Search results for: rennin enzyme

Authors: Chiun-C.R. Wang, Po-Wen Yen, Chien-Chun Huang

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Banana is produced in large quantities in tropical and subtropical areas. Banana is one of the important fruits which constitute a valuable source of energy, vitamins and minerals. The ripening and maturity standards of banana vary from country to country depending on the expected shelf life of market. The compositions of bananas change dramatically during ethylene-induced ripening that are categorized as nutritive values and commercial utilization. Nevertheless, there is few study reporting the changes of physicochemical properties of banana starch during ethylene-induced ripening of green banana. The objectives of this study were to investigate the changes of chemical composition and enzyme activity of banana and physicochemical properties of banana starch during ethylene-induced ripening. Green bananas were harvested and ripened by ethylene gas at low temperature (15℃) for seven stages. At each stage, banana was sliced and freeze-dried for banana flour preparation. The changes of total starch, resistant starch, chemical compositions, physicochemical properties, activity of amylase, polyphenolic oxidase (PPO) and phenylalanine ammonia lyase (PAL) of banana were analyzed each stage during ripening. The banana starch was isolated and analyzed for gelatinization properties, pasting properties and microscopic appearance each stage of ripening. The results indicated that the highest total starch and resistant starch content of green banana were 76.2% and 34.6%, respectively at the harvest stage. Both total starch and resistant starch content were significantly declined to 25.3% and 8.8%, respectively at the seventh stage. Soluble sugars content of banana increased from 1.21% at harvest stage to 37.72% at seventh stage during ethylene-induced ripening. Swelling power of banana flour decreased with the progress of ripening stage, but solubility increased. These results strongly related with the decreases of starch content of banana flour during ethylene-induced ripening. Both water insoluble and alcohol insoluble solids of banana flour decreased with the progress of ripening stage. Both activity of PPO and PAL increased, but the total free phenolics content decreased, with the increases of ripening stages. As ripening stage extended, the gelatinization enthalpy of banana starch significantly decreased from 15.31 J/g at the harvest stage to 10.55 J/g at the seventh stage. The peak viscosity and setback increased with the progress of ripening stages in the pasting properties of banana starch. The highest final viscosity, 5701 RVU, of banana starch slurry was found at the seventh stage. The scanning electron micrograph of banana starch showed the shapes of banana starch appeared to be round and elongated forms, ranging in 10-50 μm at the harvest stage. As the banana closed to ripe status, some parallel striations were observed on the surface of banana starch granular which could be caused by enzyme reaction during ripening. These results inferred that the highest resistant starch was found in the green banana at the harvest stage could be considered as a potential application of healthy foods. The changes of chemical composition and physicochemical properties of banana could be caused by the hydrolysis of enzymes during the ethylene-induced ripening treatment.

Keywords: ethylene-induced ripening, banana starch, resistant starch, soluble sugars, physicochemical properties, gelatinization enthalpy, pasting characteristics, microscopic appearance

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Authors: O. R. Raimi, A. Lateef

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Inoculum developments of A. niger were used for inoculation of medium for submerged fermentation and solid state fermentation. The filtrate obtained were used as sources of the extra-cellular enzymes. The FTase activities and the course of pH in submerged fermentation ranged from 7.53-24.42µ/ml and 4.4-4.8 respectively. The maximum FTase activity was obtained at 48 hours fermentation. In solid state fermentation, FTase activities ranged from 2.41-27.77µ/ml. Using ripe plantain peel and kola nut pod respectively. Both substrates supported the growth of the fungus, producing profuse growth during fermentation. In the control experiment (using kolanut pod) that lack supplementation, appreciable FTase activity of 16.92µ/ml was obtained. The optimum temperature range was 600C. it was also active at broad pH range of 1-9 with optimum obtain at pH of 5.0. FTase was stable within the range of investigated pH showing more than 60% activities. FTase can be used in the production of fructooligosaccharide, a functional food.

Keywords: Aspergillus niger, solid state fermentation, kola nut pods, Fructosyltransferase (FTase)

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Authors: Shahram Nanekarani, Majid Goodarzi, Majid Khosravi

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The present study aimed at analyzing of ovine major histocompatibility complex class II (Ovar II) DRB1 gene second exon in Lori Sheep breed. The MHC plays a central role in the control of disease resistance and immunological response. Genomic DNA from blood samples of 124 sheep was extracted and a 296 bp MHC exon 2 fragment was amplified using polymerase chain reaction. PCR products were characterized by the restriction fragment length polymorphism technique using Hin1I restriction enzyme. The PCRRFLP patterns showed three genotypes, AA, AB and BB with frequency of 0.282, 0.573 and 0.145, respectively. There was no significant (P > 0.05) deviation from Hardy–Weinberg equilibrium for this locus in this population. The results of the present study indicate that exon 2 of the Ovar-DRB1 gene is highly polymorphic in Lori sheep and could be considered as an important marker assisted selection, for improvement of immunity in sheep.

