Search results for: enzyme linked immunosorbent assay

Authors: Mustafa M. Donma, Orkide Donma

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Iron is physiologically essential. However, it also participates in the catalysis of free radical formation reactions. Its deficiency is associated with amplified health risks. This trace element establishes some links with another physiological process related to cell death, apoptosis. Both iron deficiency and iron overload are closely associated with apoptosis. Soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) has the ability to trigger apoptosis and plays a dual role in the physiological versus pathological inflammatory responses of tissues. The aim of this study was to investigate the status of these parameters as well as the associations among them in children with obesity, a low-grade inflammatory state. The study was performed on groups of children with normal body mass index (N-BMI) and obesity. Forty-three children were included in each group. Based upon age- and sex-adjusted BMI percentile tables prepared by World Health Organization, children whose values varied between 85 and 15 were included in N-BMI group. Children whose BMI percentile values were between 99 and 95 comprised obese (OB) group. Institutional ethical committee approval and informed consent forms were taken prior to the study. Anthropometric measurements (weight, height, waist circumference, hip circumference, head circumference, neck circumference) and blood pressure values (systolic blood pressure and diastolic blood pressure) were recorded. Routine biochemical analysis including serum iron, total iron binding capacity (TIBC), transferrin saturation percent (Tf Sat %), and ferritin were performed. Soluble tumor necrosis factor-like weak inducer of apoptosis levels were determined by enzyme-linked immunosorbent assay. Study data was evaluated using appropriate statistical tests performed by the statistical program SPSS. Serum iron levels were 91±34 mcrg/dl and 75±31 mcrg/dl in N-BMI and OB children, respectively. The corresponding values for TIBC, Tf Sat %, ferritin were 265 mcrg/dl vs 299 mcrg/dl, 37.2±19.1 % vs 26.7±14.6 %, and 41±25 ng/ml vs 44±26 ng/ml. in N-BMI and OB groups, sTWEAK concentrations were measured as 351 ng/L and 325 ng/L, respectively (p>0.05). Correlation analysis revealed significant associations between sTWEAK levels and iron related parameters (p<0.05) except ferritin. In conclusion, iron contributes to apoptosis. Children with iron deficiency have decreased apoptosis rate in comparison with that of healthy children. sTWEAK is inducer of apoptosis. Obese children had lower levels of both iron and sTWEAK. Low levels of sTWEAK are associated with several types of cancers and poor survival. Although iron deficiency state was not observed in this study, the correlations detected between decreased sTWEAK and decreased iron as well as Tf Sat % values were valuable findings, which point out decreased apoptosis. This may induce a proinflammatory state, potentially leading to malignancies in the future lives of obese children.

Keywords: apoptosis, children, iron-related parameters, obesity, soluble tumor necrosis factor-like weak inducer of apoptosis

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Authors: Mustafa M. Donma, Orkide Donma

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Zinc is a vital element required for growth and development. This fact makes zinc important, particularly for children. It maintains normal cellular structure and functions. This essential element appears to have protective effects against coronary artery disease and cardiomyopathy. Higher serum zinc levels are associated with lower risk of cardiovascular diseases (CVDs). There is a significant association between low serum zinc levels and heart failure. Zinc may be a potential biomarker of cardiovascular health. High sensitive cardiac troponin T (hs-cTnT) and cardiac myosin binding protein C (cMyBP-C) are new generation markers used for prediagnosis, diagnosis, and prognosis of CVDs. The aim of this study is to determine zinc as well as new generation cardiac markers profiles in children with normal body mass index (N-BMI), obese (OB), morbid obese (MO) children, and children with metabolic syndrome (MetS) findings. The association among them will also be investigated. Four study groups were constituted. The study protocol was approved by the institutional Ethics Committee of Tekirdag Namik Kemal University. Parents of the participants filled informed consent forms to participate in the study. Group 1 is composed of 44 children with N-BMI. Group 2 and Group 3 comprised 43 OB and 45 MO children, respectively. Forty-five MO children with MetS findings were included in Group 4. World Health Organization age- and sex-adjusted BMI percentile tables were used to constitute groups. These values were 15-85, 95-99, and above 99 for N-BMI, OB, and MO, respectively. Criteria for MetS findings were determined. Routine biochemical analyses, including zinc, were performed. High sensitive-cTnT and cMyBP-C concentrations were measured by kits based on enzyme-linked immunosorbent assay principle. Appropriate statistical tests within the scope of SPSS were used for the evaluation of the study data. p<0.05 was accepted as statistically significant. Four groups were matched for age and gender. Decreased zinc concentrations were measured in Groups 2, 3, and 4 compared to Group 1. Groups did not differ from one another in terms of hs-cTnT. There were statistically significant differences between cMyBP-C levels of MetS group and N-BMI as well as OB groups. There was an increasing trend going from N-BMI group to MetS group. There were statistically significant negative correlations between zinc and hs-cTnT as well as cMyBP-C concentrations in MetS group. In conclusion, inverse correlations detected between zinc and new generation cardiac markers (hs-TnT and cMyBP-C) have pointed out that decreased levels of this physiologically essential trace element accompany increased levels of hs-cTnT as well as cMyBP-C in children with MetS. This finding emphasizes that both zinc and these new generation cardiac markers may be evaluated as biomarkers of cardiovascular health during severe childhood obesity precipitated with MetS findings and also suggested as the messengers of the future risk in the adulthood periods of children with MetS.

Keywords: cardiac myosin binding protein-C, cardiovascular diseases, children, high sensitive cardiac troponin T, obesity

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Authors: Ashish Shukla, K. P. Mishra, Pushplata Tripathi

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This paper investigates the production and optimization of β-galactosidase enzyme using synthetic medium by isolated wild strains (S1, S2) mutated strains (M1, M2) through SSF and SmF. Among the different cell disintegration methods used, the highest specific activity was obtained when the cells were permeabilized using isoamyl alcohol. Wet lab experiments were performed to investigate the effects of carbon and nitrogen substrates present in Vogel’s medium on β-galactosidase enzyme activity using S1, S2, and M1, M2 strains through SSF. SmF experiments were performed for effects of carbon and nitrogen sources in YLK2Mg medium on β-galactosidase enzyme activity using S1, S2 and M1, M2 strains. Effect of pH on β-galactosidase enzyme production was also done using S1, S2, and M1, M2 strains. Results were found to be very appreciable in all the cases.

