Search results for: angiotensin converting enzyme

Authors: Jung-En Kuan, Whei-Fen Wu

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In this study, a symbiotic bacteria from yellow mealworm's (Tenebrio molitor) mid-gut was isolated with characteristics of growth on minimal-tributyrin medium. After a PCR-amplification of its 16s rDNA, the resultant nucleotide sequences were then analyzed by schemes of the phylogeny trees. Accordingly, it was designated as Pseudomonas nitroreducens D-01. Next, by searching the lipolytic enzymes in its protein data bank, one of those potential lipolytic α/β hydrolases was identified, again using PCR-amplification and nucleotide-sequencing methods. To construct an expression of this lipolytic gene in plasmids, the target-gene primers were then designed, carrying the C-terminal his-tag sequences. Using the vector pET21a, a recombinant lipolytic hydrolase D gene with his-tag nucleotides was successfully cloned into it, of which the lipolytic D gene is under a control of the T7 promoter. After transformation of the resultant plasmids into Eescherichia coli BL21 (DE3), an IPTG inducer was used for the induction of the recombinant proteins. The protein products were then purified by metal-ion affinity column, and the purified proteins were found capable of forming a clear zone on tributyrin agar plate. Shortly, its enzyme activities were determined by degradation of p-nitrophenyl ester(s), and the substantial yellow end-product, p-nitrophenol, was measured at O.D.405 nm. Specifically, this lipolytic enzyme efficiently targets p-nitrophenyl butyrate. As well, it shows the most reactive activities at 40°C, pH 8 in potassium phosphate buffer. In thermal stability assays, the activities of this enzyme dramatically drop when the temperature is above 50°C. In metal ion assays, MgCl₂ and NH₄Cl induce the enzyme activities while MnSO₄, NiSO₄, CaCl₂, ZnSO₄, CoCl₂, CuSO₄, FeSO₄, and FeCl₃ reduce its activities. Besides, NaCl has no effects on its enzyme activities. Most organic solvents decrease the activities of this enzyme, such as hexane, methanol, ethanol, acetone, isopropanol, chloroform, and ethyl acetate. However, its enzyme activities increase when DMSO exists. All the surfactants like Triton X-100, Tween 80, Tween 20, and Brij35 decrease its lipolytic activities. Using Lineweaver-Burk double reciprocal methods, the function of the enzyme kinetics were determined such as Km = 0.488 (mM), Vmax = 0.0644 (mM/min), and kcat = 3.01x10³ (s⁻¹), as well the total efficiency of kcat/Km is 6.17 x10³ (mM⁻¹/s⁻¹). Afterwards, based on the phylogenetic analyses, this lipolytic protein is classified to type IV lipase by its homologous conserved region in this lipase family.

Keywords: enzyme, esterase, lipotic hydrolase, type IV

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Authors: Khalida Boutemak, Nasssima Benali, Nadji Moulai-Mostefa

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Effect of enzyme on the yield and chemical composition of Artemisia campestris essential oil is reported in the present study. It was demonstrated that enzyme facilitated the extraction of essential oil with increase in oil yield and did not affect any noticeable change in flavour profile of the volatile oil. Essential oil was tested for antibacterial activity using Escherichia coli; which was extremely sensitive against control with the largest inhibition (29mm), whereas Staphylococcus aureus was the most sensitive against essential oil obtained from enzymatic pre-treatment with the largest inhibition zone (25mm). The antioxidant activity of the essential oil with hemicellulase pre-treatment (EO2) and control sample (EO1) was determined through reducing power. It was significantly lower than the standard drug (vitamin C) in this order: vitamin C˃EO2˃EO1.

Keywords: Artemisia campestris, enzyme pre-treatment, hemicellulase, antibacterial activity, antioxidant activity

Procedia PDF Downloads 298

Authors: Wei-Ju Huang, Hung-Pin Hsu

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[Background] In congestive heart failure (CHF), it has always been the principle of clinical treatment to control the water retention mechanism in the body to prevent excessive fluid retention. Early control of sympathetic nerves, Renin-Angiotensin-Aldosterone system (RAA system, RAAS), or strengthening of Atrial Natriuretic Peptide (ANP) was the point. In RAA system, related hormones, such as angiotensin, or enzymes in the pathway, such as ACE-I, can be used with corresponding inhibitors to reduce water content.[Aim] In recent years, clinical studies have pointed out that if different mechanisms are combined, the control effect seems to be better. For example, recent studies showed that ENTRESTO, a combination of Sacubitril and Valsartan, is a good new drug for CHF. Sacubitril is a prodrug. After activation, it can inhibit neprilysin and act as a neprilysin inhibitor (ARNI) to reduce the breakdown of natriuretic peptides(ANP). Valsartan is a kind of angiotensin receptor blocker (ARB), both of which are used to treat heart failure at the same time, have excellent curative effects.[Materials and Methods] Considering the side effects of this drug, coughing and a few cases of diarrhea were observed. However, the effect of this drug on the patient's intestinal tract has not been confirmed. On the other hand, studies have pointed out that ANP supplement can improve the CHF and increase the inhibitory effect on cancer cells. Therefore, the purpose of this study is to use a special microbial detection method to prove that whether oral drugs have an effect on microorganisms.The experimental method uses Nissui Compact Dry to observe the situation in different types of microorganisms. After the drug is dissolved in water, it is implanted in a petri dish, and the presence of different microorganisms is detected through different antibody reactions to confirm whether the drug has some toxicology in the gut.[Results and Discussion]From the above experimental results, it can be known that among the effects of Sacubitril and Valsartan on the basic microbial flora of the human body, low doses had no significant effect on Escherichia coli or intestinal bacteria. If Sacubitril or Valsartan with a high concentration of 3mg/ml is used alone or under the stimulation of a high concentration of the two drugs, it has a significant inhibitory effect on Escherichia coli. However, in terms of the effect on intestinal bacteria, high concentration of Sacubitril has a more significant inhibitory effect on intestinal bacteria, while high concentration of Valsartan has a less significant inhibitory effect on intestinal bacteria. The inhibitory effect of the combination of the two drugs on intestinal bacteria is also less significant.[Conclusion]The results of this study can be used as a further reference for the possible side effects of the clinical use of Sacubitril and Valsartan on the intestinal tract of patients,