Keywords: MHC-DRB1 gene, polymorphism, PCR-RFLP, lori sheep

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Authors: Hiroshi Nakayama, Yuji Ito

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Synthetic cannabinoids have attracted much public attention recently in Japan. 1-pentyl-3-(1-naphthoyl)-indole (JWH-018), 1-pentyl-2-methyl-3-(1-naphthoyl) indole (JWH-015), 1-(5-fluoropentyl)-3- (1-(2,2,3,3- tetramethylcyclopropyl)) indole (XLR-11) and 1-methyl-3- (1-admantyl) indole (JWH-018 adamantyl analog) are known as synthetic cannabinoids and are also considered dangerous illegal drugs in Japan. It has become necessary to develop sensitive and useful methods for detection of synthetic cannabinoids. We produced two monoclonal antibodies (MAb) against synthetic cannabinoids, named NT1 (IgG1) and NT2 (IgG1), using Hybridoma technology. The cross-reactivity of these produced MAbs was evaluated using a competitive enzyme-linked immunosorbent assay (ELISA). In the results, we found both of these antibodies recognize many kinds of synthetic cannabinoids analog. However, neither of these antibodies recognizes naphtoic acid, 1-methyl-indole and indole known as a raw material of synthetic cannabinoid. Thus, the MAbs produced in this study could be a useful tool for the detection of synthetic cannabinoids.

Keywords: ELISA, monoclonal antibody, sensor, synthetic cannabinoid

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Authors: Hassan Waseem

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Cytomegalovirus (CMV) is ubiquitously distributed viral agent responsible for different clinical manifestations that may vary according to the immunologic status of the patient. CMV can cause morbidity and mortality among fetuses and patients with compromised immune system. A cross-sectional study was carried out in Islamabad to investigate the prevalence and risk factors associated with CMV infection among pregnant women. Blood samples of 172 pregnant women visiting Mother and Child Healthcare, Pakistan Institute of Medical Sciences (PIMS) Islamabad were taken. In present study, serum samples of the women were checked for CMV-specific IgG and IgM antibodies by enzyme linked immunosorbent assay (ELISA). Clinical, obstetrical and socio-demographical characteristics of the women were collected by using structured questionnaires. Out of 172 pregnant women included in the study, 171 (99.4%) were CMV specific IgG positive and 30 (17.4%) were found positive for CMV-IgM antibodies. The CMV has taken an endemic form in Pakistan so, routine screening of CMV among pregnant women is recommended.

Keywords: Cytomegalovirus, blood transfusion, ELISA, seroprevalence

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Authors: Mustafa Ümi̇t Ünal, Nafi̇z Çeli̇ktaş, Aysun Şener, Sara Betül Dolgun, Duygu Keser

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The aim of this study was to compare acid and base pretreatment of switchgrass for bioethanol production. Switchgrass was pretreated with sulfuric acid and sodium hydroxide at 0.5, 1.0 and 1.5% (v/v) at 120, 140, 180 °C for 10, 60 and 90. Optimization of enzymatic hydrolysis of the pretreated switchgrass samples were carried out using three different enzyme mixtures (22.5 mg cellulase and 75 mg cellobiase /g biomass; 45 mg cellulase and 150 mg cellobiase /g biomass; 90 mg cellulase and 300 mg cellobiase /g biomass). Samples were removed at 24-h interval for fermentable sugar analyses with HPLC. The results showed that use of 90 mg cellulase and 300 mg cellobiase/g biomass resulted in the highest fermentable sugar formation. Furthermore, the highest fermentable sugar yield was obtained by pretreatment at 120 °C for 10 min using 1.0 % sodium hydroxide.

Keywords: switchgrass, acid pretreatment, enzymatic hydrolysis, base pretreatment, ethanol production

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Authors: Ibrahim Alshunaibir

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Infection with Helicobacter pylori is common worldwide but few studies focus on the prevalence and spread of the infection in Saudi Arabia. This study was undertaken to observe the epidemiology of Helicobacter pylori infection in patients suffering from gastrointestinal sign and symptoms in one of the largest hospitals in the capital of Saudi Arabia, Riyadh. Methods: Enzyme-linked immunosorbent assay (ELISA) was undertaken for this study with nearly 6000 samples collected and examined for patients suffering from (dyspeptic) symptoms ranging in their age from 5 to 75 years. Results: The prevalence of helicobacter infection was 67% increasing with age. Female shows higher percentage of H. pylori infection than male. Conclusions: The percentage rate was higher in female than male. This study shows a high percentage of helicobacter infection in Saudi Arabia.