Keywords: β-galactosidase, cell disintegration, permeabilized, SSF, SmF

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Authors: Nuha Mansour Alhazmi

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Replacement of petro-based plastics by a biodegradable plastic are vastly growing process. Poly-β-hydroxybutyrate (PHB) is a biodegradable biopolymer, synthesized by some bacterial genera. The objective of the current study is to explore the ability of some fungi to biodegrade PHB. The degradation of (PHB) was detected in Petri dish by the formation of a clear zone around the fungal colonies due to the production of depolymerase enzyme which has an interesting role in the PHB degradation process. Among 10 tested fungi, the most active PHB biodegraded fungi were identified as Trichoderma asperellum using morphological and molecular characters. The highest PHB degradation was at 25°C, pH 7.5 after 7 days of incubation for the tested fungi. Finally, the depolymerase enzyme was isolated, purified using column chromatography and characterized. In conclusion, PHB can be biodegraded in solid and liquid medium using depolymerase enzyme from T. asperellum.

Keywords: degradation, depolymerase enzyme, PHB, Trichoderma asperellum

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Authors: Jamal Abd Awadallak, Thiago Olinek Reinehr, Eduardo Raizer, Deise Molinari, Edson Antonio, Camila da Silva da Silva

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The hydrolysis of soy oil catalyzed by 1,3-specific enzyme (Lecitase Ultra) in a well-stirred bioreactor was studied. Two forms of applications of the ultrasound were evaluated aiming to increase reaction rates, wherein the use of probe ultrasound associated with the use of surfactant to pre-emulsify the substrate showed the best results. Two different reaction periods were found: the first where the ultrasound has great influence on reaction rates, and the second where ultrasound influence is minimal. Studies on the time of pre-emulsification, surfactant concentration and enzyme concentration showed that the initial rate of hydrolysis depends on the interfacial area between the oil phase and the aqueous phase containing the enzyme.

Keywords: specific enzyme, free fatty acids, Hydrolysis, lecitase ultra, ultrasound

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Authors: Misha Ali, Qayyum Husain, Nida Alam, Masood Ahmad

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Improving the activity and stability of the enzyme is an important aspect in bioremediation processes. Immobilization of enzyme is an efficient approach to amend the properties of biocatalyst required during wastewater treatment. The present study was done to immobilize partially purified ginger peroxidase on amino functionalized silica coated titanium dioxide nanocomposite. Interestingly there was an enhancement in enzyme activity after immobilization on nanosupport which was evident from effectiveness factor (η) value of 1.76. Immobilized enzyme was characterized by transmission electron microscopy, scanning electron microscopy and Fourier transform infrared spectroscopy. Immobilized peroxidase exhibited higher activity in a broad range of pH and temperature as compared to free enzyme. Also, the thermostability of peroxidase was strikingly improved upon immobilization. After six repeated uses, the immobilized peroxidase retained around 62% of its dye decolorization activity. There was a 4 fold increase in Vmax of immobilized peroxidase as compared to free enzyme. Circular dichroism spectroscopy demonstrated conformational changes in the secondary structure of enzyme, a possible reason for the enhanced enzyme activity after immobilization. Immobilized peroxidase was highly efficient in the removal of acid yellow 42 dye in a stirred batch process. Our study shows that this bio-remediating system has remarkable potential for treatment of aromatic pollutants present in wastewater.

Keywords: acid yellow 42, decolorization, ginger peroxidase, immobilization

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Authors: Suphalucksana, Wichai, Sangsoponjit Settasit, Soytong Kasem

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A study on the efficiency for enzyme production of fungi isolated from stomach of buffalo was conducted. The fungi were collected from 4 parts of stomach as rumen, reticulum, omasum and abomasums. The objective to study the efficiency of fungi from stomach of buffalo had effected to produced enzyme and to selected fungi for their ability to produced enzyme cellulase, hemicellulase and ligninase. Results shown that the fungi isolated from rumen were: Eupenicillium sp. (B-RU-01-1), Eupenicillium sp. (B-RU-02-3G), Rhyzopus stolonifer (B-RU-01-4) and Trichoderma sp. (B-RU-01-2). From the reticulum, Aspergillus glaucus (B-RET-02-3), Aspergillus orchraceus (B-RET-02-2) and Penicillium sp. (B-RET-02-4) were found. In the omasum Aspergillus fumigatus (B-OMA-01-1G), Eurotium sp. (B-OMA-01-4) and Rhizopus stolonifer (B-OMA-02-3) were isolated and in the abomasums Aspergillus flavas (B-ABO-02-3), Aspergillus fumigatus (B-ABO-02-1), Aspergillus niger (B-ABO-01-3G), Aspergillius terreus (B-ABO-02-4) and Mucor sp. (B-ABO-02-4G). Results of enzyme analysis revealed that cellulase was produced by isolated: Eupenicillium sp. (B-RU-02-3G), Eupenicillium sp. (B-RU-01-1), Penicillium sp. (B-RET-02-4), Aspergillius glaucus (B-RET-02-3), Aspergillus ochraceus (B-RET-02-2), Aspergillius fumigatus (B-OMA-01-1G), Eurotium sp. (B-OMA-01-4), Aspergillius flavus (B-ABO-02-3), Aspergillius fumigatus (B-ABO-02-1), Aspergillius niger (B-ABO-01-3G), Aspergillius terreus (B-ABO-02-4). Hemicellulase was produced Eupenicillium sp. (B-RU-02-3G), Eupenicillium sp. (B-RU-01-1), Rhizopus stolonifer (B-RU-01-4), Trichoderma sp. (B-RU-01-2), Aspergillius glaucus (B-RET-02-3), Aspergillus ochraceus (B-RET-02-2), Penicillium sp. (B-RET-02-4), Aspergillius fumigatus (B-OMA-01-1G), Eurotium sp. (B-OMA -01-4), Aspergillius flavus (B-ABO-02-3), Aspergillius fumigatus (B-ABO-02-1) Aspergillius niger (B-ABO-01-3G), Aspergillius terreus (B-ABO-02-4), Mucor sp. (B-ABO-02-4G). For the enzyme ligninase, two isolates were found to produced this enzyme namely : Trichoderma sp. (B-RU-01-2) and Mucor sp. (B-ABO-02-4G).

