) at a flow rate of 1.0 ml/min (pH 3.5 adjusted with 0.1 % TEA in Water) isocratic pump mode, Injection volume 20 µl and wavelength of detection was kept at 283 nm. Retention time for Amiloride Hydrochloride and Furosemide was 1.810 min and 4.269 min respectively. Linearity of the proposed method was obtained in the range of 40-60 µg/ml and 320-480 µg/ml and Correlation coefficient was 0.999 and 0.998 for Amiloride hydrochloride and Furosemide, respectively. Forced degradation study was carried out on combined dosage form with various stress conditions like hydrolysis (acid and base hydrolysis), oxidative and thermal conditions as per ICH guideline Q2 (R1). The RP- HPLC method has shown an adequate separation for Amiloride hydrochloride and Furosemide from its degradation products. Proposed method was validated as per ICH guidelines for specificity, linearity, accuracy; precision and robustness for estimation of Amiloride hydrochloride and Furosemide in commercially available tablet dosage form and results were found to be satisfactory and significant. The developed and validated stability indicating RP-HPLC method can be used successfully for marketed formulations. Forced degradation studies help in generating degradants in much shorter span of time, mostly a few weeks can be used to develop the stability indicating method which can be applied later for the analysis of samples generated from accelerated and long term stability studies. Further, kinetic study was also performed for different forced degradation parameters of the same combination, which help in determining order of reaction.

Keywords: amiloride hydrochloride, furosemide, kinetic study, stability indicating RP-HPLC method validation

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Authors: Catherine Joke Adeseko

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Enzymatic browning is an economically important disorder that degrades organoleptic properties and prevent the consumer from purchasing fresh fruit and vegetables. Prevention and control of enzymatic browning in fruit and its product is imperative. Therefore, this study sought to investigate the catalytic effect of polyphenol oxidase (PPO) in the adverse browning of African bush mango (Irvingia gabonensis) fruit peel and pulp. PPO was isolated and purified, and its physicochemical properties, such as the effect of pH with SDS, temperature, and thermodynamic studies, which invariably led to thermal inactivation of purified PPO at 80 °C, were evaluated. The pH and temperature optima of PPO were found at 7.0 and 50, respectively. There was a gradual increase in the activity of PPO as the pH increases. However, the enzyme exhibited a higher activity at neutral pH 7.0, while enzymatic inhibition was observed at acidic region, pH 2.0. The presence of SDS at pH 5.0 downward was found to inhibit the activity of PPO from the peel and pulp of I. gabonensis. The average value of enthalpy (ΔH), entropy (ΔS), and Gibbs free energy (ΔG) obtained at 20 min of incubation and temperature 30 – 80 °C were respectively 39.93 kJ.mol-1, 431.57 J.mol-1 .K-1 and -107.99 kJ.mol-1 for peel PPO, and 37.92 kJ.mol-1, -442.51J.mol-1.K-1, and -107.22 kJ.mol-1 for pulp PPO. Thermal inactivation of PPO from I. gabonensis exhibited a reduction in catalytic activity as the temperature and duration of heat inactivation increases using catechol, reflected by an increment in k value. The half-life of PPO (t1/2) decreases as the incubation temperature increases due to the instability of the enzyme at high temperatures and was higher in pulp than peel. Both D and Z values decrease with increase in temperature. The information from this study suggests processing parameters for controlling PPO in the potential industrial application of I. gabonensis fruit in order to prolong the shelf-life of this fruit for maximum utilization.

Keywords: enzymatic, browning, characterization, activity

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Authors: Fatih Sonmez

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Alzheimer’s disease (AD) is a progressive neurodegenerative disease and it is the most common form of dementia that affects aged people. Acetylcholinesterase is a hydrolase involved in the termination of impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter ACh in the central and peripheral nervous system. Carvacrol (5-iso-propyl-2-methyl-phenol) is a main bioactive monoterpene isolated from many medicinal herbs, such as Thymus vulgaris, Monarda punctate and Origanum vulgare spp. It is known that carvacrol has been widely used as an active anti-inflammatory ingredient, which can inhibit the isoproterenol induced inflammation in myocardial infarcted rats. In this paper, a series of 12 carvacrol substituted carbamate derivatives (2a-l) was synthesized and their inhibitory activities on AChE and BuChE were evaluated. Among them, 2d exhibited the strongest inhibition against AChE with an IC50 value of 2.22 µM, which was 130-fold more than that of carvacrol (IC50 = 288.26 µM).