Keywords: Helicobacter pylori, percentage, dyspeptic, Saudi Arabia

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Authors: Tauseef Ahmad, Muhammad Ishaq, Mathew Eapen, Ahyoung Park, Sam Karpiniec, Vanni Caruso, Rajaraman Eri

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Fucoidans are complex, fucose-rich sulfated polymers discovered in brown seaweeds. Fucoidans are popular around the world, particularly in the nutraceutical and pharmaceutical industries, due to their promising medicinal properties. Fucoidans have been shown to have a variety of biological activities, including anti-inflammatory effects. They are known to inhibit inflammatory processes through a variety of mechanisms, including enzyme inhibition and selectin blockade. Inflammation is a part of the complicated biological response of living systems to damaging stimuli, and it plays a role in the pathogenesis of a variety of disorders, including arthritis, inflammatory bowel disease, cancer, and allergies. In the current investigation, various fucoidan extracts from Undaria pinnatifida, Fucus vesiculosus, Macrocystis pyrifera, Ascophyllum nodosum, and Laminaria japonica were assessed for inhibition of pro-inflammatory cytokine production (TNF-α, IL-1β, and IL-6) in LPS induced human macrophage cell line (THP-1) and human peripheral blood mononuclear cells (PBMCs). Furthermore, we also sought to catalogue these extracts based on their anti-inflammatory effects in the current in-vitro cell model. Materials and Methods: To assess the cytotoxicity of fucoidan extracts, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5, -diphenyltetrazolium bromide) cell viability assay was performed. Furthermore, a dose-response for fucoidan extracts was performed in LPS induced THP-1 cells and PBMCs after pre-treatment for 24 hours, and levels of TNF-α, IL-1β, and IL-6 cytokines were measured using Enzyme-Linked Immunosorbent Assay (ELISA). Results: The MTT cell viability assay demonstrated that fucoidan extracts exhibited no evidence of cytotoxicity in THP-1 cells or PBMCs after 48 hours of incubation. The results of the sandwich ELISA revealed that all fucoidan extracts suppressed cytokine production in LPS-stimulated PBMCs and human THP-1 cells in a dose-dependent manner. Notably, at lower concentrations, the lower molecular fucoidan (5-30 kDa) extract from Macrocystis pyrifera was a highly efficient inhibitor of pro-inflammatory cytokines. Fucoidan extracts from all species including Undaria pinnatifida, Fucus vesiculosus, Macrocystis pyrifera, Ascophyllum nodosum, and Laminaria japonica exhibited significant anti-inflammatory effects. These findings on several fucoidan extracts provide insight into strategies for improving their efficacy against inflammation-related diseases. Conclusion: In the current research, we have successfully catalogued several fucoidan extracts based on their efficiency in LPS-induced macrophages and PBMCs in downregulating the key pro-inflammatory cytokines (TNF-, IL-1 and IL-6), which are prospective targets in human inflammatory illnesses. Further research would provide more information on the mechanism of action, allowing it to be tested for therapeutic purposes as an anti-inflammatory medication.

Keywords: fucoidan, PBMCs, THP-1, TNF-α, IL-1β, IL-6, inflammation

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Authors: W. Soufi, F. Boukli Hacene, S. Ghalem

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Alzheimer's disease (AD) is a neurodegenerative disease that leads to a progressive and permanent deterioration of nerve cells. This disease is progressively accompanied by an intellectual deterioration leading to psychological manifestations and behavioral disorders that lead to a loss of autonomy. It is the most frequent of degenerative dementia. Alzheimer's disease (AD), which affects a growing number of people, has become a major public health problem in a few years. In the context of the study of the mechanisms governing the evolution of AD disease, we have found that natural flavonoids are good acetylcholinesterase inhibitors that reduce the rate of ßA secretion in neurons. This work is to study the inhibition of acetylcholinesterase (AChE) which is an enzyme involved in Alzheimer's disease, by methods of molecular modeling. These results will probably help in the development of an effective therapeutic tool in the fight against the development of Alzheimer's disease. Our goal of the research is to study the inhibition of acetylcholinesterase (AChE) by molecular modeling methods.

Keywords: Alzheimer's disease, acetylcholinesterase, flavonoids, molecular modeling

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Authors: Aliyu Adamu, Fahrul Huyop, Roswanira Abdul Wahab, Mohd Shahir Shamsir

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Halogenated organic compounds occur in huge amount in biosphere. This is attributable to the diverse use of halogen-based compounds in the synthesis of various industrially important products. Halogenated compound is toxic and may persist in the environment, thereby causing serious health and environmental pollution problems. L-2-haloacid dehalogenases (EC 3.8.1.2) catalyse the specific cleavage of carbon-halogen bond in L-isomers of halogenated compounds, which consequently reverse the effects of environmental halogen-associated pollution. To enhance the efficiency and utility of these enzymes, this study investigates the catalytic amino acid residues and the molecular functional mechanism of DehL, by classical molecular dynamic simulations, MM-PBSA and ab initio fragments molecular orbital (FMO) calculations. The results of the study will serve as the basis for the molecular engineering of the enzyme.

Keywords: DehL, Functional mechanism, Catalytic residues, L-2-haloacid dehalogenase

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Authors: I. Savitskaya, A. Kistaubayeva, A. Zhubanova, I. Blavachinskaiya, D. Ibrayeva, M. Abdulzhanova, A. Otarbay, A.Isabekova

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This study was conducted for the investigation of number of cellulolytic bacteria and their ability in decomposition. Seven samples surface soil were collected on cellulose Zailiskii Alatau slopes. Cellulolitic activity of new strains of Bacillus, isolated from soil is determined. Isolated cellulose degrading bacteria were screened for determination of the highest cellulose activity by quantitative assay using Congo red, gravimetric assay and colorimetric DNS method trough of the determination of the parameters of sugar reduction. Strains are assigned to: B.subtilis, B.licheniformis, B. cereus and, В. megaterium. Bacillus strains consisting of several different types of cellulases have broad substrate specificity of cellulase complexes formed by them. Cellulolitic bacteria were recorded to have highest cellulase activity and selected for optimization of cellulase enzyme production.

