Enzymatic Parameters Research Articles

Effects of Pulsed Electric Field (PEF) Treatment on Enhancing Activity and Conformation of α-Amylase

To explore an efficient, safe, and speedy application of pulsed electric field (PEF) technology for enzymatic modification, effects of PEF treatment on the enzymatic activity, property and kinetic parameters of α-amylase were investigated. Conformational transitions were also studied with the aid of circular dichroism (CD) and fluorescence spectra. The maximum enzymatic activity of α-amylase was obtained under 15 kV/cm electric field intensity and 100 mL/min flow velocity PEF treatment, in which the enzymatic activity increased by 22.13 ± 1.14% compared with control. The activation effect could last for 18 h at 4 °C. PEF treatment could widen the range of optimum temperature for α-amylase, however, it barely exerted any effect on the optimum pH. On the other hand, α-amylase treated by PEF showed an increase of Vmax, t1/2 and ΔG, whereas a decrease of Km and k were observed. Furthermore, it can be observed from fluorescence and CD spectra that PEF treatment had increased the number of amino acid residues, especially that of tryptophan, on α-amylase surface with enhanced α-helices by 34.76% and decreased random coil by 12.04% on α-amylase when compared with that of untreated. These changes in structure had positive effect on enhancing α-amylase activity and property.

In Vitro Study on Hepatoprotective Effect of Phyllanthus fraternus Against Lead Induced Toxicity

Lead is one of the most abundant and hazardous environmental contaminants of the global concern. Environmental and occupational exposure to lead has continued to pose a serious threat to the health of living organisms due to its propensity to induce toxicity in mammalian systems. The major aim of the present in vitro study was to investigate ameliorative potential of Phyllanthus fraternus against lead induced hepatotoxicity. Current synergistic study involves exposure of goat liver homogenate to different concentrations of lead acetate and specific concentration of Phyllanthus fraternus aqueous extract for particular duration. During the study, biochemical indices like lipid peroxidation (LPO), Protein levels and enzymatic activities of alkaline phosphatase (ALPase), acid phosphatase (ACPase) and succinate dehydrogenase (SDH) were measured. Results of the present study revealed that protein levels and enzymatic parameters of alkaline phosphatase as well as succinate dehydrogenase declined significantly while levels of lipid peroxidation and acid phosphatase increased significantly in lead acetate exposed goat liver homogenates as compared to control groups. The results also emphasized that effect of lead acetate was dose dependent. As the dosage of lead was increased, the intensity of damage to liver also found to be increased. Maximum alterations in all biochemical parameters were found at the dosage of 5 ppm. The results of the current study strongly suggested that lead had affected antioxidant system, protein content and activities of specific metabolic enzymes of liver homogenate. Co-administration of herbal aqueous extract of Phyllanthus fraternus (5 mg/ml) along with 5 ppm lead acetate to liver homogenate exerted an ameliorative effect and maintained the studied parameters closed to control group, which confers its protective role against lead induced hepatotoxicity.

Evaluation of Genotoxic Effects of Asbestos on Occupationally Exposed Workers in Brazil

Abstract Genotoxic effects of occupational workers exposed to asbestos can be evaluated using different biomarkers as oxidative stress enzymes in conjuction with comet assay. This study assessed changes to oxidative stress enzymatic parameters and genotoxic damage in workers occupationally exposed and non-exposed to chrysotile asbestos, who attended the outpatient Clinic of the Center for Worker Health Studies and Human Ecology (CESTEH/ENSP/FIOCRUZ) in Brazil. Chest radiography and spirometry were performed to assess clinical progression of symptoms. The traditional visual score comet assay in peripheral whole blood cells was used to assess DNA damage, and oxidative stress was evaluated by measuring catalase (CAT) and glutathione S-transferase (GST) activities. Respiratory alterations were observed in 53% of workers exposed, as determined by pulmonary function and bronchodilation, and 6 workers were diagnosed with asbestosis. The comet assay was statistically significantly higher in the exposed group and individuals with asbestosis compared to the non-exposed group and individuals without asbestosis, respectively. Linear regression analysis showed that 28,4% and 50,5% of comet assay results were increased by exposure to asbestos and developed asbestosis. The results of CAT and GST were not difference between the groups. These results supports the association of genotoxic damage and the onset of asbestosis by chrysotile asbestos exposure in workers of this study.

Quality Assessment of a Battery of Organic Wastes and Composts Using Maturity, Stability and Enzymatic Parameters

Chemical and biological parameters (NH4 +–N/NO3 −–N ratio, humification indices, and the activities of hydrolytic exoenzymes), commonly used to assess compost maturity and/or stability, were considered for the quality evaluation of a battery of organic materials, intended to be land applied. Acid and alkaline phosphatases, β-glucosidase, proteases and β-glucosaminidase activities proved to be reliable tests to distinguish the organic materials that were in an active stage of microbial activity, highly correlated to the chemical parameters NH4 +–N content and NH4 +–N/NO3 −–N ratio. In fact, these chemical parameters evidenced as important in the quality assessment of an organic material, strongly correlated with the biological parameters. The same was not true for the majority of the humification indices, which proved inadequate to compare the quality of such diverse organic materials. This was demonstrated by a multivariate statistical treatment of data, performed with these results in combination with results from the Dewar self-heating test, respiration activity and germination index, obtained in previous studies. Concluding, in a similar scenario, where the organic materials in evaluation are varied, both in the raw material an in the stability of the organic matter, the quality should be assessed by the integrated use of both chemical and biological parameters.

Optimization of Process Parameters and Kinetic Model of Enzymatic Extraction of Polyphenols from Lonicerae Flos.