Keywords: Acetylcholinesterase, Butyrylcholinesterase, Carbamate, Carvacrol

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Authors: Vadim Makarov, Stewart T.Cole

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PBTZ169 is novel drug candidate with high efficacy in animals models, and its combination treatment of PBTZ169 with BDQ and pyrazinamide was shown to be more efficacious than the standard treatment for tuberculosis in a mouse model. The target of PBTZ169 is famous DprE1, an essential enzyme in cell wall biosynthesis. The crystal structure of the DprE1-PBTZ169 complex reveals formation of a semimercaptal adduct with Cys387 in the active site and explains the irreversible inactivation of the enzyme. Furthermore, this drug candidate demonstrated during preclinical research ‘drug like’ properties what made it an attractive drug candidate to treat tuberculosis in humans. During first clinical trials several cohorts of the healthy volunteers were treated by the single doses of PBTZ169 as well as two weeks repeated treatment was chosen for two maximal doses. As expected PBTZ169 was well tolerated, and no significant toxicity effects were observed during the trials. The study of the metabolism shown that human metabolism of PBTZ169 is very different from microbial or animals compound transformation. So main pathway of microbial, mice and less rats metabolism connected with reduction processes, but human metabolism mainly connected with oxidation processes. Due to this difference we observed several metabolites of PBTZ169 in humans with antitubercular activity, and now we can conclude that animal antituberculosis activity of PBTZ169 is a result not only activity of the drug itself, but it is a result of the sum activity of the drug and its metabolites. Direct antimicrobial plasma activity was studied, and such activity was observed for 24 hours after human treatment for some doses. This data gets high chance for good efficacy of PBTZ169 in human for treatment TB infection. Second phase of clinical trials was started summer of 2017 and continues to the present day. Available data will be presented.

Keywords: clinical trials, DprE1, PBTZ169, metabolism

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Authors: Parastou Kharazmi, Folke Björk

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Some of the main causes for degradation of polymeric materials are thermal aging, hydrolysis, oxidation or chemical degradation by acids, alkalis or water. The first part of this paper provides a brief summary of advances in technology, methods and specification of composite materials for relining as a rehabilitation technique for sewage systems. The second part summarizes an investigation on frequently used composite materials for relining in Sweden, the rubber filled epoxy composite and reinforced polyester composite when they were immersed in deionized water or in dry conditions, and elevated temperatures up to 80°C in the laboratory. The tests were conducted by visual inspection, microscopy, Dynamic Mechanical Analysis (DMA), Differential Scanning Calorimetry (DSC) as well as mechanical testing, three point bending and tensile testing.

Keywords: composite, epoxy, polyester, relining, sewage

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Authors: Mei Han, Jinkou Zhao, Yuan Yao, Liangui Feng, Xianbin Ding, Guohui Wu, Chao Zhou, Lin Ouyang, Rongrong Lu, Bo Zhang

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To compare the HIV incidence estimated using BED capture enzyme linked immunosorbent assay (BED-CEIA) and observed in a cohort against the HIV incidence among men who have sex with men (MSM) measured by pooling polymerase chain reaction (pooling-PCR). A total of 617 MSM subjects were included in a respondent driven sampling survey in Chongqing in 2008. Among the 129 that were tested HIV antibody positive, 102 were defined with long-term infection, 27 were assessed for recent HIV infection (RHI) using BED-CEIA. The remaining 488 HIV negative subjects were enrolled to the prospective cohort and followed-up every 6 months to monitor HIV seroconversion. All of the 488 HIV negative specimens were assessed for acute HIV infection (AHI) using pooling-PCR. Among the 488 negative subjects in the open cohort, 214 (43.9%) were followed-up for six months, with 107 person-years of observation and 14 subjects seroconverted. The observed HIV incidence was 12.5 per 100 person-years (95% CI=9.1-15.7). Among the 488 HIV negative specimens, 5 were identified with acute HIV infection using pooling-PCR at an annual rate of 14.02% (95% CI=1.73-26.30). The estimated HIV-1 incidence was 12.02% (95% CI=7.49-16.56) based on BED-CEIA. The HIV incidence estimated with three different approaches was different among subgroups. In the highly HIV prevalent MSM, it costs US$ 1724 to detect one AHI case, while detection of one case of RHI with BED assay costs only US$ 42. Three approaches generated comparable and high HIV incidences, pooling PCR and prospective cohort are more close to the true level of incidence, while BED-CEIA seemed to be the most convenient and economical approach for at-risk population’s HIV incidence evaluation at the beginning of HIV pandemic. HIV-1 incidences were alarmingly high among MSM population in Chongqing, particularly within the subgroup under 25 years of age and those migrants aged between 25 to 34 years.