Keywords: cellulose-degrading bacteria, cellulase complex, foothills soil, screening

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Authors: Ahmad Manbohi, Seyyed Hamid Ahmadi

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A low-cost paper-based microfluidic device (PAD) for the multiplex electrochemical determination of glucose, uric acid, and dopamine in biological fluids was developed. Using wax printing, PAD containing a central zone, six channels, and six detection zones was fabricated, and the electrodes were printed on detection zones using pre-made electrodes template. For each analyte, two detection zones were used. The carbon working electrode was coated with chitosan-BSA (and enzymes for glucose and uric acid). To detect glucose and uric acid, enzymatic reactions were employed. These reactions involve enzyme-catalyzed redox reactions of the analytes and produce free electrons for electrochemical measurement. Calibration curves were linear (R^² > 0.980) in the range of 0-80 mM for glucose, 0.09–0.9 mM for dopamine, and 0–50 mM for uric acid, respectively. Blood samples were successfully analyzed by the proposed method.

Keywords: biological fluids, biomarkers, microfluidic paper-based electrochemical biosensors, Multiplex

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Authors: Achmad Buhori, Reza Imam Pratama, Tissa Wiraatmaja, Wanti Megawati

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Data from many countries indicates that there is a marked increase of dental caries. The increases in caries appear to occur in lower socioeconomic groups. It is possible that the benefits of prevention of dental caries are not reaching these groups. However, there is a way to decrease dental caries by adding 5% of bromelain and calcium as an active agent in toothpaste. Bromelain can break glutamine-alanine bond and arginine-alanine bond which is a constituent of amino acid that causes dental plague which is one of the factors of dental caries. Calcium help rebuilds the teeth by strengthening and repairing enamel. Bromelain can be found from the extraction of pineapple (Ananas comosus) cobs (88.86-94.22 % of bromelain recovery during extraction based on the enzyme unit) and calcium can be taken from eggshell (95% of dry eggshell consist of calcium). The aim of this experiment is to make a toothpaste which contains bromelain and calcium as an effective, cheap, and healthy way to decrease dental caries around the world.

Keywords: bromelain, calcium, dental caries, dental plague, toothpaste

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Authors: C. Makwana, S. R. Dave

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Arsenic is toxic to almost all living cells. Its contamination in natural sources affects the growth of microorganisms. The presence of arsenic is associated with various human disorders also. The attempt of this sort of study provides information regarding the performance of our isolated microorganisms in the presence of Arsenic, which have ample scope for bioremediation. Six isolates were selected from the polluted sample of industrial zone Vatva, Ahmedabad, Gujarat, India, out of which two were Thermophilic organisms. The thermophilic exopolysaccharide (EPS) producing Bacillus was used for microbial enhance oil recovery (MEOR) and in the bio beneficiation. Inorganic arsenic primarily exists in the form of arsenate or arsenite. This arsenic resistance isolate was capable of transforming As +3 to As+5. This isolate would be useful for arsenic remediation standpoint from aquatic systems. The study revealed that the thermophilic microorganism was growing at 55 degree centigrade showed considerable remediation property. The results on the growth and enzyme catalysis would be discussed in response to Arsenic remediation.

Keywords: aquatic systems, thermophilic, exopolysacchride, arsenic

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Authors: Jung-Feng Hsieh, Chu Ze Chen

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Curcumin is the main active compound of turmeric that can inhibit the activities of α-glucosidase and xanthine oxidase (XO). α-Glucosidase and XO inhibitors are widely used to treat patients with diabetes mellitus and gout, respectively; therefore, the objective of this research was to evaluate the inhibitory activities of curcumin and its analogs against α-glucosidase and XO. Our results demonstrated that CM-F had the strongest antioxidant activity with a half-maximal effective concentration (EC50) of 9.39 ± 0.16 μM, which was superior to vitamin E (EC50=17.03 ± 0.09 μM). CM-F also exhibited potent inhibitory activity against XO with an IC50 value of 6.14 ± 0.38 μM and enzyme kinetic results revealed competitive inhibition of XO. We also found that CM-1 and CM-2 inhibited α-glucosidase with IC50 values of 21.06 ± 0.92 μM and 5.95 ± 0.09 μM, respectively, and kinetic studies indicated that both CM-1 and CM-2 are mixed competitive inhibitors of α-glucosidase. Furthermore, docking simulation identified five hydrogen bonds between XO and CM-F; however, only one and two hydrogen bonds are involved in CM-1 and CM-2 binding to α-glucosidase, respectively. Accordingly, curcumin and its analogs have the potential to be used in the treatment of patients with diabetes mellitus and gout.