Keywords: enzyme production from fungi, enzyme production, fungi, agricultural technology

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Authors: Kashif Ahmed

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Recent years researchers have been motivated towards extensive exploring of living organism, which could be utilized effectively in intense industrial conditions. The present study shows enhanced production, purification and characterization of industrial enzyme, invertase (Beta-D-fructofuranosidase) from Penicillium lilacinum. Various agricultural based by-products (cotton stalk, sunflower waste, rice husk, molasses and date syrup) were used as energy source. The highest amount of enzyme (13.05 Units/mL) was produced when the strain was cultured on growth medium containing date syrup as energy source. Yeast extract was used as nitrogen source after 96 h of incubation at incubation temperature of 40º C. Initial pH of medium was 8.0, inoculum size 6x10⁶ conidia and 200 rev/min agitation rate. The enzyme was also purified (7 folds than crude) and characterized. Molecular mass of purified enzyme (65 kDa) was determined by 10 % SDS-PAGE. Lineweaver-Burk Plot was used to determine Kinetic constants (Vmax 178.6 U/mL/min and Km 2.76 mM). Temperature and pH optima were 55º C and 5.5 respectively. MnCl₂ (52.9 %), MgSO₄ (48.9 %), BaCl₂ (24.6 %), MgCl₂ (9.6 %), CoCl₂ (5.7 %) and NaCl (4.2 %) enhanced the relative activity of enzyme and HgCl₂ (-92.8 %), CuSO₄ (-80.2 %) and CuCl₂ (-76.6 %) were proved inhibitors. The strain was showing enzyme activity even at extreme conditions of temperature (up to 60º C) and pH (up to 9), so it can be used in industries.

Keywords: invertase, Penicillium lilacinum, submerged fermentation, industrial enzyme

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Authors: Sibashish Baksi, Ujjaini Sarkar, Sudeshna Saha

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In the present study, hydrolysis of Pinus roxburghi wood powder was carried out with Viscozyme, and kinetics of the hydrolysis has been investigated. Finely ground sawdust is submerged into 2% aqueous peroxide solution (pH=11.5) and pretreated through autoclaving, probe sonication, and alkaline peroxide pretreatment. Afterward, the pretreated material is subjected to hydrolysis. A chain of experiments was executed with delignified biomass (50 g/l) and varying enzyme concentrations (24.2–60.5 g/l). In the present study, 14.32 g/l of glucose, along with 7.35 g/l of xylose, have been recovered with a viscozyme concentration of 48.8 g/l and the same condition was treated as optimum condition. Additionally, thermal deactivation of viscozyme has been investigated and found to be gradually decreasing with escalated enzyme loading from 48.4 g/l (dissociation constant= 0.05 h⁻¹) to 60.5 g/l (dissociation constant= 0.02 h⁻¹). The hydrolysis reaction is a pseudo first-order reaction, and therefore, the rate of the hydrolysis can be expressed as a fractal-like kinetic equation that communicates between the product concentration and hydrolytic time t. It is seen that the value of rate constant (K) increases from 0.008 to 0.017 with augmented enzyme concentration from 24.2 g/l to 60.5 g/l. Greater value of K is associated with stronger enzyme binding capacity of the substrate mass. However, escalated concentration of supplied enzyme ensures improved interaction with more substrate molecules resulting in an enhanced de-polymerization of the polymeric sugar chains per unit time which eventually modifies the physiochemical structure of biomass. All fractal dimensions are in between 0 and 1. Lower the value of fractal dimension, more easily the biomass get hydrolyzed. It can be seen that with increased enzyme concentration from 24.2 g/l to 48.4 g/l, the values of fractal dimension go down from 0.1 to 0.044. This indicates that the presence of more enzyme molecules can more easily hydrolyze the substrate. However, an increased value has been observed with a further increment of enzyme concentration to 60.5g/l because of diffusional limitation. It is evident that the hydrolysis reaction system is a heterogeneous organization, and the product formation rate depends strongly on the enzyme diffusion resistances caused by the rate-limiting structures of the substrate-enzyme complex. Value of the rate constant increases from 1.061 to 2.610 with escalated enzyme concentration from 24.2 to 48.4 g/l. As the rate constant is proportional to Fick’s diffusion coefficient, it can be assumed that with a higher concentration of enzyme, a larger amount of enzyme mass dM diffuses into the substrate through the surface dF per unit time dt. Therefore, a higher rate constant value is associated with a faster diffusion of enzyme into the substrate. Regression analysis of time curves with various enzyme concentrations shows that diffusion resistant constant increases from 0.3 to 0.51 for the first two enzyme concentrations and again decreases with enzyme concentration of 60.5 g/l. During diffusion in a differential scale, the enzyme also experiences a greater resistance during diffusion of larger dM through dF in dt.

Keywords: viscozyme, glucose, fractal kinetics, thermal deactivation

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Authors: G. K. Sruthi, Sila Bhattacharya

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Sorghum is an important food crop in the dry tropical areas of the world, and possesses significant levels of phytochemicals and dietary fiber to offer health benefits. However, the absence of gluten is a limitation for converting the sorghum dough into sheeted/flattened/rolled products. Chapathi/roti (flat unleavened bread prepared conventionally from whole wheat flour dough) was attempted from sorghum as wheat gluten causes allergic reactions leading to celiac disease. Dynamic oscillatory rheology of sorghum flour dough (control sample) and enzyme treated sorghum doughs were studied and linked to the attributes of the finished ready-to-eat product. Enzymes like amylase, xylanase, and a mix of amylase and xylanase treated dough affected drastically the rheological behaviour causing a lowering of dough consistency. In the case of amylase treated dough, marked decrease of the storage modulus (G') values from 85513 Pa to 23041 Pa and loss modulus (G") values from 8304 Pa to 7370 Pa was noticed while the phase angle (δ) increased from 5.6 to 10.1o for treated doughs. There was a 2 and 3 fold increase in the total sugar content after α-amylase and xylanase treatment, respectively, with simultaneous changes in the structure of the dough and finished product. Scanning electron microscopy exhibited enhanced extent of changes in starch granules. Amylase and mixed enzyme treatment produced a sticky dough which was difficult to roll/flatten. The dough handling properties were improved by the use of xylanase and quality attributes of the chapath/roti. It is concluded that enzyme treatment can offer improved rheological status of gluten free doughs and products.

Keywords: sorghum dough, amylase, xylanase, dynamic oscillatory rheology, sensory assessment

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Authors: Shweta Sinha, Mukesh Doble, Manju S. L.

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Pyrazole scaffold is an important group of compound in heterocyclic chemistry and is found to possess numerous uses in chemistry. Pyrazole derivatives are also known to possess important biological activities including antitumor, antimicrobial, antiviral, antifungal, anticancer and anti-inflammatory. Inflammation is associated with pain, allergy and asthma. Leukotrienes are mediators of various inflammatory and allergic disorders. 5-Lipoxygenase (5-LOX) is an important enzyme involved in the biosynthesis of leukotrienes and metabolism of arachidonic acid (AA) and thus targeted for anti-inflammation. In vitro inhibitory activity of pyrazol-3-yl thiazole 4-carboxylic acid derivatives is tested against enzyme 5-LOX. Most of these compounds exhibit good inhibitory activity against this enzyme. Binding mode study of these compounds is determined by computational tool. Further experiments are being done to understand the mechanism of action of these compounds in inhibiting this enzyme. To conclude, these compounds appear to be a promising target in drug design against 5-LOX.