Keywords: BED-CEIA, HIV, incidence, pooled PCR, prospective cohort

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Authors: Ahmed F. Azmy, Amal E. Saafan, Tamer M. Essam, Magdy A. Amin, Shaban H. Ahmed

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Bacterial strains capable of degradation of malathion from the domestic sewage were isolated by an enrichment culture technique. Three bacterial strains were screened and identified as Acinetobacter baumannii (AFA), Pseudomonas aeruginosae (PS1),andPseudomonas mendocina (PS2) based on morphological, biochemical identification and 16S rRNA sequence analysis. Acinetobacter baumannii AFA was the most efficient malathion degrading bacterium, so used for further biodegradation study. AFA was able to grow in mineral salt medium (MSM) supplemented with malathion (100 mg/l) as a sole carbon source, and within 14 days, 84% of the initial dose was degraded by the isolate measured by high performance liquid chromatography. Strain AFA could also degrade other organophosphorus compounds including diazenon, chlorpyrifos and fenitrothion. The effect of different culture conditions on the degradation of malathion like inoculum density, other carbon or nitrogen sources, temperature and shaking were examined. Degradation of malathion and bacterial cell growth were accelerated when culture media were supplemented with yeast extract, glucose and citrate. The optimum conditions for malathion degradation by strain AFA were; an inoculum density of 1.5x 1012CFU/ml at 30°C with shaking. A specific polymerase chain reaction primers were designed manually using multiple sequence alignment of the corresponding carboxylesterase enzymes of Acinetobacter species. Sequencing result of amplified PCR product and phylogenetic analysis showed low degree of homology with the other carboxylesterase enzymes of Acinetobacter strains, so we suggested that this enzyme is a novel esterase enzyme. Isolated bacterial strains may have potential role for use in bioremediation of malathion contaminated.

Keywords: Acinetobacter baumannii, biodegradation, malathion, organophosphate pesticides

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Authors: Vagolu S. Krishna, Shan Zheng, Estharla M. Rekha, Luke W. Guddat, Dharmarajan Sriram

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Tuberculosis (TB) remains a major threat to human health. This due to the fact that current drug treatments are less than optimal as well as the rising occurrence of multi drug-resistant and extensively drug-resistant strains of the etiological agent, Mycobacterium tuberculosis (Mt). Given the wide-spread significance of this disease, we have undertaken a design and evaluation program to discover new anti-TB drug leads. Here, our attention is focused on ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid biosynthesis pathway. Importantly, this enzyme is present in bacteria but not in humans, making it an attractive proposition for drug discovery. In the present work, we used high-throughput virtual screening to identify seventeen potential inhibitors of KARI using the Birla Institute of Technology and Science in-house database. Compounds were selected based on high docking scores, which were assigned as the result of favourable interactions between the compound and the active site of KARI. The Ki values for two leads, compounds 14 and 16 are 3.71 and 3.06 µM, respectively for Mt KARI. To assess the mode of binding, 100 ns molecular dynamics simulations for these two compounds in association with Mt KARI were performed and showed that the complex was stable with an average RMSD of less than 2.5 Å for all atoms. Compound 16 showed an MIC of 2.06 ± 0.91 µM and a 1.9 fold logarithmic reduction in the growth of Mt in an infected macrophage model. The two compounds exhibited low toxicity against murine macrophage RAW 264.7 cell lines. Thus, both compounds are promising candidates for development as an anti-TB drug leads.