Keywords: curcumin, α-glucosidase, inhibitor, xanthine oxidase

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Authors: Fengmei Li, Li Xu, Guoliang Xia

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Lysozyme is a catalytic enzyme that performs bacterial cell lysis by cleaving the β-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine of peptidoglycan in cell walls. In the present study, an invertebrate-type (i-type) lysozyme gene was cloned from Chinese mitten crab Eriocheir sinensis (designated as EsLysozyme) based on PCR-based rapid amplification of cDNA ends (RACE) technology. The full-length cDNA of EsLysozyme was of 831 bp. SMART and SIGNALP 3.0 program analysis revealed that EsLysozyme contained a signal peptide and a destabilase domain. The five amino acid residues (Tyr63, Trp64, Tyr91, His110, Pro114) and the conserved motif GSLSCG(P/Y)FQI and CL(E/L/R/H)C(I/M)C in i-type lysozymes were also found in EsLysozyme. The high similarity of EsLysozyme with L. vannamei lysozymes and phylogenetic analysis suggested that EsLysozyme should be a new member of i-type lysozyme family.

Keywords: i-type lysozyme, Eriocheir sinensis, cloning, characterization

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Authors: Stephy Saavedra, Annsy C. Arredondo, Gisele Monteiro, Adalberto Pessoa Jr, Carol N. Flores-Fernandez, Amparo I. Zavaleta

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L-asparaginase from bacterial sources is used in leukemic treatment and food industry. This enzyme is classified based on its affinity towards L-asparagine and L-glutamine. Likewise, ansZ genes express L-asparaginase with higher affinity to L-asparagine. The aim of this work was to clone and express of ansZ gene from Bacillus sp. CH11 isolated from Chilca salterns in Peru. The gene encoding L-asparaginase was cloned into pET15b vector and transformed in Escherichia coli BL21 (DE3) pLysS. The expression was carried out in a batch culture using LB broth and 0.5 mM IPTG. The recombinant L-asparaginase showed a molecular weight of ~ 39 kDa by SDS PAGE and a specific activity of 3.19 IU/mg of protein. The cloning and expression of ansZ gene from this halotolerant Bacillus sp. CH11 allowed having a biological input to improve a future scaling-up.

Keywords: ansZ gene, Bacillus sp, Chilca salterns, recombinant L-asparaginase

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Authors: Yun-Hsuan Chang, Chih-Chun Huang, Ru-Band Lu

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Dopamine, metabolized to 3,4-dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenase 2 (ALDH2), ALDH2*1/*1, and ALDH2*1/*2+ALDH*2/*2 equally carried in Han Chinese. The relationship between dopamine metabolic enzyme and cognitive performance in bipolar II disorder comorbid with anxiety disorder (AD) remains unclear. This study proposed to explore the association between ALDH2 polymorphisms, anxiety comorbidity in bipolar II disorder. One hundred and ninety-seven BPII with or without AD comorbidity were recruited and compared with 130 Health controls (HC). A polymerase chain reaction and restriction fragment length polymorphism analysis was used to determine genotypes for ALDH2, and neuropsychological battery was performed. Two factor analyses with AD comorbidity and ALDH2 showed a significant main effect of ALDH2 on attention and marginally significant interaction between AD and ALDH2 memory performance. The ALDH2 polymorphisms may play a different role in the neuropsychological performance on varied neuropsychological performance in BPII comorbid with and without AD.

Keywords: anxiety disorder, bipolar II disorder, comorbidity, genetic

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Authors: M. A. Siti Faridah, K. Muhammad, H. M. Ghazali, Y. A. Yusof

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This study was conducted to investigate the effects of three variables namely types of cell wall degrading enzymes (Viscozyme L, Pectinex Ultra SP-L, Rapidase PAC, Rohament CL and Rohapect PTE) at varying concentrations (0.25-3% v/w) and times (30 min-24 h) on the zinc (Zn-) amaranth purees. Liquefaction treatment of the Zn-amaranth purees with Viscozyme (1% v/w at pH 5 and 45ºC for 3 h) was found to be the best procedure, which produced Zn-amaranth puree with low viscosity (8.60 mPas). Zn-amaranth purees were also found to have the highest metallo-chlorophyll derivative contents (0.16 mg/g), free radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH) values (12.49 mM (TE)/g fresh weight) and ferric reducing antioxidant power (FRAP) values (4.57 mM (TE)/g fresh weight) within 3 h of liquefaction. Other physicochemical properties of the enzyme-liquefied Zn-amaranth purees indicated that lightness (L*) (12.54), greenness a*/b* (-0.30), reducing sugar (103.88 mg/mL) and soluble dietary fibre (5.94%) of the purees were higher compared to that of nonenzyme-liquefied amaranth purees.