Keywords: inflammation, inhibition, 5-lipoxygenase, pyrazole

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Authors: Parmjit S. Panesar, Rupinder Kaur, Ram S. Singh

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Enzymes are the biocatalysts which catalyze the biochemical processes and thus have a wide variety of applications in the industrial sector. β-Galactosidase (E.C. 3.2.1.23) also known as lactase, is one of the prime enzymes, which has significant potential in the dairy and food processing industries. It has the capability to catalyze both the hydrolytic reaction for the production of lactose hydrolyzed milk and transgalactosylation reaction for the synthesis of prebiotics such as lactulose and galactooligosaccharides. These prebiotics have various nutritional and technological benefits. Although, the enzyme is naturally present in almonds, peaches, apricots and other variety of fruits and animals, the extraction of enzyme from these sources increases the cost of enzyme. Therefore, focus has been shifted towards the production of low cost enzyme from the microorganisms such as bacteria, yeast and fungi. As compared to yeast and bacteria, fungal β-galactosidase is generally preferred as being extracellular and thermostable in nature. Keeping the above in view, the present study was carried out for the isolation of the β-galactosidase producing fungal strain from the food as well as the agricultural wastes. A total of more than 100 fungal cultures were examined for their potential in enzyme production. All the fungal strains were screened using X-gal and IPTG as inducers in the modified Czapek Dox Agar medium. Among the various isolated fungal strains, the strain exhibiting the highest enzyme activity was chosen for further phenotypic and genotypic characterization. The strain was identified as Rhizomucor pusillus on the basis of 5.8s RNA gene sequencing data.

Keywords: beta-galactosidase, enzyme, fungal, isolation

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Authors: Leila Mosafa, Majid Moghadam, Mohammad Shahedi

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In this work, pectinase was immobilized on the surface of silica-coated magnetite nanoparticles via covalent attachment. The magnetite-immobilized enzyme was characterized by Fourier transform infrared spectroscopy, X-ray powder diffraction, scanning electron microscopy and vibrating sample magnetometry techniques. Response surface methodology using Minitab Software was applied for statistical designing of operating conditions in order to immobilize pectinase on magnetic nanoparticles. The optimal conditions were obtained at 30°C and pH 5.5 with 42.97 µl pectinase for 2 h. The immobilization yield was 50.6% at optimized conditions. Compared to the free pectinase, the immobilized pectinase was found to exhibit enhanced enzyme activity, better tolerance to the variation of pH and temperature, and improved storage stability. Both free and immobilized samples reduced the viscosity of apple juice from 1.12 to 0.88 and 0.92 mm2s-1, respectively, after 30 min at their optimum temperature. Furthermore, the immobilized enzyme could be reused six consecutive cycles and the efficiency loss in viscosity reduction was found to be only 8.16%.

Keywords: magnetite nanoparticles, pectinase enzyme, immobilization, juice clarification, enzyme activity

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Authors: Mohammad Alkhatatbeh, Nehad Ayoub, Nizar Mhaidat, Nesreen Saadeh, Lisa Lincz

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CD36 is involved in the development of atherosclerosis by enhancing macrophage endocytosis of oxidized-low density lipoproteins and foam cell formation. Soluble CD36 (sCD36) was found to be elevated in type 2 diabetic patients and was supposed to act as a marker of insulin resistance and atherosclerosis. In young subjects, sCD36 was associated with cardiovascular risk factors including obesity and hypertriglyceridemia. This study was conducted to further investigate the relationship between plasma sCD36 and cardiovascular risk factors among middle-aged patients with metabolic syndrome (MetS) and healthy controls. SCD36 concentrations were determined by enzyme-linked immunosorbent assays (ELISA) for 41 patients with MetS and 36 healthy controls. Data for other variables were obtained from patients' medical records. SCD36 concentrations were relatively low compared to most other studies and were not significantly different between the MetS group and controls (P-value=0.17). SCD36 was also not correlated with age, body mass index, glucose, lipid profile, serum electrolytes and blood counts. SCD36 was not significantly different between subjects with obesity, hyperglycemia, dyslipidemia, hypertension or cardiovascular disease and those without these abnormalities (P-value > 0.05). The inconsistency between results reported in this study and other studies may be unique to the study population or be a result of the lack of a reliable standardized method for determining absolute sCD36 concentrations. However, further investigations are required to assess CD36 tissue expression in the study population and to assess the accuracy of various commercially available sCD36 ELISA kits. Thus, the availability of a standardized simple sCD36 ELISA that could be performed in any basic laboratory would be more favorable to the specialized flow cytometry methods that detect CD36+ microparticles if it was to be used as a biomarker.

Keywords: metabolic syndrome, CD36, cardiovascular risk, obesity, type 2 diabetes mellitus

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Authors: Z. Yousefniayejahr, S. Bolognini, A. Bonini, C. Campobasso, N. Poma, F. Vivaldi, M. Di Luca, A. Tavanti, F. Di Francesco

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Pathogenic bacteria represent a threat to healthcare systems and the food industry, because their rapid detection remains challenging. Electrochemical biosensors are gaining prominence as a novel technology for the detection of pathogens due to intrinsic features such as low cost, rapid response time, and portability, which make them a valuable alternative to traditional methodologies. These sensors use biorecognition elements that are crucial for the identification of specific bacteria. In this context, bacteriophages are promising tools for their inherent high selectivity towards bacterial hosts, which is of fundamental importance when detecting bacterial pathogens in complex biological samples. In this study, we present the development of a low-cost and portable sensor based on the Zeno phage for the rapid detection of Staphylococcus aureus. Screen-printed gold electrodes functionalized with the Zeno phage were used, and electrochemical impedance spectroscopy was applied to evaluate the change of the charge transfer resistance (Rct) as a result of the interaction with S. aureus MRSA ATCC 43300. The phage-based biosensor showed a linear range from 101 to 104 CFU/mL with a 20-minute response time and a limit of detection (LOD) of 6 CFU/mL under physiological conditions. The biosensor’s ability to recognize various strains of staphylococci was also successfully demonstrated in the presence of clinical isolates collected from different geographic areas. Assays using S. epidermidis were also carried out to verify the species-specificity of the phage sensor. We only observed a remarkable change of the Rct in the presence of the target S. aureus bacteria, while no substantial binding to S. epidermidis occurred. This confirmed that the Zeno phage sensor only targets S. aureus species within the genus Staphylococcus. In addition, the biosensor's specificity with respect to other bacterial species, including gram-positive bacteria like Enterococcus faecium and the gram-negative bacterium Pseudomonas aeruginosa was evaluated, and a non-significant impedimetric signal was observed. Notably, the biosensor successfully identified S. aureus bacterial cells in a complex matrix such as a nasal swab, opening the possibility of its use in a real-case scenario. We diluted different concentrations of S. aureus from 108 to 100 CFU/mL with a ratio of 1:10 in the nasal swap matrices collected from healthy donors. Three different sensors were applied to measure various concentrations of bacteria. Our sensor indicated high selectivity to detect S. aureus in biological matrices compared to time-consuming traditional methods, such as enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and radioimmunoassay (RIA), etc. With the aim to study the possibility of using this biosensor to address the challenge associated with pathogen detection, ongoing research is focused on the assessment of the biosensor’s analytical performances in different biological samples and the discovery of new phage bioreceptors.