Keywords: ketol-acid reductoisomerase, macrophage, molecular docking and dynamics, tuberculosis

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Authors: Anju Joshi, C. N. Tharamani

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Diabetes is one of the leading cause of death at present and remains an important concern as the prevalence of the disease is increasing at an alarming rate. Therefore, it is crucial to diagnose the accurate levels of glucose for developing an efficient therapeutic for diabetes. Due to the availability of convenient and compact self-testing, continuous monitoring of glucose is feasible nowadays. Enzyme based electrochemical sensing of glucose is quite popular because of its high selectivity but suffers from drawbacks like complicated purification and immobilization procedures, denaturation, high cost, and low sensitivity due to indirect electron transfer. Hence, designing a robust enzyme free platform using transition metal oxides remains crucial for the efficient and sensitive determination of glucose. In the present work, manganese doped nickel oxide nanoparticles (Mn-NiO) has been synthesized onto the surface of multiwalled carbon nanotubes using a simple microwave assisted approach for non-enzymatic electrochemical sensing of glucose. The morphology and structure of the synthesized nanostructures were characterized using scanning electron microscopy (SEM) and X-Ray diffraction (XRD). We demonstrate that the synthesized nanostructures show enormous potential for electrocatalytic oxidation of glucose with high sensitivity and selectivity. Cyclic voltammetry and square wave voltammetry studies suggest superior sensitivity and selectivity of Mn-NiO decorated carbon nanotubes towards the non-enzymatic determination of glucose. A linear response between the peak current and the concentration of glucose has been found to be in the concentration range of 0.01 μM- 10000 μM which suggests the potential efficacy of Mn-NiO decorated carbon nanotubes for sensitive determination of glucose.

Keywords: diabetes, glucose, Mn-NiO decorated carbon nanotubes, non-enzymatic

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Authors: Reginald Anyanwu

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Enzymes are biological molecules that significantly regulate the rate of almost all of the chemical reactions that take place within cells, and have been widely used for products’ innovations. They are vital for life and serve a wide range of important functions in the body, such as aiding in digestion and metabolism. The present study was aimed at finding out the extent to which biological molecules have been utilized by pharmaceutical, food and beverage, and biofuel industries in commercial and scale up applications. Taking into account the escalating business opportunities in this vertical, biotech firms have also been penetrating enzymes industry especially that of food. The aim of the study therefore was to find out how biocatalysis can be successfully deployed; how enzyme application can improve industrial processes. To achieve the purpose of the study, the researcher focused on the analytical tools that are critical for the scale up implementation of enzyme immobilization to ascertain the extent of increased product yield at minimum logistical burden and maximum market profitability on the environment and user. The researcher collected data from four pharmaceutical companies located at Anambra state and Imo state of Nigeria. Questionnaire items were distributed to these companies. The researcher equally made a personal observation on the applicability of these biological molecules on innovative Products since there is now shifting trends toward the consumption of healthy and quality food. In conclusion, it was discovered that enzymes have been widely used for products’ innovations but there are however variations on their applications. It was also found out that pivotal contenders of enzymes market have lately been making heavy investments in the development of innovative product solutions. It was recommended that the applications of enzymes on innovative products should be widely practiced.

Keywords: enzymes, pharmaceuticals, process development, quality food consumption, scale-up applications

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Authors: Lopamudra Ray, Malla Padma, Dibya Bhol, Samir Ranjan Mishra, A. N. Panda, Gurdeep Rastogi, T. K. Adhya, Ajit Kumar Pattnaik, Mrutyunjay Suar, Vishakha Raina

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Chilika Lake is the largest coastal estuarine brackish water lagoon in Asia situated on the east coast of India and is a designated Ramsar site. In the current study, several chitinolytic microorganisms were isolated and screened by appearance of clearance zone on 0.5% colloidal chitin agar plate. A strain designated as RC 1830 displayed maximum colloidal chitin degradation by release of 112 μmol/ml/min of N-acetyl D-glucosamine (GlcNAc) in 48h. The strain was taxonomically identified by polyphasic approach based on a range of phenotypic and genotypic properties and was found to be a novel species named Streptomyces chilikensis RC1830. The organism was halophilic (12% NaCl w/v), alkalophilic (pH10) and was capable of hydrolyzing chitin, starch, cellulose, gelatin, casein, tributyrin and tween 80. The partial purification of chitinase enzymes from RC1830 was performed by DEAE Sephacel anion exchange chromatography which revealed the presence of a very low molecular weight chitinase(10.5kD) which may be a probable chitobiosidase enzyme. The study reports the presence of a low MW chitinase (10.5kD) and a chitin decaetylase from a novel Streptomyces strain RC1830 isolated from Chilika Lake. Previously chitinases less than 20.5kD have not been reported from any other Streptomyces species. The enzymes was characterized with respect to optimum pH, temperature, and substrate specificity and temperature stability.