Keywords: sacubitril, valsartan, entresto, congestive heart failure (CHF)

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Authors: Itti Bist, Snehasis Bhakta, Di Jiang, Tia E. Keyes, Aaron Martin, Robert J. Forster, James F. Rusling

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DNA damage from metabolites of lipophilic drugs and pollutants, generated by enzymes, represents a major toxicity pathway in humans. These metabolites can react with DNA to form either 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG), which is the oxidative product of DNA or covalent DNA adducts, both of which are genotoxic and hence considered important biomarkers to detect cancer in humans. Therefore, detecting reactions of metabolites with DNA is an effective approach for the safety assessment of new chemicals and drugs. Here we describe a novel electrochemiluminescent (ECL) sensor array which can detect DNA oxidation and chemical DNA damage in a single array, facilitating a more accurate diagnostic tool for genotoxicity screening. Layer-by-layer assembly of DNA and enzyme are assembled on the pyrolytic graphite array which is housed in a microfluidic device for sequential detection of two type of the DNA damages. Multiple enzyme reactions are run on test compounds using the array, generating toxic metabolites in situ. These metabolites react with DNA in the films to cause DNA oxidation and chemical DNA damage which are detected by ECL generating osmium compound and ruthenium polymer, respectively. The method is further validated by the formation of 8-oxodG and DNA adduct using similar films of DNA/enzyme on magnetic bead biocolloid reactors, hydrolyzing the DNA, and analyzing by liquid chromatography-mass spectrometry (LC-MS). Hence, this combined DNA/enzyme array/LC-MS approach can efficiently explore metabolic genotoxic pathways for drugs and environmental chemicals.

Keywords: biosensor, electrochemiluminescence, DNA damage, microfluidic array

Procedia PDF Downloads 337

Authors: Amtul Jamil Sami

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Apis mellifera is an insect of immense economic importance lives on rich carbohydrate diet including cellulose, nectar, honey and pollen. The carbohydrate metabolism in A mellifera has not been understood fully, as there are no data available, on the functional expression of cellulase gene. The cellulose hydrolyzing enzyme is required for the digestion of pollen cellulose wall, to release the important nutrients (amino acids, minerals, vitamins etc.) from the pollen. A dissection of Apis genome had revealed that there is one gene present for the expression of endo-beta-1,4-glucanase, for cellulose hydrolysis. In the presented work, functional expression of endo-beta-1,4 glucanase gene is reported. Total soluble proteins of the honey bee were isolated and were tested cellulose hydrolyzing enzyme activity, using carboxy-methyl cellulose, as a substrate. A mellifera proteins were able to hydrolyze carboxy-methyl cellulose, confirming its endo- type mode of action. Endo beta-1,4 glucanase enzyme was only present in the gut tissues, no activity was detected in the salivary glands. The pH optima of the enzyme were in the acidic pH range of 4-5-5-0, indicating its metabolic role in the acidic stomach of A mellifera. The reported enzyme is unique, as endo-beta- 1,4 glucanase was able to generate non reducing sugar, as an end product. The results presented, are supportive to the information that the honey bee is capable of producing its novel endo-beta-1,4 glucanase. Further it could be helpful, in understanding, the carbohydrate metabolism in A mellifera.

Keywords: honey bees, Endo-beta 1, 4- glucanase, Apis mellifera, functional expression

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Authors: Kanchan Vishwakarma, Shivesh Sharma, Nitin Kumar

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Soil is incoherent matter on Earth’s surface having organic and mineral content. The spatial variation of 4 soil enzyme activities and microbial biomass were assessed for two seasons’ viz. monsoon and winter along the latitudinal gradient in North-central India as the area of this study is fettered with respect to national status. The study was facilitated to encompass the effect of climate change, enzyme activity and biomass on nutrient cycling. Top soils were sampled from 4 sites in North-India. There were significant correlations found between organic C, N & P wrt to latitude gradient in two seasons. This distribution of enzyme activities and microbial biomass was consequence of alterations in temperature and moisture of soil because of which soil properties change along the latitude transect.

Keywords: latitude gradient, microbial biomass, moisture, soil, organic carbon, temperature

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Authors: Lee A. Browne, K. Rumbold

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The first fluorinating enzyme, 5'-fluoro-5'-deoxyadenosine synthase (fluorinase) was isolated from the soil bacterium Streptomyces cattleya. Such an enzyme, with the ability to catalyze a C-F bond, presents great potential as a biocatalyst. Naturally fluorinated compounds are extremely rare in nature. As a result, the number of fluorinases identified remains relatively few. The field of fluorination is almost completely synthetic. However, with the increasing demand for fluorinated organic compounds of commercial value in the agrochemical, pharmaceutical and materials industries, it has become necessary to utilize biologically based methods such as biocatalysts. A key step in this crucial process is the large-scale production of the fluorinase enzyme in considerable quantities for industrial applications. Thus, this study aimed to optimize expression of the fluorinase enzyme in both prokaryotic and eukaryotic expression systems in order to obtain high protein yields. The fluorinase gene was cloned into the pET 41b(+) and pPinkα-HC vectors and used to transform the expression hosts, E.coli BL21(DE3) and Pichia pastoris (PichiaPink™ strains) respectively. Expression trials were conducted to select optimal conditions for expression in both expression systems. Fluorinase catalyses a reaction between S-adenosyl-L-Methionine (SAM) and fluoride ion to produce 5'-fluorodeoxyadenosine (5'FDA) and L-Methionine. The activity of the enzyme was determined using HPLC by measuring the product of the reaction 5'FDA. A gradient mobile phase of 95:5 v/v 50mM potassium phosphate buffer to a final mobile phase containing 80:20 v/v 50mM potassium phosphate buffer and acetonitrile were used. This resulted in the complete separation of SAM and 5’-FDA which eluted at 1.3 minutes and 3.4 minutes respectively. This proved that the fluorinase enzyme was active. Optimising expression of the fluorinase enzyme was successful in both E.coli and PichiaPink™ where high expression levels in both expression systems were achieved. Protein production will be scaled up in PichiaPink™ using fermentation to achieve large-scale protein production. High level expression of protein is essential in biocatalysis for the availability of enzymes for industrial applications.