Keywords: amaranth, antioxidant, chlorophyll derivative, enzymatic liquefaction

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Authors: Hamide Amani, Afshin FarahBakhsh, Iman Farahbakhsh

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In this paper, an amprometric biosensor based on surface modified polypyrrole (PPy) has been developed for the quantitative estimation of urea in aqueous solutions. The incorporation of urease (Urs) into a bipolymeric substrate consisting of PPy was performed by entrapment to the polymeric matrix, PPy acts as amperometric transducer in these biosensors. To increase the membrane conductivity, multi-walled carbon nanotubes (MWCNT) were added to the PPy solution. The entrapped MWCNT in PPy film and the bipolymer layers were prepared for construction of Pt/PPy/MWCNT/Urs. Two different configurations of working electrodes were evaluated to investigate the potential use of the modified membranes in biosensors. The evaluation of two different configurations of working electrodes suggested that the second configuration, which was composed of an electrode-mediator-(pyrrole and multi-walled carbon nanotube) structure and enzyme, is the best candidate for biosensor applications.

Keywords: urea biosensor, polypyrrole, multi-walled carbon nanotube, urease

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Authors: Margarita Ivanova, Julia Dao, Andrew Friedman, Neil Kasaci, Rekha Gopal, Ozlem Goker-Alpan

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In Fabry disease (FD), α-galactosidase A (α-Gal A) deficiency leads to the accumulation of globotriaosylceramide (Lyso-Gb3 and Gb3), triggering a pathologic cascade that causes the severity of organs damage. The heart is one of the several organs with high sensitivity to the α-Gal A deficiency. A subgroup of patients with significant residual of α-Gal A activity with primary cardiac involvement is occasionally referred to as “cardiac variant.” The cardiovascular complications are most frequently encountered, contributing substantially to morbidity, and are the leading cause of premature death in male and female patients with FD. The deposition of Lyso-Gb-3 and Gb-3 within the myocardium affects cardiac function with resultant progressive cardiovascular pathology. Gb-3 and Lyso-Gb-3 accumulation at the cellular level trigger a cascade of events leading to end-stage fibrosis. In the cardiac tissue, Lyso-Gb-3 deposition is associated with the increased release of inflammatory factors and transforming growth factors. Infiltration of lymphocytes and macrophages into endomyocardial tissue indicates that inflammation plays a significant role in cardiac damage. Moreover, accumulated data suggest that chronic inflammation leads to multisystemic FD pathology even under enzyme replacement therapy (ERT). NF-κB activation plays a subsequent role in the inflammatory response to cardiac dysfunction and advanced heart failure in the general population. TNFalpha/NF-κB signaling protects the myocardial evoking by ischemic preconditioning; however, this protective effect depends on the concentration of TNF-α. Thus, we hypothesize that TNF-α is a critical factor in determining the grade of cardio-pathology. Cardiac hypertrophy corresponds to the expansion of the coronary vasculature to maintain a sufficient supply of nutrients and oxygen. Coronary activation of angiogenesis and fibrosis plays a vital role in cardiac vascularization, hypertrophy, and tissue remodeling. We suggest that the interaction between the inflammatory pathways and cardiac vascularization is a bi-directional process controlled by secreted cytokines and growth factors. The co-coordination of these two processes has never been explored in FD. In a cohort of 40 patients with FD, biomarkers associated with inflammation and cardio hypertrophic remodeling were studied. FD patients were categorized into three groups based on LVmass/DSA, LVEF, and ECG abnormalities: FD with no cardio complication, FD with moderate cardio complication, and severe cardio complication. Serum levels of NF-kB, TNFalpha, Il-6, Il-2, MCP1, ING-gamma, VEGF, IGF-1, TGFβ, and FGF2 were quantified by enzyme-linked immunosorbent assays (ELISA). Among the biomarkers, MCP-1, INF-gamma, VEGF, TNF-alpha, and TGF-beta were elevated in FD patients. Some of these biomarkers also have the potential to correlate with cardio pathology in FD. Conclusion: The study provides information about the role of inflammatory pathways and biomarkers of cardio hypertrophic remodeling in FD patients. This study will also reveal the mechanisms that link intracellular accumulation of Lyso-GB-3 and Gb3 to the development of cardiomyopathy with myocardial thickening and resultant fibrosis.

Keywords: biomarkers, Fabry disease, inflammation, growth factors

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Authors: Manju Verma, Parag A. Deshpande

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Electronic structure calculations of hydrogen terminated metal-porphyrin graphene were carried out to explore the catalytic activity for CO2 hydration reaction. A ruthenium atom was substituted in place of carbon atom of graphene and ruthenium chelated carbon atoms were replaced by four nitrogen atoms in metal-porphyrin graphene system. Ruthenium atom created the active site for CO2 hydration reaction. Ruthenium-porphyrin graphene followed the mechanism of carbonic anhydrase enzyme for CO2 conversion to HCO3- ion. CO2 hydration reaction over ruthenium-porphyrin graphene proceeded via the elementary steps: OH- formation from H2O dissociation, CO2 bending in presence of nucleophilic attack of OH- ion, HCO3- ion formation from proton migration, HCO3- ion desorption by H2O addition. Proton transfer to yield HCO3- ion was observed as a rate limiting step from free energy landscape.