Keywords: chitinases, chitobiosidase, Chilika Lake, India

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Authors: Idan Chiyanzu, Maruping Mangena

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Crude glycerol, a major by-product from the transesterification of triacylglycerides with alcohol to biodiesel, is known to have a broad range of applications. For example, its bioconversion can afford a wide range of chemicals including alcohols, organic acids, hydrogen, solvents and intermediate compounds. In bacteria, the 2-oxoglutarate dioxygenase (2-OGD) enzymes are widely found among the Pseudomonas syringae species and have been recognized with an emerging importance in ethylene formation. However, the use of optimized enzyme function in recombinant systems for crude glycerol conversion to ethylene is still not been reported. The present study investigated the production of ethylene from crude glycerol using engineered E. coli MG1655 and JM109 strains. Ethylene production with an optimized expression system for 2-OGD in E. coli using a codon optimized construct of the ethylene-forming gene was studied. The codon-optimization resulted in a 20-fold increase of protein production and thus an enhanced production of the ethylene gas. For a reliable bioreactor performance, the effect of temperature, fermentation time, pH, substrate concentration, the concentration of methanol, concentration of potassium hydroxide and media supplements on ethylene yield was investigated. The results demonstrate that the recombinant enzyme can be used for future studies to exploit the conversion of low-priced crude glycerol into advanced value products like light olefins, and tools including recombineering techniques for DNA, molecular biology, and bioengineering can be used to allowing unlimited the production of ethylene directly from the fermentation of crude glycerol. It can be concluded that recombinant E.coli production systems represent significantly secure, renewable and environmentally safe alternative to thermochemical approach to ethylene production.

Keywords: crude glycerol, bioethylene, recombinant E. coli, optimization

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Authors: Ozlem Bahadir Acikara, Mert Ilhan, Ekin Kurtul, Karel Smejkal, Esra Kupeli Akkol

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Present study is aimed to investigate in vitro inhibitory effects of the extracts prepared from the aerial parts of Podospermum canum (Asteraceae) on hyaluronidase, collagenase, and elastase enzymes using a bioassay-guided fractionation. Inhibitory effects of the extract, sub-extracts, fractions obtained by column chromatography, and isolated compounds on collagenase, elastase, and hyaluronidase were performed by using in vitro enzyme inhibitory assays based on spectrophotometric evaluation. The ethyl acetate and remaining water extracts prepared from the plant displayed significant inhibitory activities on collagenase and elastase, while petroleum ether and chloroform extracts did not show any inhibitory activity. Eleven known compounds: arbutin, 6'-O-caffeoylarbutin, cichoriin, 3,5-dicaffeoylquinic acid methyl ester, apigenin-7-O-β-glucoside, luteolin-7-O-β-glucoside, apigenin-7-O-β-rutinoside, isoorientin, orientin, vitexin, procatechuic acid, and compound 4-hydroxy-benzoic acid 4-(6-O-α-rhamnopyranosyl-β-glucopyranosyl) benzyl ester have been obtained from ethyl acetate sub-extract of the plant through bioassay-guided fractionation and isolation. Results of the present study have revealed that among the isolated compounds, apigenin-7-O-β-glucoside, luteolin-7-O-β-glucoside, apigenin-7-O-β-rutinoside and isoorientin showed potent enzyme inhibitory activities. However, methanolic extract of P. canum displayed a greater inhibitory activity than fractions and isolated compounds both on collagenase and elastase.