Keywords: biocatalyst, expression, fluorinase, PichiaPink™

Procedia PDF Downloads 523

Authors: Alabi Olushola John, Ogbiko Anthonia, Tsado Daniel Nma, Mbajiorgu Ejike Felix, Adama Theophilus Zubairu

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A total of thirty (30) goats weighting between 5.8 and 7.3 kg were used to determine the growth performance, body linear measurements and body condition score of Semi intensively manged Savanna Brown goats fed enzyme treated sawdust diets (ETSD). They divided into five dietary treatments (T) groups with three replications using a completely randomized design. Treatment one (1) comprises of animals fed diet on 0 % enzyme treated sawdust while Treatment 2 (T2), Treatment 3 (T3), Treatment 4 (T4) and Treatment 5 (T5) comprises of animals fed diets containing 10, 20, 30 and 40 % enzyme treated sawdust diets, respectively. The study lasted 16 weeks. Data on growth performance parameters, body linear measurement (height at wither, body length, chest girth, hind leg length, foreleg length, facial length) and body condition score were collected and analyzed using one way analysis of variance. No significant difference (p>0.05) was observed in the all growth performance parameters and linear body measurements. However, significant difference was observed in body length and daily body length gains with highest value observed in animals fed the control diets (7.38 and 0.08 cm respectively) and animals on 30 % ETSD (7.25 and 0.07 cm respectively) and lowest values (4.75 and 0.05 cm respectively) were observed in animals fed 10 % ETSD among the treatment groups. It was, therefore, concluded that enzyme treated sawdust can be used in the diets of Savanna Brown goats up to 40 % replacement for maize offal since this treatment improved the body length and daily body length gains.

Keywords: performance, sawdust, enzyme treated, semi-intensively, replacement

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Authors: Ayuko Itsuki, Sachiyo Aburatani

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Soil enzyme activities in Kasuga-yama Hill Primeval Forest (Nara, Japan) were examined to determine levels of mineralization and metabolism. Samples were selected from the soil surrounding laurel-leaved (B_B-1) and Carpinus japonica (B_B-2 and P_w) trees for analysis. Cellulase, β-xylosidase, and protease activities were higher in B_B-1 samples those in B_B-2 samples. These activity levels corresponded to the distribution of cellulose and hemicellulose in the soil horizons. Cellulase, β-xylosidase, and chymotrypsin activities were higher in soil from the P_w forest than in that from the B_B-2 forest. The relationships between the soil enzymes calculated by Spearman’s rank correlation indicate that the interactions between enzymes in B_B-2 samples were more complex than those in P_w samples.

Keywords: comparative analysis, enzyme activities, forest soil, Spearman's rank correlation

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Authors: Esther Friedlander, Philip Friedlander

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The use of immunotherapy that inhibits programmed death-1 (PD-1) or its ligand PD-L1 confers survival benefits in patients with non-small cell lung cancer (NSCLC). However, approximately 45% of patients experience primary treatment resistance, necessitating the development of strategies to improve efficacy. While the renin-angiotensin system (RAS) has systemic hemodynamic effects, tissue-specific regulation exists along with modulation of immune activity in part through regulation of myeloid cell activity, leading to the hypothesis that RAS inhibition may improve anti-PD-1/L-1 efficacy. A retrospective analysis was conducted that included 173 advanced solid tumor cancer patients treated at Valley Hospital, a community Hospital in New Jersey, USA, who were treated with a PD-1/L-1 inhibitor in a defined time period showing a statistically significant relationship between RAS pathway inhibition (RASi through concomitant treatment with an ACE inhibitor or angiotensin receptor blocker) and positive efficacy to the immunotherapy that was independent of age, gender and cancer type. Subset analysis revealed strong numerical benefit for efficacy in both patients with squamous and nonsquamous NSCLC as determined by documented clinician assessment of efficacy and by duration of therapy. A PUBMED literature search was now conducted to identify studies assessing the effect of RAS pathway inhibition on anti-PD-1/L1 efficacy in advanced solid tumor patients and compare these findings to those seen in the Valley Hospital retrospective study with a focus on NSCLC specifically. A total of 11 articles were identified assessing the effects of RAS pathway inhibition on the efficacy of checkpoint inhibitor immunotherapy in advanced cancer patients. Of the 11 studies, 10 assessed the effect on survival of RASi in the context of treatment with anti-PD-1/PD-L1, while one assessed the effect on CTLA-4 inhibition. Eight of the studies included patients with NSCLC, while the remaining 2 were specific to genitourinary malignancies. Of the 8 studies, two were specific to NSCLC patients, with the remaining 6 studies including a range of cancer types, of which NSCLC was one. Of these 6 studies, only 2 reported specific survival data for the NSCLC subpopulation. Patient characteristics, multivariate analysis data and efficacy data seen in the 2 NSLCLC specific studies and in the 2 basket studies, which provided data on the NSCLC subpopulation, were compared to that seen in the Valley Hospital retrospective study supporting a broader effect of RASi on anti-PD-1/L1 efficacy in advanced NSLCLC with the majority of studies showing statistically significant benefit or strong statistical trends but with one study demonstrating worsened outcomes. This comparison of studies extends published findings to the community hospital setting and supports prospective assessment through randomized clinical trials of efficacy in NSCLC patients with pharmacodynamic components to determine the effect on immune cell activity in tumors and on the composition of the tumor microenvironment.