Keywords: electrochemical impedance spectroscopy, bacteriophage, biosensor, Staphylococcus aureus

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Authors: Munisath Khandoker, Stephan Haefele, Andy Gregory

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Extracellular enzymes play a key role in soil organic carbon (SOC) decomposition and nutrient cycling and are known indicators for soil health; however, it is not understood how these enzymes respond to different land uses and their relationships to other soil properties have not been extensively reviewed. The relationships among the activities of three soil enzymes: β-glucosaminidase (NAG), phosphomonoesterase (PHO) and β-glucosidase (GLU), were examined. The impact of soil organic amendments, soil types and land management on soil enzyme activities were reviewed, and it was hypothesized that soils with increased SOC have increased enzyme activity. Long-term experiments at Rothamsted Research Woburn and Harpenden sites in the UK were used to evaluate how different management practices affect enzyme activity involved in carbon (C) and nitrogen (N) cycling in the soil. Samples were collected from soils with different organic treatments such as straw, farmyard manure (FYM), compost additions, cover crops and permanent grass cover to assess whether SOC can be linked with increased levels of enzymatic activity and what influence, if any, enzymatic activity has on total C and N in the soil. Investigating the interactions of important enzymes with soil characteristics and SOC can help to better understand the health of soils. Studies on long-term experiments with known histories and large datasets can better help with this. SOC tends to decrease during land use changes from natural ecosystems to agricultural systems; therefore, it is imperative that agricultural lands find ways to increase and/or maintain SOC in the soil.

Keywords: biological soil health indicators, extracellular enzymes, soil health, soil, microbiology

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Authors: Kazam Ali, Muhammad Sagheer, Mansoor-Ul- Hasan, Abdul Rashid, Chaudhary Muhammad Shahid Hanif, Fawad Zafar Ahmad Khan, Hafiz Muhammad Aatif

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Natural extracts of two medicinal plants Nicotiana tabacum and Eucalyptus globulus were tested for their toxic and enzyme inhibition effects against three insects species of stored grains Tribolium castaneum, Trogoderma granarium and Sitophilus granarius. Responses of insects varied with exposure periods and dilution levels of acetone extracts of plants. Both plant extracts were lethal to insects but the crude leaf extract of N. tabacum evidenced strong toxic action against three tested insect species. Maximum mortality 36.30% in S. granarius, 25.96% in T. castaneum, and 21.88% in T. granarium were found at 20% dilution level, after 10 days exposure to botanical extract of N. tabacum. The impact of N. tabacum and E. globulus on the activity of esterases; acetylcholinesterase (AChE), α-carboxylesterase (α-CE), β-carboxylesterase (β-CE) and phosphatses; acid phosphatase (AcP), alkaline phosphatase (AlP) of three stored grain insect species were also studied in the survivors of toxicity assay. Whole body homogenates of insects were used for enzyme determination and consumption of high dose rate N. tabacum extract containing diet resulted in maximum 55.33% inhibition of AChE and 26.17% AlP inhibition in T. castaneum, while 44.17% of α-CE and 31.67% inhibition of β-CE activity were noted in S. granarius. Maximum inhibition 23.44% of AcP activity was found in T. granarium exposed to diet treated with the extract of E. globulus. The findings indicate that acetone extracts of N. tabacum and E. globulus are naturally occurring pesticide and facts of the enzyme inhibition relations specify that their effect changes with the insect species.

Keywords: natural extract, medicinal plant, toxic effects, enzyme inhibition, acetone extract

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Authors: Afsheen Aman, Zainab Bibi, Shah Ali Ul Qader

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Introduction: Xylan is a heteropolysaccharide composed of xylose monomers linked together through 1,4 linkages within a complex xylan network. Owing to wide applications of xylan hydrolytic products (xylose, xylobiose and xylooligosaccharide) the researchers are focusing towards the development of various strategies for efficient xylan degradation. One of the most important strategies focused is the use of heat tolerant biocatalysts which acts as strong and specific cleaving agents. Therefore, the exploration of microbial pool from extremely diversified ecosystem is considerably vital. Microbial populations from extreme habitats are keenly explored for the isolation of thermophilic entities. These thermozymes usually demonstrate fast hydrolytic rate, can produce high yields of product and are less prone to microbial contamination. Another possibility of degrading xylan continuously is the use of immobilization technique. The current work is an effort to merge both the positive aspects of thermozyme and immobilization technique. Methodology: Geobacillus stearothermophilus was isolated from soil sample collected near the blast furnace site. This thermophile is capable of producing thermostable endo-β-1,4-xylanase which cleaves xylan effectively. In the current study, this thermozyme was immobilized within a synthetic and a non-synthetic matrice for continuous production of metabolites using entrapment technique. The kinetic parameters of the free and immobilized enzyme were studied. For this purpose calcium alginate and polyacrylamide beads were prepared. Results: For the synthesis of immobilized beads, sodium alginate (40.0 gL-1) and calcium chloride (0.4 M) was used amalgamated. The temperature (50°C) and pH (7.0) optima of immobilized enzyme remained same for xylan hydrolysis however, the enzyme-substrate catalytic reaction time raised from 5.0 to 30.0 minutes as compared to free counterpart. Diffusion limit of high molecular weight xylan (corncob) caused a decline in Vmax of immobilized enzyme from 4773 to 203.7 U min-1 whereas, Km value increased from 0.5074 to 0.5722 mg ml-1 with reference to free enzyme. Immobilized endo-β-1,4-xylanase showed its stability at high temperatures as compared to free enzyme. It retained 18% and 9% residual activity at 70°C and 80°C, respectively whereas; free enzyme completely lost its activity at both temperatures. The Immobilized thermozyme displayed sufficient recycling efficiency and can be reused up to five reaction cycles, indicating that this enzyme can be a plausible candidate in paper processing industry. Conclusion: This thermozyme showed better immobilization yield and operational stability with the purpose of hydrolyzing the high molecular weight xylan. However, the enzyme immobilization properties can be improved further by immobilizing it on different supports for industrial purpose.