Keywords: Asteraceae, collagenase, elastase, hyaluronidase, Podospermum canum

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Authors: Ragy Raafat Gaber Attaalla

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For cardiovascular illness, oral P2Y12 inhibitors including clopidogrel, prasugrel, and ticagrelor are frequently recommended. Each of these medications has advantages and disadvantages. In the absence of genotyping, it has been demonstrated that the stronger platelet aggregation inhibitors prasugrel and ticagrelor are superior than clopidogrel at preventing significant adverse cardiovascular events following an acute coronary syndrome and percutaneous coronary intervention (PCI). Both, nevertheless, come with a higher risk of bleeding unrelated to a coronary artery bypass. As a prodrug, clopidogrel needs to be bioactivated, principally by the CYP2C19 enzyme. A CYP2C19 no function allele and diminished or absent CYP2C19 enzyme activity are present in about 30% of people. The reduced exposure to the active metabolite of clopidogrel and reduced inhibition of platelet aggregation among clopidogrel-treated carriers of a CYP2C19 no function allele likely contributed to the reduced efficacy of clopidogrel in clinical trials. Clopidogrel's pharmacogenetic results are strongest when used in conjunction with PCI, but evidence for other indications is growing. One of the most typical examples of clinical pharmacogenetic application is CYP2C19 genotype-guided antiplatelet medication following PCI. Guidance is available from expert consensus groups and regulatory bodies to assist with incorporating genetic information into P2Y12 inhibitor prescribing decisions. Here, we examine the data supporting genotype-guided P2Y12 inhibitor selection's effects on clopidogrel response and outcomes and discuss tips for pharmacogenetic implementation. We also discuss procedures for using genotype data to choose P2Y12 inhibitor therapies as well as any unmet research needs. Finally, choosing a P2Y12 inhibitor medication that optimally balances the atherothrombotic and bleeding risks may be influenced by both clinical and genetic factors.

Keywords: inhibitors, cardiovascular events, coronary intervention, pharmacogenetic implementation

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Authors: F. Ambreen, A.Naheed

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Osteoarthritis (OA) is a degenerative disorder affecting millions of people worldwide. Nitric oxide (NO) was found to play a catabolic role in the development of osteoarthritis. It is a toxic free radical gas generated during the metabolism of L-arginine by the enzyme Nitric oxide synthase (NOS). Inducible Nitric Oxide Synthase (iNOS) is one of the isoform of NOS, and its overexpression leads to the excessive formation of NO that results in pathophysiological joint conditions. Several synthetic anti-inflammatory drugs and inhibitors are present to date, but all showed side effects and complications. Therefore, the pursuit of natural disease-modifying drugs remains a top priority. Curcumin is an active component of turmeric, and the past few decades have witnessed intense research devoted to the antioxidant and anti-inflammatory properties of curcumin. The present study focused on curcumin and its derivatives in the search for new iNOS inhibitors for the treatment of osteoarthritis. We conducted a molecular docking study on curcumin and its four derivatives; cyclocurcumin, tetrahydrocurcumin, demethoxycurcumin and curcumin monoglucoside with iNOS using CLC Drug discovery work bench 3.02. We selected two co-crystallized ligands for this study; tetrahydrobiopterin and N-omega-propyl-L-arginine present in complex with the enzyme iNOS. Results showed the best binding affinity of N-omega-propyl-L-arginine with cyclocurcumin and curcumin monoglucoside that exhibit binding energies of -65.2 kcal/mol and -68 kcal/mol respectively. Whereas with tetrahydrobiopterin, best binding scores of -64.7 kcal/mol and -62.2 kcal/mol were found with tetrahydrocurcumin and demethoxycurcumin respectively. This information could open doors of research for the designing of novel drugs using herbs such as curcumin for the treatment of inflammatory joint diseases.

Keywords: curcumin, iNOS, molecular docking, osteoarthritis

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Authors: Klaudia Chmielewska, Krystyna Dzierzbicka, Iwona Inkielewicz-Stepniak

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Carnosine, an endogenous peptide consisting of β-alanine and L-histidine has a variety of functions to mention: antioxidant, antiglycation, and reducing the toxicity of metal ions. It has therefore been proposed to act as a therapeutic agent for many pathological states, although its therapeutic index is limited by quick enzymatic cleavage. To overcome this limitation, there’s an urge to create new derivatives which might become less potent to hydrolysis, while preserving the therapeutic effect. The poster summarizes the efficiency of two peptide synthesis methods, which were: (1) the mixed anhydride with isobutyl chloroformate and N-methylmorpholine (NMM) and (2) carbodiimide - mediated coupling method via appropriate reagent condensing, here – CDI. The methods were used to obtain dipeptides which were the derivatives of carnosine. Obtained dipeptides were made in the form of methyl esters and their structures will be confirmed 1H NMR, 13C NMR, MS and elemental analysis techniques. Later on, they will be analyzed for their antioxidant properties, in comparison to carnosine.