Keywords: immunotherapy, cancer, angiotensin, efficacy, PD-1, lung cancer, NSCLC

Procedia PDF Downloads 37

Authors: Patsarawadee Paojinda, Waranya Imprasittichai, Sudaratana R. Krungkrai, Nirianne Marie Q. Palacpac, Toshihiro Horii, Jerapan Krungkrai

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Fusion of the last two enzymes in the pyrimidine biosynthetic pathway in the inversed order by having COOH-terminal orotate phosphoribosyltransferase (OPRT) and NH2-terminal orotidine 5'-monophosphate decarboxylase (OMPDC), as OMPDC-OPRT, are described in many organisms. Here, we produced gene fusions of Plasmodium falciparum OMPDC-OPRT and expressed the bifunctional protein in Escherichia coli. The enzyme was purified to homogeneity using affinity and anion-exchange chromatography, exhibited enzymatic activities and functioned as a dimer. The activities, although unstable, can be stabilized by its substrate and product during purification and long-term storage. Furthermore, the enzyme expressed a perfect catalytic efficiency (kcat/Km). The kcat was selectively enhanced up to 3 orders of magnitude, while the Km was not much affected and remained at low µM levels when compared to the monofunctional enzymes. The fusion of the two enzymes, creating a “super-enzyme” with perfect catalytic power and more flexibility, reflects cryptic relationship of enzymatic reactivaties and metabolic functions on molecular evolution.

Keywords: bifunctional enzyme, orotate phosphoribosyltransferase, orotidine 5'-monophosphate decarboxylase, plasmodium falciparum

Procedia PDF Downloads 258

Authors: Qing Cao, Fei Wang, Yu-Qiang Wang, Li-Ya Huang, Tian-Tian Sang, Shu-Yan Chen

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Aims: TNF Receptor-Associated Factor 6 (TRAF6) acts as a multifunctional regulator of the Transforming Growth Factor (TGF)-β signaling pathway, and mediates Smad-independent JNK and p38 activation via TGF-β. This study was performed to test the hypothesis that TGF-β/TRAF6 is essential for angiotensin-II (Ang II)-induced differentiation of rat c-kit+ Cardiac Stem Cells (CSCs). Methods and Results: c-kit+ CSCs were isolated from neonatal Sprague Dawley (SD) rats, and their c-kit status was confirmed with immunofluorescence staining. A TGF-β type I receptor inhibitor (SB431542) or the small interfering RNA (siRNA)-mediated knockdown of TRAF6 were used to investigate the role of TRAF6 in TGF-β signaling. Rescue of TRAF6 siRNA transfected cells with a 3'UTR deleted siRNA insensitive construct was conducted to rule out the off target effects of the siRNA. TRAF6 dominant negative (TRAF6DN) vector was constructed and used to infect c-kit+ CSCs, and western blotting was used to assess the expression of TRAF6, JNK, p38, cardiac-specific proteins, and Wnt signaling proteins. Physical interactions between TRAF6 and TGFβ receptors were studied by coimmunoprecipitation. Cardiac differentiation was suppressed in the absence of TRAF6. Forced expression of TRAF6 enhanced the expression of TGF-β-activated kinase1 (TAK1), and inhibited Wnt signaling. Furthermore, TRAF6 increased the expression of cardiac-specific proteins (cTnT and Cx-43) but inhibited the expression of Wnt3a. Conclusions: Our data suggest that TRAF6 plays an important role in Ang II induced differentiation of c-kit+ CSCs via the non-canonical signaling pathway.

Keywords: cardiac stem cells, differentiation, TGF-β, TRAF6, ubiquitination, Wnt

Procedia PDF Downloads 363

Authors: M. S. Tajul Islam, Md. Zahangir Alam

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To enhance biogas production through anaerobic digestion, the application of various type of pre-treatment method has some limitations in terms of sustainable environmental management. Many studies on pretreatments especially chemical and physical processes are carried out to evaluate the anaerobic digestion for enhanced biogas production. Among the pretreatment methods acid and alkali pre-treatments gained the highest importance. Previous studies have showed that although acid and alkali pretreatment has significant effect on degradation of biomass, these methods have some negative impact on environment due to their hazard in nature while enzymatic pre-treatment is environmentally friendly. One of the constrains to use of enzyme in pretreatment process for biogas production is high cost which is currently focused to reduce cost through fermentation of waste-based media. As such palm oil mill effluent (POME) as an abundant resource generated during palm oil processing at mill is being used a potential fermentation media for enzyme production. This low cost of enzyme could be an alternative to biogas pretreatment process. This review is to focus direct application of enzyme as enzymatic pre-treatment on POME to enhanced production of biogas.

Keywords: POME, enzymatic pre-treatment, biogas, lignocellulosic biomass, anaerobic digestion