Keywords: ruthenium-porphyrin graphene, CO2 hydration, carbonic anhydrase, heterogeneous catalyst, density functional theory

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Authors: Ben Amar Cheba, Taha Ibrahim Zaghloul, Mohamad Hisham El-Massry, Ahmad Rafik El-Mahdy

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Seventy two bacterial strains with ability to degrade chitin were isolated during a screening program. One of the most potent isolates (strain R2) was identified as Bacillus sp. using conventional methods as well as 16S rRNA technique and submitted in the Gen Bank sequence database as Bacillus sp. R2 with a given accession number DQ 923161. This strain was able to produce high levels of extracellular chitinase. The chitinase of Bacillus sp. R2 hydrolyzed several chitinous substrates preferentially and showed a maximum activity toward the β chitin such as Calmar pen and squid bone chitins with the folds 1.47 and 1.23 respectively. The enzyme also exhibited a substrate binding capacity of more than 70% for squid chitin, shrimp shell colloidal chitin, chitosan and prawn shell chitin. The chitinase showed a moderate antifungal activity against many phytopathogenic fungi such as Aspergillus niger, A. flavus, Penicillium degitatum and Fusarium calmorum.This strain could be a suitable candidate for chitinase production on an industrial scale for using as promising antifungal biopestecide.

Keywords: antifungal activity, Bacillus sp. R2, chitinase, substrate specificity

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Authors: Rafayel Azizyan, Valeri Arakelyan, Aram Gevorgyan, Varduhi Balayan, Emil Gevorgyan

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Currently, mathematical and computer modeling are widely used in different biological studies to predict or assess behavior of such complex systems as biological ones. This study deals with mathematical and computer modeling of bi-substrate enzymatic reactions, which play an important role in different biochemical pathways. The main objective of this study is to represent the results from in silico investigation of bi-substrate enzymatic reactions in the presence of uncompetitive inhibitors, as well as to describe in details the inhibition effects. Four models of uncompetitive inhibition were designed using different software packages. Particularly, uncompetitive inhibitor to the first [ES1] and the second ([ES1S2]; [FS2]) enzyme-substrate complexes have been studied. The simulation, using the same kinetic parameters for all models allowed investigating the behavior of reactions as well as determined some interesting aspects concerning influence of different cases of uncompetitive inhibition. Besides that shown, that uncompetitive inhibitors exhibit specific selectivity depending on mechanism of bi-substrate enzymatic reaction.

Keywords: mathematical modeling, bi-substrate enzymatic reactions, reversible inhibition

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Authors: Yazun Jarrar

Abstract:

Smoking has effect on platelet aggregation and the activity of anti-platelet drugs. The chemical 20-hydroxyeicosatetraenoic acid (20-HETE) is a cardiotoxic arachidonic acid metabolite which increases platelet aggregation. In this study, we investigated the influence of cigarette smoking on 20-HETE levels and protein expression of 20-HETE producing enzyme CYP4A11 in isolated platelets from smoker and non-smoker volunteers. The protein expression and 20-HETE levels were analyzed using immunoblot and High-Performance Liquid Chromatography with Mass Spectrometry (HPL-MS) assays. The results showed that 20-HETE level was higher significantly among smokers than non-smokers (t-test, p-value<0.05). The protein expression of CYP4A11 was significantly higher (t-test, p-value<0.05) among the platelets of smokers. We concluded that cigarette smoking increased the level of platelet activator 20-HETE through increasing the protein expression of CYP4A11. These findings may increase the understanding of smoking-drug interaction during antiplatelets therapy.

Keywords: smoking, 20-HETE, CYP4A11, platelet

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Authors: Tesfaye Alamirew, Delele Worku, Solomon W. Fanta, Nigus Gabbiye

Abstract:

Recently graphen based nanocomposites are become an emerging research areas for fabrication of enzymatic biosensors due to their property of large surface area, conductivity and biocompatibility. This review summarizes recent research reports of graphen based nanocomposites for the fabrication of glucose and ethanol enzymatic biosensors. The newly fabricated enzyme free microwave treated nitrogen doped graphen (MN-d-GR) had provided highest sensitivity towards glucose and GCE/rGO/AuNPs/ADH composite had provided far highest sensitivity towards ethanol compared to other reported graphen based nanocomposites. The MWCNT/GO/GOx and GCE/ErGO/PTH/ADH nanocomposites had also enhanced wide linear range for glucose and ethanol detection respectively. Generally, graphen based nanocomposite enzymatic biosensors had fast direct electron transfer rate, highest sensitivity and wide linear detection ranges during glucose and ethanol sensing.