Keywords: carnosine, method, peptide, synthesis

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Authors: Jyoti Kumari, Pavuluri Srinivasa Rao

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Jackfruit (ArtocarpusheterophyllusL.) is an underutilized yet highly nutritious fruit with unique flavour, known for its therapeutic and culinary properties. Fresh jackfruit bulb has a very short shelf life due to high moisture and sugar content leading to microbial and enzymatic browning, hindering its consumer acceptability and marketability. An attempt has been made for the preservation of the ripe jackfruit bulbs, by the application of high pressure (HP) over a range of 200-500 MPa at ambient temperature for dwell times ranging from 5 to 20 min. The physicochemical properties of jackfruit bulbs such as the pH, TSS, and titrable acidity were not affected by the pressurization process. The ripening index of the fruit bulb also decreased following HP treatment. While the ascorbic acid and antioxidant activity of jackfruit bulb were well retained by high pressure processing (HPP), the total phenols and carotenoids showed a slight increase. The HPP significantly affected the colour and textural properties of jackfruit bulb. High pressure processing was highly effective in reducing the browning index of jackfruit bulbs in comparison to untreated bulbs. The firmness of the bulbs improved upon the pressure treatment with longer dwelling time. The polyphenol oxidase has been identified as the most prominent oxidative enzyme in the jackfruit bulb. The enzymatic activity of polyphenol oxidase and peroxidase were significantly reduced by up to 40% following treatment at 400 MPa/15 min. HPP of jackfruit bulbs at ambient temperatures is shown to be highly beneficial in improving the shelf stability, retaining its nutrient profile, color, and appearance while ensuring the maximum inactivation of the spoilage enzymes.

Keywords: antioxidant capacity, ascorbic acid, carotenoids, color, HPP-high pressure processing, jackfruit bulbs, polyphenol oxidase, peroxidase, total phenolic content

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Authors: K. S. Zaytsev, Y. R. Nartsissov

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Glycine is one of the key inhibitory neurotransmitters in Central nervous system (CNS) meanwhile glycinergic transmission is highly dependable on its appropriate reuptake from synaptic cleft. Glycine transporters (GlyT) of types 1 and 2 are the enzymes providing glycine transport back to neuronal and glial cells along with Na⁺ and Cl⁻ co-transport. The distribution and stoichiometry of GlyT1 and GlyT2 differ in details, and GlyT2 is more interesting for the research as it reuptakes glycine to neuron cells, whereas GlyT1 is located in glial cells. In the process of GlyT2 activity, the translocation of the amino acid is accompanied with binding of both one chloride and three sodium ions consequently (two sodium ions for GlyT1). In the present study, we developed a computer simulator of GlyT2 and GlyT1 activity based on known experimental data for quantitative estimation of membrane glycine transport. The trait of a single protein functioning was described using the probability approach where each enzyme state was considered separately. Created scheme of transporter functioning realized as a consequence of elemental steps allowed to take into account each event of substrate association and dissociation. Computer experiments using up-to-date kinetic parameters allowed receiving the number of translocated glycine molecules, Na⁺ and Cl⁻ ions per time period. Flexibility of developed software makes it possible to evaluate glycine reuptake pattern in time under different internal characteristics of enzyme conformational transitions. We investigated the behavior of the system in a wide range of equilibrium constant (from 0.2 to 100), which is not determined experimentally. The significant influence of equilibrium constant in the range from 0.2 to 10 on the glycine transfer process is shown. The environmental conditions such as ion and glycine concentrations are decisive if the values of the constant are outside the specified range.

Keywords: glycine, inhibitory neurotransmitters, probability approach, single protein functioning

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Authors: Sarah Gaßmeyer, Nadine Hülsemann, Raphael Stoll, Kenji Miyamoto, Robert Kourist

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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

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Authors: Amin Abbasi, Fariborz Shekari, Seyed Bahman Mousavi

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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

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Authors: V. Karuppaiah, J. C. Padaria, C. Srivastava

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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

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Authors: Ali Aydin, Mert Sudagidan, Aysen Coban, Alparslan Kadir Devrim

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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

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Authors: Evgeny A. Ermakov, Lyudmila P. Smirnova, Valentina N. Buneva