Procedia PDF Downloads 525

Authors: Sasangka Prasetyawan, Anna Roosdiana, Diah Mardiana, Suratmo

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Pectinase are enzymes that hydrolyze pectin compounds. The use of this enzyme is primarily to reduce the viscosity of the beverage thus simplifying the purification process. Pectinase activity influenced by microbial sources . Exploration of two types of microbes that Aspergillus spp. and Bacillus spp. pectinase give different performance, but the use of local strain is still not widely studied. The aim of this research is exploration of pectinase from A. niger and B. firmus include production conditions and characterization. Bacillus firmus incubated and shaken at a speed of 200 rpm at pH variation (5, 6, 7, 8, 9, 10), temperature (30, 35, 40, 45, 50) °C and incubation time (6, 12, 18, 24, 30, 36 ) hours. Media was centrifuged at 3000 rpm, pectinase enzyme activity determined. Enzyme production by A. niger determined to variations in temperature and pH were similar to B. firmus, but the variation of the incubation time was 24, 48, 72, 96, 120 hours. Pectinase crude extract was further purified by precipitation using ammonium sulfate saturation in fraction 0-20 %, 20-40 %, 40-60 %, 60-80 %, then dialyzed. Determination of optimum conditions pectinase activity performed by measuring the variation of enzyme activity on pH (4, 6, 7, 8, 10), temperature (30, 35, 40, 45, 50) °C, and the incubation time (10, 20, 30, 40, 50) minutes . Determination of kinetic parameters of pectinase enzyme reaction carried out by measuring the rate of enzyme reactions at the optimum conditions, but the variation of the concentration of substrate (pectin 0.1 % , 0.2 % , 0.3 % , 0.4 % , 0.5 % ). The results showed that the optimum conditions of production of pectinase from B. firmus achieved at pH 7-8.0, 40-50 ⁰C temperature and fermentation time 18 hours. Purification of pectinase showed the highest purity in the 40-80 % ammonium sulfate fraction. Character pectinase obtained : the optimum working conditions of A. niger pectinase at pH 5 , while pectinase from B. firmus at pH 7, temperature and optimum incubation time showed the same value, namely the temperature of 50 ⁰C and incubation time of 30 minutes. The presence of metal ions can affect the activity of pectinase , the concentration of Zn 2 + , Pb 2 + , Ca 2 + and K + and 2 mM Mg 2 + above 6 mM inhibit the activity of pectinase .

Keywords: pectinase, Bacillus firmus, Aspergillus niger, green chemistry

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Authors: Anna Trusek-Holownia, Andrzej Noworyta

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Using an enzyme of known specificity the hydrolysis of protein was carried out in a controlled manner. The aim was to obtain oligopeptides being the so-called active peptides or their direct precursors. An original way of expression of the protein hydrolysis kinetics was introduced. Peptide bonds contained in the protein were recognized as a diverse-quality substrate for hydrolysis by the applied protease. This assumption was positively verified taking as an example the hydrolysis of albumin by thermolysin. Peptide linkages for this system should be divided into at least four groups. One of them is a group of bonds non-hydrolyzable by this enzyme. These that are broken are hydrolyzed at a rate that differs even by tens of thousands of times. Designated kinetic constants were k'F = 10991.4 L/g.h, k'M = 14.83L/g.h, k'S about 10-1 L/g.h for fast, medium and slow bonds, respectively. Moreover, a procedure for unfolding of the protein, conducive to the improved susceptibility to enzymatic hydrolysis (approximately three-fold increase in the rate) was proposed.

Keywords: peptide bond hydrolysis, kinetics, enzyme specificity, biologically active peptides

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Authors: Li Na Zhao, Arieh Warshel

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The design of enzymes is an extremely challenging task, and this is also true for metalloenzymes. In the case of naturally evolved enzymes, one may consider the active site residues as the musicians in the enzyme orchestra, while the metal can be considered as their concertmaster. Together they catalyze reactions as if they performed a masterpiece written by nature. The Lactonase can be thought as a member of the amidohydrolase family, with two concertmasters, Fe and Zn, at its active site. It catalyzes the quorum sensing signal- N-acyl homoserine lactones (AHLs or N-AHLs)- by hydrolyzing the lactone ring. This process, known as quorum quenching, provides a strategy in the treatment of infectious diseases without introducing selection pressure. However, the activity of lactonase is metal-dependent, and this dependence hampers the clinic usage. In our study, we use the empirical valence bond (EVB) approach to evaluate the catalytic contributions decomposing them to electrostatic and other components.

Keywords: enzyme redesign, empirical valence bond, lactonase, quorum quenching

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Authors: Fahimeh Khatami, Elham Sanagar Darbani, Behnosh Khir Khah, R.Khatami

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Rural and ruralism is one of the residential forms that form in special natural areas, and the Interaction between their internal and external forces cause developments and changes that are different in time and space. Over time, historical developments, social and economic changes in the political system cause developments and rapid growth of the rural to urban settlements. However, criteria for recognizing rural settlements to the city are different in every land. One of the problems in modern plan is inattention to indicators and criteria of changing these settlements to the city. The method of this research is a type of applied and compilation research and library and field methods are used in it. And also qualitative and quantitative indicators have been provided while collecting documents and studies from rural districts like Dehnow, Virani, Abardeh, Zoshk, Nowchah, Jaqarq in tourism area of Mashhad. In this research, the used tool is questionnaire and for analyzing quantitative variables by Morris and Mac Granahan examination, the importance of each factor and the development settlements are evaluated, and the rural that can convert to the city was defined. In result, according to Askalvgram curve obtained from analysis, it was found that among the mentioned villages, Virani and Nowchah rural districts have this ability to convert to the city; Zoshk rural district will be converting to the city in future and Dehnow, Abardeh and Jaqarq rural districts won’t be converting.

Keywords: rural settlements, city, indicators, Torqabe and Shandiz rural districts

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Authors: Wayde Veldman, Ozlem T. Bishop, Igor Polikarpov