Keywords: glucose, ethanol, enzymatic biosensor, graphen, nanocomposite

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Authors: Marcela Dvorakova, Lenka Langhansova, Premysl Landa

Abstract:

A series of quinazoline derivatives bearing aromatic rings in 2- and 4-positions were prepared and tested for their biological activity. Firstly, the compounds were evaluated for their potential to inhibit various kinases, such as autophagy activating kinase ULK1, 3-Phosphoinositide-dependent kinase 1, and TANK-binding kinase 1. None of the compounds displayed any activity on these kinases. Secondly, the compounds were tested for their anti-inflammatory activity expressed as cyclooxygenase (COX) isoforms and 5-lipoxygenase (5-LOX) inhibition. Three of the compounds showed significant selectivity towards COX-1 isoform (COX-2/COX-1 SI = 20-30). They inhibited COX-1 in a single-digit µM range. There was also one compound that exhibited inhibitory activity towards all three tested enzymes in µM range (IC50COX-1 = 1.9 µM; IC50COX-2 and 5-LOX = 10.1µM. COX-1 inhibition was until recently considered undesirable due to COX-1 constitutive expression in most cell types and tissues. Thus, there are not many compounds known with selective COX-1 activity. However, it is now believed that COX-1 plays an important role in the pathophysiology of several acute and chronic disorders, including cancer or neurodegenerative diseases. Thus, the discovery of effective COX-1 selective inhibitors is desirable and important.

Keywords: cyclooxygenases, kinases, lipoxygenases, quinazolines

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Authors: Kimberley Phoena Fan, Yu Zhang

Abstract:

ECOSURF™ EH-3 Surfactant (EH3) is a nonionic surfactant and has superior wetting and excellent oil removal properties. It is biodegradable with low toxicity and meets or exceeds US EPA Design for the Environment Criteria, and is widely used as a home cleaner, commercial and industrial degreaser. We have recently found that EH3 also possesses a special function which is characterized as an enhancer to Taq DNA polymerase and ameliorator to reduce the effects of PCR inhibitors, i.e., blood, urea, Guanidinium thiocyanate, Humic acids, polyphenol, and Polysaccharides. This is a new kind of PCR enhancer that does not work on relieving secondary structures of GC-rich templates. We have compared EH3’s effects on Taq DNA Polymerase along with other well-known enhancers, such as DMSO, betaine, and BSA, using GC rich or deficient template and found that, unlike DMSO and Betaine, the EH3 boosting effect on PCR reaction is not through reducing Tm. The results show the same increase of PCR products regardless of the GC contents or secondary structures. The mechanism of EH3 enhancing PCR is through its direct interaction with or stimulation of the DNA polymerase and making the enzymes more resistant to inhibitors in the presence of EH3. This phenomenon has first been observed for EH3, a new type of PCR enzyme enhancer. Subsequent research also shows that a series of similar surfactants boost Taq DNA polymerase as well.

Keywords: EH3, DNA, polymerase, enhancer, raw biological samples

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Authors: Ayodeji O. Falade, Leonard V. Mabinya, Uchechukwu U. Nwodo, Anthony I. Okoh

Abstract:

Given the high utility of peroxidase in several industrial processes, the search for novel microorganisms with enhanced peroxidase production capacity is of keen interest. This study investigated the process conditions for optimum peroxidase production by Ensifer sp, new ligninolytic proteobacteria with peroxidase production potential. Also, some agricultural residues were valorized for peroxidase production under solid state fermentation. Peroxidase production was optimum at an initial medium pH 7, incubation temperature of 30 °C and agitation speed of 100 rpm using alkali lignin fermentation medium supplemented with guaiacol as the most effective inducer and ammonium sulphate as the best inorganic nitrogen. Optimum peroxidase production by Ensifer sp. was attained at 48 h with specific productivity of 12.76 ± 1.09 U mg⁻¹. Interestingly, probable laccase production was observed with optimum specific productivity of 12.76 ± 0.45 U mg⁻¹ at 72 h. The highest peroxidase yield was observed with sawdust as solid substrate under solid state fermentation. In conclusion, Ensifer sp. possesses the capacity for enhanced peroxidase production that can be exploited for various biotechnological applications.

Keywords: catalase-peroxidase, enzyme production, peroxidase, polymerase chain reaction, proteobacteria

Procedia PDF Downloads 308

Authors: Debora B. Moretti, Wiolene M. Nordi, Thaline Maira P. Cruz, José Eurico P. Cyrino, Raul Machado-Neto

Abstract:

Enzyme activity was evaluated in the intestine of juvenile dourado (Salminus brasiliensis) fed with diets containing 0, 10 or 20% of lyophilized bovine colostrum (LBC) inclusion for either 30 or 60 days. The intestinal enzymes acid and alkaline phosphatase (ACP and ALP, respectively), non-specific esterase (NSE), lipase (LIP), dipeptidyl aminopeptidase IV (DAP IV) and leucine aminopeptidase (LAP) were studied using histochemistry in four intestinal segments (S1, S2, S3 and posterior intestine). Weak proteolitic activity was observed in all intestinal segments for DAP IV and LAP. The activity of NSE and LIP was also weak in all intestines, except for the moderate activity of NSE in the S2 of 20% LBC group after 30 days and in the S1 of 0% LBC group after 60 days. The ACP was detected only in the S2 and S3 of the 10% LBC group after 30 days. Moderate and strong staining was observed in the first three intestinal segments for ALP and weak activity in the posterior intestine. The activity of DAP IV, LAP and ALP were also present in the cytoplasm of the enterocytes. In the present results, bovine colostrum feeding did not cause alterations in activity of intestinal enzymes.

Keywords: carnivorous fish, enterocyte, intestinal epithelium, teleost

Procedia PDF Downloads 329