Keywords: immobilization, reusability, thermozymes, xylanase

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Authors: Mustafa Metin Donma, Orkide Donma

Abstract:

Childhood obesity is an important clinical problem because it may lead to chronic diseases during the adulthood period of the individual. Obesity is a metabolic disease associated with low-grade inflammation. The liver occurs at the center of metabolic pathways. Adropin, fibroblast growth factor-21 (FGF-21), and fetuin-A are hepatokines. Due to the immense participation of the liver in glucose metabolism, these liver-derived factors may be associated with insulin resistance (IR), which is a phenomenon discussed within the scope of obesity problems. The aim of this study is to determine the concentrations of adropin, FGF-21, and fetuin-A in childhood obesity, to point out possible differences between the obesity groups, and to investigate possible associations among these three hepatokines in obese and morbidly obese children. A total of one hundred and thirty-two children were included in the study. Two obese groups were constituted. The groups were matched in terms of mean ± SD values of ages. Body mass index values of obese and morbidly obese groups were 25.0 ± 3.5 kg/m² and 29.8 ± 5.7 kg/m², respectively. Anthropometric measurements including waist circumference, hip circumference, head circumference, and neck circumference were recorded. Informed consent forms were taken from the parents of the participants. The ethics committee of the institution approved the study protocol. Blood samples were obtained after overnight fasting. Routine biochemical tests, including glucose- and lipid-related parameters, were performed. Concentrations of the hepatokines (adropin, FGF-21, fetuin A) were determined by enzyme-linked immunosorbent assay. Insulin resistance indices such as homeostasis model assessment for IR (HOMA-IR), alanine transaminase-to aspartate transaminase ratio (ALT/AST), diagnostic obesity notation model assessment laboratory index, diagnostic obesity notation model assessment metabolic syndrome index as well as obesity indices such as diagnostic obesity notation model assessment-II index, and fat mass index were calculated using the previously derived formulas. Statistical evaluation of the study data as well as findings of the study was performed by SPSS for Windows. Statistical difference was accepted significant when p is smaller than 0.05. Statistically significant differences were found for insulin, triglyceride, high-density lipoprotein cholesterol levels of the groups. A significant increase was observed for FGF-21 concentrations in the morbidly obese group. Higher adropin and fetuin-A concentrations were observed in the same group in comparison with the values detected in the obese group (p > 0.05). There was no statistically significant difference between the ALT/AST values of the groups. In all of the remaining IR and obesity indices, significantly increased values were calculated for morbidly obese children. Significant correlations were detected between HOMA-IR and each of the hepatokines. The highest one was the association with fetuin-A (r=0.373, p=0.001). In conclusion, increased levels observed in adropin, FGF-21, and fetuin-A have shown that these hepatokines possess increasing potential going from obese to morbid obese state. Out of the correlations found with the IR index, the most affected hepatokine was fetuin-A, the parameter possibly used as the indicator of the advanced obesity stage.

Keywords: adropin, fetuin A, fibroblast growth factor-21, insulin resistance, pediatric obesity

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Authors: Dina Helmy El-Ghonemy, Thanaa Hamed Ali

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The aim of this study was to purify and characterize a keratinolytic enzyme produced by Aspergillus sp. DHE7 cultured in basal medium containing chicken feather as substrate. The enzyme was purified through ammonium sulfate saturation of 60%, followed by gel filtration chromatography in Sephadex G-100, with a 16.4-purification fold and recovery yield of 52.2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified enzyme is a monomeric enzyme with an apparent molecular mass of 30 kDa — the purified keratinase of Aspergillus sp. DHE7 exhibited activity in a broad range of pH (7- 9) and temperature (40℃-60℃) profiles with an optimal activity at pH eight and 50℃. The keratinolytic activity was inhibited by protease inhibitors such as phenylmethylsulfonyl fluoride and ethylenediaminetetraacetate, while no reduction of activity was detected by the addition of dimethyl sulfoxide (DMSO). Bivalent cations, Ca²⁺ and Mn²⁺, were able to greatly enhance the activity of keratinase by 125.7% and 194.8%, respectively, when used at one mM final concentration. On the other hand, Cu²⁺ and Hg²⁺ inhibited the enzyme activity, which might be indicative of essential vicinal sulfhydryl groups of the enzyme for productive catalysis. Furthermore, the purified keratinase showed significant stability and compatibility against the tested commercial detergents at 37ºC. Therefore, these results suggested that the purified keratinase from Aspergillus sp. DHE7 may have potential use in the detergent industry and should be of interest in the processing of poultry feather waste.

Keywords: Aspergillus sp. DHE7, biochemical characterization, keratinase, purification, waste management

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Authors: Ghazala Yaqub, Zubi Sadiq, Almas Hamid, Saira Iqbal

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This paper is the very first report of three dual action hybrids synthesized by covalent linkage of carbazole based novel antibacterial compounds with efflux pump inhibitors i.e., indole acetic acid/gallic acid. Novel carbazole based antibacterial compounds were prepared first and then these were covalently linked with efflux pump inhibitors which leads to the successful formation of hybrids. All prepared compounds were evaluated for their bacterial cell killing capability against Escherichia coli, Staphylococcus aureus, Pasteurella multocida and Bacillus subtilis. Compound were effective against all tested bacterial strains at different concentrations. But when these compounds were linked with efflux pump inhibitors they showed dramatic enhancement in their bacterial cell killing potential and minimum inhibitory concentration of all hybrids ranges from 7.250 µg/mL to 0.0283 µg/mL.

Keywords: antimicrobial assay, carbazole, dual action hybrids, efflux pump inhibitors

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Authors: Shanakr Bhattarai, V. S. Tiwari, Barak Akabayov