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In bacteria, mono and disaccharides are phosphorylated during uptake into the cell via the widely used phosphoenolpyruvate (PEP)-dependent phosphotransferase transport system. As an initial step in the phosphorylated disaccharide metabolism pathway, certain glycoside hydrolase family 1 (GH1) enzymes play a crucial role in releasing phosphorylated and non-phosphorylated monosaccharides. However, structural determinants for the specificity of these enzymes still need to be clarified. GH1 enzymes are known to have a wide array of functions. According to the CAZy database, there are twenty-one different enzymatic activities in the GH1 family. Here, the structure and substrate specificity of a GH1 enzyme from Bacillus licheniformis, hereafter known as BlBglH, was investigated. The sequence of the enzyme BlBglH was compared to the sequences of other characterized GH1 enzymes using sequence alignment, sequence identity calculations, phylogenetic analysis, and motif discovery. Through these various analyses, BlBglH was found to have sequence features characteristic of the 6-phospho-β-glucosidase activity enzymes. Additionally, motif and structure comparisons of the three most commonly studied GH1 enzyme-activities revealed a shared loop amongst the different structures that consist of different sequence motifs – this loop is thought to guide specific substrates (depending on activity) towards the active-site. To further affirm BlBglH enzyme activity, molecular docking and molecular dynamics simulations were performed. Docking was carried out using 6-phospho-β-glucosidase enzyme-activity positive (p-Nitrophenyl-beta-D-glucoside-6-phosphate) and negative (p-Nitrophenyl-beta-D-galactoside-6-phosphate) control ligands, followed by 400 ns molecular dynamics simulations. The positive-control ligand maintained favourable interactions within the active site until the end of the simulation. The negative-control ligand was observed exiting the enzyme at 287 ns. Binding free energy calculations showed that the positive-control complex had a substantially more favourable binding energy compared to the negative-control complex. Jointly, the findings of this study suggest that the BlBglH enzyme possesses 6-phospho-β-glucosidase enzymatic activity.

Keywords: 6-P-β-glucosidase, glycoside hydrolase 1, molecular dynamics, sequence analysis, substrate specificity

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Authors: R. Pravin Kumar, L. Roopa

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Enzymes are molecular machines used in various industries such as pharmaceuticals, cosmetics, food and animal feed, paper and leather processing, biofuel, and etc. Nevertheless, this has been possible only by the breath-taking efforts of the chemists and biologists to evolve/engineer these mysterious biomolecules to work the needful. Main agenda of this enzyme engineering project is to derive screening and selection tools to obtain focused libraries of enzyme variants with desired qualities. The methodologies for this research include the well-established directed evolution, rational redesign and relatively less established yet much faster and accurate insilico methods. This concept was initiated as a Receptor Rependent-4Dimensional Quantitative Structure Activity Relationship (RD-4D-QSAR) to predict kinetic properties of enzymes and extended here to study transaminase by a 7D QSAR approach. Induced-fit scenarios were explored using Quantum Mechanics/Molecular Mechanics (QM/MM) simulations which were then placed in a grid that stores interactions energies derived from QM parameters (QMgrid). In this study, the mutated enzymes were immersed completely inside the QMgrid and this was combined with solvation models to predict descriptors. After statistical screening of descriptors, QSAR models showed > 90% specificity and > 85% sensitivity towards the experimental activity. Mapping descriptors on the enzyme structure revealed hotspots important to enhance the enantioselectivity of the enzyme.

Keywords: QMgrid, QM/MM simulations, RD-4D-QSAR, transaminase

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Authors: Sidra Shaw, Sarenna Shaw, Chae Joon Lee, Irimpan Mathews, Eric Koehn

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One of the biggest public health concerns of our time is increasing antimicrobial resistance. As of 2019, the CDC has documented more than 2.8 million serious antibiotic resistant infections in the United States. Currently, antibiotic resistant infections are directly implicated in over 750,000 deaths per year globally. On our current trajectory, British economist Jim O’Neill predicts that by 2050, an additional 10 million people (about half the population of New York) will die annually due to drug resistant infections. As a result, new biochemical pathways must be targeted to generate next generation antibiotic drugs that will be effective against drug resistant bacteria. One enticing target is the biosynthesis of DNA within bacteria, as few drugs interrupt this essential life process. Thymidylate synthase enzymes are essential for life as they catalyze the synthesis of a DNA building block, 2′-deoxythymidine-5′-monophosphate (dTMP). In humans, the thymidylate synthase enzyme (TSase) has been shown to be distinct from the flavin-dependent thymidylate synthase (FDTS) produced by many pathogenic bacteria. TSase and FDTS have distinct structures and mechanisms of catalysis, which should allow selective inhibition of FDTS over human TSase. Currently, C. diff is one of the most antibiotic resistant bacteria, and no drugs that target thymine biosynthesis exist for C. diff. Here we present the initial biochemical characterization of FDTS from C. diff. Specifically, we examine enzyme kinetics and binding features of this enzyme to determine the nature of interaction with ligands/inhibitors and understand the molecular mechanism of catalysis. This research will provide more insight into the targetability of the C. diff FDTS enzyme for novel antibiotic drugs.

Keywords: flavin-dependent thymidylate synthase, FDTS, clostridioides difficile, C. diff, antibiotic resistance, DNA synthesis, enzyme kinetics, binding features

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Authors: Anita Jäger, Andrew Salazar, Jörg von Hagen, Harald Kolmar

Abstract:

In the treatment of individuals with sensitive and psoriatic skin, several inflammation and itch-related molecular and cellular targets have been identified, but many of these have yet to be characterized. In this study, we present two potential targets in the skin that can be linked to the inflammation and itch cycle. 11ßHSD1 is the enzyme responsible for converting inactive cortisone to active cortisol used to transmit signals downstream. The activation of the receptor NK1R correlates with promoting inflammation and the perception of itch and pain in the skin. In this study, both targets have been investigated based on their involvement in inflammation. The role of both identified targets was characterized based on the secretion of inflammation cytokine- IL6, IL-8, and CCL2, as well as phosphorylation and signaling pathways. It was found that treating skin cells with molecules able to inhibit inflammatory pathways results in the reduction of inflammatory signaling molecules secreted by skin cells and increases their proliferative capacity. Therefore, these molecular targets and their associated pathways show therapeutic potential and can be mitigated via small molecules. This research can be used for further studies in inflammation and itch pathways and can help to treat pathological symptoms.