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Schizophrenia associated with dysregulation of neurotransmitter processes in the central nervous system and disturbances in the humoral immune system resulting in the formation of antibodies (Abs) to the various components of the nervous tissue. Abs to different neuronal receptors and DNA were detected in the blood of patients with schizophrenia. Abs hydrolyzing DNA were detected in pool of polyclonal autoantibodies in autoimmune and infectious diseases, such catalytic Abs were named abzymes. It is believed that DNA-hydrolyzing abzymes are cytotoxic, cause nuclear DNA fragmentation and induce cell death by apoptosis. Abzymes with DNAase activity are interesting because of the mechanism of formation and the possibility of use as diagnostic markers. Therefore, in our work we have set following goals: to determine the level anti-DNA Abs in the serum of patients with schizophrenia and to study DNA-hydrolyzing activity of IgG of patients with schizophrenia. Materials and methods: In our study there were included 41 patients with a verified diagnosis of paranoid or simple schizophrenia and 24 healthy donors. Electrophoretically and immunologically homogeneous IgGs were obtained by sequential affinity chromatography of the serum proteins on protein G-Sepharose and gel filtration. The levels of anti-DNA Abs were determined using ELISA. DNA-hydrolyzing activity was detected as the level of supercoiled pBluescript DNA transition in circular and linear forms, the hydrolysis products were analyzed by agarose electrophoresis followed by ethidium bromide stain. To correspond the registered catalytic activity directly to the antibodies we carried out a number of strict criteria: electrophoretic homogeneity of the antibodies, gel filtration (acid shock analysis) and in situ activity. Statistical analysis was performed in ‘Statistica 9.0’ using the non-parametric Mann-Whitney test. Results: The sera of approximately 30% of schizophrenia patients displayed a higher level of Abs interacting with single-stranded (ssDNA) and double-stranded DNA (dsDNA) compared with healthy donors. The average level of Abs interacting with ssDNA was only 1.1-fold lower than that for interacting with dsDNA. IgG of patient with schizophrenia were shown to possess DNA hydrolyzing activity. Using affinity chromatography, electrophoretic analysis of isolated IgG homogeneity, gel filtration in acid shock conditions and in situ DNAse activity analysis we proved that the observed activity is intrinsic property of studied antibodies. We have shown that the relative DNAase activity of IgG in patients with schizophrenia averaged 55.4±32.5%, IgG of healthy donors showed much lower activity (average of 9.1±6.5%). It should be noted that DNAase activity of IgG in patients with schizophrenia with a negative symptoms was significantly higher (73.3±23.8%), than in patients with positive symptoms (43.3±33.1%). Conclusion: Anti-DNA Abs of patients with schizophrenia not only bind DNA, but quite efficiently hydrolyze the substrate. The data show a correlation with the level of DNase activity and leading symptoms of patients with schizophrenia.

Keywords: anti-DNA antibodies, abzymes, DNA hydrolysis, schizophrenia

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Authors: Rachid El Aidani, Phuong Nguyen-Tri, Toan Vu-Khanh

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This study intends to show the influence of the hydrolytic degradation on the properties of the e-PTFE/NOMEX® membranes used in fire-protective clothing. The modification of water vapour permeability, morphology and chemical structure was examined by MOCON Permatran, electron microscopy scanning (SEM), and ATR-FTIR, respectively. A decrease in permeability to water vapour of the aged samples was observed following closure of transpiration pores. Analysis of fiber morphology indicates the appearance of defects at the fibers surface with the presence of micro cavities as well as the of fibrils. ATR-FTIR analysis reveals the presence of a new absorption band attributed to carboxylic acid terminal groups generated during the amide bond hydrolysis.

Keywords: hydrolytic ageing, moisture membrane, water vapor permeability, morphology

Procedia PDF Downloads 277

Authors: Chukwuma S. Ezeonu, Ikechukwu N. E. Onwurah, Uchechukwu U. Nwodo, Chibuike S. Ubani, Chigozie M. Ejikeme

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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

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Authors: Massimo Mozzon, Nadia Raffaelli

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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 126

Authors: M. I. Tyletc, O. I. Podgornya, T. G. Shaposhnikova, S. V. Shabelnikov, A. G. Mittenberg, M. A. Daugavet

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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

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Authors: Mohammad Faheem, M. Tabish Rehman, Mohd Danishuddin, Asad U. Khan

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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

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