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The plummeting number of infections and death is due to the development of drug-resistant bacteria. In addition, the number of approved antibiotic drugs by the Food and Drug Administration (FDA) is insufficient. Therefore, developing new drugs and finding novel targets for central metabolic pathways in bacteria is urgently needed. One of the promising targets is DNA replication machinery which consists of many essential proteins and enzymes. DnaG primase is an essential enzyme and a central part of the DNA replication machinery. DnaG primase synthesizes short RNA primers that initiate the Okazaki fragments by the lagging strand DNA polymerase. Therefore, it is reasonable to assume that inhibition of primase activity will stall DNA replication and prevent bacterial proliferation. We did the expression and purification of eight different bacterial DnaGs (Mycobacterium tuberculosis(Mtb), Bacillus anthracis (Ba), Mycobacterium smegmatis (Msmeg), Francisella tularencis (Ft), Vibrio cholerae (Vc) and Yersinia pestis (Yp), Staphylococcus aureus(Saureus), Escherichia coli(Ecoli)) followed by the radioactive activity assay. After obtaining the pure and active protein DnaG, we synthesized the inhibitors for them. The inhibitors were divided into five different groups, each containing five molecules, and the cocktail inhibition assay was performed against each DnaGs. The groups of molecules inhibiting the DnaGs were further tested with individual molecules belonging to inhibiting groups. Each molecule showing inhibition was titrated against the corresponding DnaGs to find IC50. We got a molecule(VS167) that acted as broad inhibitors, inhibiting all eight DnaGs. Molecules VS180 and VS186 inhibited seven DnaGs (except Saureus). Similarly, two molecules(VS 173, VS176) inhibited five DnaGs (Mtb, Ba, Ft, Yp, Ecoli). VS261 inhibited four DnaGs (Mtb, Ba, Ft, Vc). MS50 inhibited Ba and Vc DnaGs. And some of the inhibitors inhibited only one DnaGs. Thus we found the broad and specific inhibitors for different bacterial DnaGs, and their Structure-activity analysis(SAR) was done. Further, We tried to explain the similarities among the enzyme DnaGs from different bacteria based on their inhibition pattern.

Keywords: DNA replication, DnaG, okazaki fragments, antibiotic drugs

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Authors: Kriti Upadhyay, Ashraf Ali, Puja Sohal, Randeep Guleria

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Background: In Chronic Obstructive Pulmonary diseases (COPD) pathogenesis oxidative stress plays an important role. Hypoxia-Inducible factor (HIF-1α) is a dimeric protein complex which Functions as a master transcriptional regulator of the adaptive response to hypoxiaand is a risk factor that increases when oxidative stress triggers. The role ofHIF-1αin COPD due to smoking is lacking. Aim: This study aims to evaluate the role of HIF-1α in smoker COPD patients comparing its association with diseases severity. Method: In this cross-sectional study, we recruited 87 subjects, 57 were smokers with COPD,15 were smokers without COPD and other 15 were non-smoker healthy controls. The mean age was 54.6± 9.32 (cases 57.08±8.15; controls 50.0± 9.8). There were 62%smokers, 25% non-smokers,7% tobacco chewers and 6% ex-smokers. Enzyme-linked immune sorbent assay (ELISA) method was used for analyzing serum samples wherein HIF-1α was analyzed by Sandwich-ELISA. Results: In smoker COPD patients, a significantly higher HIF-1α level showed positive association with hypoxia, smoking status and severity of disease (p=0.03). The mean value of HIF-1α was not significantly different in smokers without COPD and healthy controls. Conclusion: It is found that HIF-1α level was increased in smoker COPD, but not in smokers without COPD. This suggests that development of COPD drive the HIF-1α pathway and it correlates with the severity of diseases.

Keywords: COPD, chronic obstructive pulmonary diseases, smokers, nonsmokers, hypoxia

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Authors: Sehrish Iftikhar, Ahmad Ali Shahid, Kiran Nawaz, Waheed Anwar

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Discovery and development of new compounds require intense studies in chemistry, biochemistry. Numerous experiments under laboratory-, greenhouse- and field conditions can be performed to select suitable candidates and to understand their full potential. Novel fungicides are fundamental to combat plant diseases. Fusarium solani is important plant pathogen. New broad spectrum foliar fungicides against complex II were designed in this study. Complex II, namely succinate dehydrogenase (SDH), or succinate quinone oxidoreductase (SQR) is a multi-subunit enzyme at the crossroads of TCA and ETC at the inner mitochondrial membrane. The need for new and innovative fungicides is driven by resistance management, regulatory hurdles and increasing customer expectations amongst others. Fungicidal activity was assessed for the effect on mycelial growth and spore germination of the fungi using fungicide amended media assay. In mycelial growth assay compounds C10 and C6 were highly active against all the isolates. The compounds C1 and C10 were found most potent in spore germination test. It fully proved that the SDHIs designed in this paper displayed as good inhibitory effects against Fusarium solani.

Keywords: Wilt, Fusarium, SDH, antifungal

Procedia PDF Downloads 226

Authors: Anahita Izadyar, My Ni Van, Kayleigh Amber Rodriguez, Ilwoo Seok, Elizabeth E. Hood

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Using a recombinant enzyme derived from corn and a simple modification, we fabricated a facile, fast, and cost-beneficial biosensor to measure glucose. The Nafion/ Plant Produced Mn Peroxidase (PPMP)– glucose oxidase (GOx)- Bovine serum albumin (BSA) /Au electrode showed an excellent amperometric response to detect glucose. This biosensor is capable of responding to a wide range of glucose—20.0 µM−15.0 mM and has a lower detection limit (LOD) of 2.90µM. The reproducibility response using six electrodes is also very substantial and indicates the high capability of this biosensor to detect a wide range of 3.10±0.19µM to 13.2±1.8 mM glucose concentration. Selectivity of this electrode was investigated in an optimized experimental solution contains 10% diet green tea with citrus containing ascorbic acid (AA), and citric acid (CA) in a wide concentration of glucose at 0.02 to 14.0mM with an LOD of 3.10µM. Reproducibility was also investigated using 4 electrodes in this sample and shows notable results in the wide concentration range of 3.35±0.45µM to of 13.0 ± 0.81 mM. We also used other voltammetry methods to evaluate this biosensor. We applied linear sweep voltammetry (LSV) and this technique shows a wide range of 0.10−15.0 mM to detect glucose with a lower detection limit of 19.5µM. The performance and strength of this enzyme biosensor were the simplicity, wide linear ranges, sensitivities, selectivity, and low limits of detection. We expect that the modified biosensor has the potential for monitoring various biofluids.

Keywords: plant-produced manganese peroxidase, enzyme-based biosensors, glucose, modified gold electrode, glucose oxidase

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Authors: Ashish Kumar Singh, Mehak Gulati, Neelam Verma

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Arginine majorly acts as a substrate for the enzyme nitric oxide synthase (NOS) for the production of nitric oxide, a strong vasodilator. Current study demonstrated a novel amperometric approach for estimation of arginine using nitric oxide synthase. The enzyme was co-immobilized in carbon paste electrode with NADP+, FAD and BH4 as cofactors. The detection principle of the biosensor is enzyme NOS catalyzes the conversion of arginine into nitric oxide. The developed biosensor could able to detect up to 10-9M of arginine. The oxidation peak of NO was observed at 0.65V. The developed arginine biosensor was used to monitor arginine content in fruit juices.