Keywords: inflammation, itch, signaling pathway, skin

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Authors: Tomokazu Shirai, Akihiko Kondo

Abstract:

Technologies of metabolic engineering or synthetic biology are essential for effective microbial bioproduction. It is especially important to develop an in silico tool for designing a metabolic pathway producing an unnatural and valuable chemical such as fossil materials of fuel or plastics. We here demonstrated two in silico tools for designing novel metabolic pathways: BioProV and HyMeP. Furthermore, we succeeded in creating an artificial metabolic pathway by enzyme engineering.

Keywords: bioinformatics, metabolic engineering, synthetic biology, genome scale model

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Authors: Sirous Eydivandi

Abstract:

Diacylglycerol-O-acyltransferase (DGAT1) gene encodes diacylglycerol transferase enzyme that plays an important role in glycerol lipid metabolism. DGAT1 is considered to be the key enzyme in controlling the synthesis of triglycerides in adipocytes. This enzyme catalyzes the final step of triglyceride synthesis (transform triacylglycerol (DAG) into triacylglycerol (TAG). A total of 20 DGAT1 gene sequences and corresponding amino acids belonging to 4 species include cattle, goats, sheep and yaks were analyzed, and the differentiation within and among the species was also studied. The length of the DGAT1 gene varies greatly, from 1527 to 1785 bp, due to deletion, insertion, and stop codon mutation resulting in elongation. Observed genetic diversity was higher among species than within species, and Goat had more polymorphisms than any other species. Novel amino acid variation sites were detected within several species which might be used to illustrate the functional variation. Differentiation of the DGAT1 gene was obvious among species, and the clustering result was consistent with the taxonomy in the National Center for Biotechnology Information.

Keywords: DGAT1gene, bioinformatic, ruminnants, biotechnology information

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Authors: T. H. Derdzyan, K. A. Ghazaryan, G. A. Gevorgyan

Abstract:

Beta-glucosidase, chitinase, leucine-aminopeptidase, acid phosphomonoestearse and acetate-esterase enzyme activities in the soils under the impact of metallurgical industrial activity in Lori marz (district) were investigated. The results of the study showed that the activities of the investigated enzymes in the soils decreased with increasing distance from the Shamlugh copper mine, the Chochkan tailings storage facility and the ore transportation road. Statistical analysis revealed that the activities of the enzymes were positively correlated (significant) to each other according to the observation sites which indicated that enzyme activities were affected by the same anthropogenic factor. The investigations showed that the soils were polluted with heavy metals (Cu, Pb, As, Co, Ni, Zn) due to copper mining activity in this territory. The results of Pearson correlation analysis revealed a significant negative correlation between heavy metal pollution degree (Nemerow integrated pollution index) and soil enzyme activity. All of this indicated that copper mining activity in this territory causing the heavy metal pollution of the soils resulted in the inhabitation of the activities of the enzymes which are considered as biological catalysts to decompose organic materials and facilitate the cycling of nutrients.

Keywords: Armenia, metallurgical industrial activity, heavy metal pollutionl, soil enzyme activity

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Authors: Md. Z. Alam, Devi R. Asih, Md. N. Salleh

Abstract:

Immobilization of lipase enzyme produced from palm oil mill effluent (POME) by the activated carbon (AC) among the low cost support materials was optimized. The results indicated that immobilization of 94% was achieved by AC as the most suitable support material. A sequential optimization strategy based on a statistical experimental design, including one-factor-at-a-time (OFAT) method was used to determine the equilibrium time. Three components influencing lipase immobilization were optimized by the response surface methodology (RSM) based on the face-centered central composite design (FCCCD). On the statistical analysis of the results, the optimum enzyme concentration loading, agitation rate and carbon active dosage were found to be 30 U/ml, 300 rpm and 8 g/L respectively, with a maximum immobilization activity of 3732.9 U/g-AC after 2 hrs of immobilization. Analysis of variance (ANOVA) showed a high regression coefficient (R2) of 0.999, which indicated a satisfactory fit of the model with the experimental data. The parameters were statistically significant at p<0.05.

Keywords: activated carbon, POME based lipase, immobilization, adsorption

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Authors: Shweta Kumari, Parmjit S. Panesar, Manab B. Bera

Abstract:

The hydrolysis of lactose using β-galactosidase is one of the most promising biotechnological applications, which has wide range of potential applications in food processing industries. However, due to intracellular location of the yeast enzyme, and expensive extraction methods, the industrial applications of enzymatic hydrolysis processes are being hampered. The use of permeabilization technique can help to overcome the problems associated with enzyme extraction and purification of yeast cells and to develop the economically viable process for the utilization of whole cell biocatalysts in food industries. In the present investigation, standardization of permeabilization process of novel yeast isolate was carried out using a statistical model approach known as Response Surface Methodology (RSM) to achieve maximal b-galactosidase activity. The optimum operating conditions for permeabilization process for optimal β-galactosidase activity obtained by RSM were 1:1 ratio of toluene (25%, v/v) and ethanol (50%, v/v), 25.0 oC temperature and treatment time of 12 min, which displayed enzyme activity of 1.71 IU /mg DW.

Keywords: β-galactosidase, optimization, permeabilization, response surface methodology, yeast

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Authors: Delgado-Meza M., Minor-Pérez H.