Keywords: arginine, biosensor, carbon paste elctrode, nitric oxide

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Authors: Ruolan Zhang, Ryo Imai, Naoko Senda, Tomoyuki Sakai

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Enzymes have attracted increasing attentions in industrial manufacturing for their applicability in catalyzing complex chemical reactions under mild conditions. Directed evolution has become a powerful approach to optimize enzymes and exploit their full potentials under the circumstance of insufficient structure-function knowledge. With the incorporation of cell-free synthetic biotechnology, rapid enzyme synthesis can be realized because no cloning procedure such as transfection is needed. Its open environment also enables direct enzyme measurement. These properties of cell-free biotechnology lead to excellent throughput of enzymes generation. However, the capabilities of current screening methods have limitations. Fluorescence-based assay needs applicable fluorescent label, and the reliability of acquired enzymatic activity is influenced by fluorescent label’s binding affinity and photostability. To acquire the natural activity of an enzyme, another method is to combine pre-screening step and high-performance liquid chromatography (HPLC) measurement. But its throughput is limited by necessary time investment. Hundreds of variants are selected from libraries, and their enzymatic activities are then identified one by one by HPLC. The turn-around-time is 30 minutes for one sample by HPLC, which limits the acquirable enzyme improvement within reasonable time. To achieve the real high-throughput enzyme screening, i.e., obtain reliable enzyme improvement within reasonable time, a widely applicable high-throughput measurement of enzymatic reactions is highly demanded. Here, a high-throughput screening method using broadband coherent anti-Stokes Raman spectroscopy (CARS) was proposed. CARS is one of coherent Raman spectroscopy, which can identify label-free chemical components specifically from their inherent molecular vibration. These characteristic vibrational signals are generated from different vibrational modes of chemical bonds. With the broadband CARS, chemicals in one sample can be identified from their signals in one broadband CARS spectrum. Moreover, it can magnify the signal levels to several orders of magnitude greater than spontaneous Raman systems, and therefore has the potential to evaluate chemical's concentration rapidly. As a demonstration of screening with CARS, alcohol dehydrogenase, which converts ethanol and nicotinamide adenine dinucleotide oxidized form (NAD+) to acetaldehyde and nicotinamide adenine dinucleotide reduced form (NADH), was used. The signal of NADH at 1660 cm⁻¹, which is generated from nicotinamide in NADH, was utilized to measure the concentration of it. The evaluation time for CARS signal of NADH was determined to be as short as 0.33 seconds while having a system sensitivity of 2.5 mM. The time course of alcohol dehydrogenase reaction was successfully measured from increasing signal intensity of NADH. This measurement result of CARS was consistent with the result of a conventional method, UV-Vis. CARS is expected to have application in high-throughput enzyme screening and realize more reliable enzyme improvement within reasonable time.

Keywords: Coherent Anti-Stokes Raman Spectroscopy, CARS, directed evolution, enzyme screening, Raman spectroscopy

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Authors: Zohreh Bayat, Dariush Minai-Tehrani

Abstract:

Lipases constitute one of the most important groups of industrial enzymes that catalyze the hydrolysis of triacylglycerol to glycerol and fatty acids. Muscarinic antagonist relieves smooth muscle spasm of the gastrointestinal tract and effect on the cardiovascular system. In this research, the effect of a muscarinic antagonist on the lipase activity of Pseudomonas aeruginosa was studied. Lineweaver–Burk plot showed that the drug inhibited the enzyme by competitive inhibition. The IC50 value (60 uM) and Ki (30 uM) of the drug revealed the drug bound to the enzyme with high affinity. Determination of enzyme activity in various pH and temperature showed that the maximum activity of lipase was at pH 8 and 60°C both in presence and absence of the drug.

Keywords: bacteria, inhibition, kinetics, lipase

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Authors: Justina I. R. Udotong, Essien U. Essien

Abstract:

Diagnostic enzymes like aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were determined as indices of heavy metal pollution in Tilapia guinensis. Three different sets of fishes treated with lead (Pb), iron (Fe) and copper (Cu) were used for the study while a fourth group with no heavy metal served as a control. Fishes in each of the groups were exposed to 2.65 mg/l of Pb, 0.85 mg/l of Fe and 0.35 mg/l of Cu in aerated aquaria for 96 hours. Tissue fractionation of the liver tissues was carried out and the three diagnostic enzymes (AST, ALT, and ALP) were estimated. Serum levels of the same diagnostic enzymes were also measured. The mean values of the serum enzyme activity for ALP in each experimental group were 19.5±1.62, 29.67±2.17 and 1.15±0.27 IU/L for Pb, Fe and Cu groups compared with 9.99±1.34 IU/L enzyme activity in the control. This result showed that Pb and Fe caused increased release of the enzyme into the blood circulation indicating increased tissue damage while Cu caused a reduction in the serum level as compared with the level in the control group. The mean values of enzyme activity obtained in the liver were 102.14±6.12, 140.17±2.06 and 168.23±3.52 IU/L for Pb, Fe and Cu groups, respectively compared to 91.20±9.42 IU/L enzyme activity for the control group. The serum and liver AST and ALT activities obtained in Pb, Fe, Cu and control groups are reported. It was generally noted that the presence of the heavy metal caused liver tissues damage and consequent increased level of the diagnostic enzymes in the serum.

Keywords: diagnostic enzymes, enzyme activity, heavy metals, tissues investigations

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Authors: He Yuhai, Ahmad Ziad Bin Sulaiman

Abstract:

Tongkat Ali, or Eurycoma longifolia, is a traditional Malay and Orang Asli herb used as aphrodisiac, general tonic, anti-Malaria, and anti-Pyretic. It has been recognized as a cashcrop by Malaysia due to its high value for the pharmaceutical use. In Tongkat Ali, eurycomanone, a quassinoid is usually chosen as a marker phytochemical as it is the most abundant phytochemical. In this research, ultrasound and enzyme were used to enhance the extraction of Eurycomanone from Tongkat Ali. Ultrasonic assisted extraction (USE) enhances extraction by facilitating the swelling and hydration of the plant material, enlarging the plant pores, breaking the plant cell, reducing the plant particle size and creating cavitation bubbles that enhance mass transfer in both the washing and diffusion phase of extraction. Enzyme hydrolyses the cell wall of the plant, loosening the structure of the cell wall, releasing more phytochemicals from the plant cell, enhancing the productivity of the extraction. Possible effects of ultrasound on the activity of the enzyme during the hydrolysis of the cell wall is under the investigation by this research. The extracts was analysed by high performance liquid chromatography for the yields of Eurycomanone. In this whole process, the conventional water extraction was used as a control of comparing the performance of the ultrasound and enzyme assisted extraction.

Keywords: ultrasound, enzymatic, extraction, Eurycoma longifolia

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