Abstract:

Protease is the generic name of enzymes that hydrolyze proteins. These are classified in the subgroup EC3.4.11-99X of the classification enzymes. In food technology the proteolysis is used to modify functional and nutritional properties of food, and in some cases this proteolysis may cause food spoilage. In general, seafood and rainbow trout have accelerated decomposition process once it has done its capture, due to various factors such as the endogenous enzymatic activity that can result in loss of structure, shape and firmness, besides the release of amino acid precursors of biogenic amines. Some studies suggest the use of protease inhibitors from legume as biological regulators of proteolytic activity. The enzyme inhibitors are any substance that reduces the rate of a reaction catalyzed by an enzyme. The objective of this study was to evaluate the reduction of the proteolytic activity of enzymes in extracts of rainbow trout with protease inhibitors obtained from chickpea flour. Different proportions of rainbow trout enzyme extract (75%, 50% and 25%) and extract chickpea enzyme inhibitors were evaluated. Chickpea inhibitors were obtained by mixing 5 g of flour in 30 mL of pH 7.0 phosphate buffer. The sample was centrifuged at 8000 rpm for 10 min. The supernatant was stored at -15°C. Likewise, 20 g of rainbow trout were ground in 20 mL of phosphate buffer solution at pH 7.0 and the mixture was centrifuged at 5000 rpm for 20 min. The supernatant was used for the study. In each treatment was determined the specific enzymatic activity with the technique of Kunitz, using hemoglobin as substrate for the enzymes acid fraction and casein for basic enzymes. Also biuret protein was quantified for each treatment. The results showed for fraction of basic enzymes in the treatments evaluated, that were inhibition of endogenous enzymatic activity. Inhibition values compared to control were 51.05%, 56.59% and 59.29% when the proportions of endogenous enzymes extract rainbow trout were 75%, 50% and 25% and the remaining volume used was extract with inhibitors. Treatments with acid enzymes showed no reduction in enzyme activity. In conclusion chickpea flour reduced the endogenous enzymatic activity of rainbow trout, which may favor its application to increase the half-life of this food. The authors acknowledge the funding provided by the CONACYT for the project 131998.

Keywords: rainbouw trout, enzyme inhibitors, proteolysis, enzyme activity

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Authors: Sidra Shaheen, Nadir Naveed Siddiqui, Shah Ali Ul Qader

Abstract:

In recent years, significant progress has been made in discovering and developing new bacterial polysaccharides producing organisms possessing extremely functional properties. Levan is a natural biopolymer of fructose which is produced by transfructosylation reaction in the presence of levansucrase. It is one of the industrially promising enzymes that offer a variety of industrial applications in the field of cosmetics, foods and pharmaceuticals. Although levan has significant applications but the yield of levan produced is not equal to other biopolymers due to the inefficiency of producer microorganism. Among wide range of levansucrase producing microorganisms, Zymomonas mobilis is considered as a potential candidate for large scale production of this natural polysaccharide. The present investigation is concerned with the isolation of levansucrase producing natural isolate having maximum enzyme production. Furthermore, production parameters were optimized to get higher enzyme yield. Levansucrase was partially purified and characterized to study its applicability on industrial scale. The results of this study revealed that the bacterial strain Z. mobilis KIBGE-IB14 was the best producer of levansucrase. Bacterial growth and enzyme production was greatly influenced by physical and chemical parameters. Maximum levansucrase production was achieved after 24 hours of fermentation at 30°C using modified medium of pH-6.5. Contrary to other levansucrases, the one presented in the current study is able to produce high amount of products in relatively short period of time with optimum temperature at 35°C. Due to these advantages, this enzyme can be used on large scale for commercial production of levan and other important metabolites.

Keywords: levansucrase, metabolites, polysaccharides, transfructosylation

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Authors: Ndidi F. Amulu, Calistus N. Ude, Patrick E. Amulu, Nneka N. Uchegbu

Abstract:

The effects of temperature and enzyme concentration on the quality of mixed pineapple and pawpaw blended fruits juice were studied. Extracts of the two fruit juices were separately treated at 70  for 15 min each so as to inactivate micro-organisms. They were analyzed and blended in different proportions of 70% pawpaw and 30% pineapple, 60% pawpaw and 40% pineapple, 50% pineapple and 50% pawpaw, 40% pawpaw and 60% pineapple. The characterization of the fresh pawpaw and pineapple juice before blending showed that the juices have good quality. The high water content of the product may have affected the viscosity, vitamin C content and total soluble solid of the blended juice to be low. The effects of the process parameters on the quality showed that better quality of the blended juice can be obtained within the optimum temperature range of (50-70 °C) and enzyme concentration range (0.12-0.18 w/v). The ratio of mix 60% pineapple juice: 40% pawpaw juice has better quality. This showed that pawpaw and pineapple juices can blend effectively to produce a quality juice.

Keywords: clarification, pawpaw, pineapple, viscosity, vitamin C

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Authors: Ankita Kushwaha, M. P. Singh

Abstract:

Background: In the present investigation the efficiency of laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) from Pleurotus citrinopileatus was assessed for the decolorization of azo dyes. Aim: Enzyme production, characterization and kinetics of a partially purified laccase from Pleurotus citrinopileatus were determined for its application in bioremediation of azo dyes. Methods & Results: Laccase has been partially purified by using 80% ammonium sulphate solution. Total activity, total protein, specific activity and purification fold for partially purified laccase were found to be 40.38U, 293.33mg/100ml, 0.91U/mg and 2.84, respectively. The pH and temperature optima of laccase were 5.0 and 50ºC, respectively, while the enzyme was most stable at pH 4.0 and temperature 30ºC when exposed for one hour. The Km of the partially purified laccase for substrates guaiacol, DMP (2,6-dimethoxyphenol) and syringaldazine (3,5-dimethoxy-4-hydroxybenzaldehyde azine) were 60, 95 and 26, respectively. This laccase has been tested for the use in the bioremediation of azo dyes in the absence of mediator molecules. Two dyes namely congo red and bromophenol blue were tested. Discussion: It was observed that laccase enzyme was very effective in the decolorization of these two dyes. More than 80% decolorization was observed within half an hour even in the absence of mediator and their lower Km value indicates that efficiency of the enzyme is very high. The results were promising due to quicker decolorization in the absence of mediators showing that it can be used as a valuable biocatalyst for quick bioremediation of azo dyes. Conclusion: The enzymatic properties of laccase from P. citrinopileatus should be considered for a potential environmental (biodegradation and bioremediation) or industrial applications.

Keywords: azo dyes, decolorization, laccase, P.citrinopileatus

Procedia PDF Downloads 186