Objective:To optimize and verify the cellulase extraction of polyphenols from honeysuckle and provide a reference for enzymatic extracting polyphenols from honeysuckle.Materials and Methods:The uniform design was used According to Fick's first law and kinetic model, fitting analysis of the dynamic process of enzymatic extracting polyphenols was conducted.Results:The optimum enzymatic extraction parameters for polyphenols from honeysuckle are found to be 80% (v/v) of alcohol, 35:1 (mL/g) of liquid-solid ratio, 80°C of extraction temperature, 8.5 of pH, 6.0 mg of enzyme levels, and 130 min of extraction time. Under the optimal conditions, the extraction rate of polyphenols was 3.03%. The kinetic experiments indicated kinetic equation had a good linear relationship with t even under the conditions of different levels of enzyme and temperature, which means fitting curve tallies well with the experimental values.Conclusion:The results of quantification showed that the results provide a reference for enzymatic extracting polyphenols from honeysuckle.SUMMARY Lonicerae flos (Lonicera japonica Thunb.) is a material of traditional Chinese medicine and healthy drinks, of which active compounds mainly is polyphenols. At present, plant polyphenols are the hotspots centents of food, cosmetic and medicine, because it has strong bioactivity. Several traditional methods are available for the extraction of plant polyphenols including impregnation, solvent extraction, ultrasonic extraction, hot-water extraction, alkaline dilute alcohol or alkaline water extraction, microwave extraction and Supercritical CO2 extraction. But now, an increasing number of research on using cellulase to extract active ingredients from plants. Enzymatic method is widely used for enzyme have excellent properties of high reaction efficiency and specificity, moderate reaction conditions, shorter extraction time and easier to control, less damage to the active ingredient. At present, the enzymatic extraction of polyphenols from honeysuckle and dynamic had not been reported. In this study, using cellulase to extract polyphenols from honeysuckle is first applied. Moreover, uniform design was used to optimize process and kinetic model of extraction was established to analyze the characteristics of enzymatic extraction, in order to improve the yield of polyphenols from honeysuckle and make maximum use of Lonicerae flos, which provide references for industrial production.

Acute and Repeated Oral Toxicity of Antidiabetic Polyherbal Formulation Flax Seed, Fenugreek and Jamun Seeds in Wistar Albino Rat

Polyherbal formulations treating for various diseases bring attention to the human society. The toxicity and its safe dosage should be studied for dose optimization and formulation. The aim of this study was to evaluate acute and repeated oral toxic effect of the hydroalcoholic extract of polyherbal formulation such as flax seed, fenugreek and jamun seed (FFJ) on wistar albino rats. Acute toxicity was tested by single oral stepwise administration of the drug at the dose of 300 mg, 2000 mg and 5000 mg/kg bw. The toxicity and mortality were observed for every 30 minutes for first 24 hours and then day by day for 14 days. At the 29th day, the blood was collected by Sino orbital puncture for analysis of hematological and biochemical parameters. Then the animals were killed by cervical decapitation and organs were separated and weighed. The results showed that no toxic symptoms were observed up to the dose of 2000 mg/kgbw. But slight changes in the stool consistency, tremor, lethargy and sleep were observed at the dose of 5000 mg/kg bw. There is no significant change in the biochemical and liver enzymatic parameters of different treated groups of polyherbal formulations. The control group animals was observed as Blood Glucose (99.7 ± 4.8), Urea (30.6 ± 1.7), Creatinine (1.08 ± 0.07) and Aspartate aminotransferase (AST) (32 ± 2.36), Alanine aminotransferases (ALT) (46 ± 1.80), Alkaline phosphatase (ALP) (164 ± 1.30). Similarly, hematological parameters such as RBC (5.63 ± 0.23), WBC (6.52 ± 0.32), ESR (8 ± 0.53), PCV (44.8 ± 2.314), MCV (79.6 ± 4.18), MCH (26.4 ± 1.2) and MCHC (33.2 ± 2.5) don’t show any significant alterations.

Efficacy Study of Livartho against Paracetamol Induced Hepatotoxicity in Adult Sprague Dawley Rats

Liver disease is a worldwide problem. It is an exposed to many kinds of xenobiotics and therapeutic agents. The purpose of this study was to evaluate therapeutic effect of Livartho drug in paracetamol induced hepatic damage Sprague dawley rats. Paracetamol at the dose level of 900mg/kg body weight administered through intraperitoneal route once daily for one week to produced liver damage in rats as manifested by the significantly decrease body weight and feed consumption along with significantly increase liver organ weight and serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), total bilirubin, cholesterol and triglyceride and decrease the total protein and albumin levels along with significantly increase myloperoxidase enzyme activity, lipid peroxidation and protein carbonyl levels as compared to control group. The antioxidant parameters such as reduced glutathione (GSH), oxidized glutathione (GSSG), total thiol and GSH/ GSSG ratio were also found lowered in paracetamol treated group as compared with control. After treatment with Livartho drug (206.6 mg/kg body weight) once daily for 16 days to paracetamol treated rats shows significantly lowered the afore mentioned biochemical parameter whereas protein and albumin levels were increased along with significant increased above mentioned antioxidant enzymatic parameters. The results of this study strongly indicate that Livartho drug is most effective medicine which has potent hepatoprotective action and free radical scavenger against paracetamol induced hepatic damage in Sprague dawley rats.

Enzymatic saccharification of high pressure assist-alkali pretreated cotton stalk and structural characterization

Cotton stalk is a potential biomass for bioethanol production, while the conversion of direct saccharification or biotransformation of cotton stalk is extremely low due to the recalcitrant nature of lignocellulose. To enhance the enzymatic conversion of cotton stalks, the enzymatic saccharification parameters of high pressure assist-alkali pretreatment (HPAP) cotton stalk were optimized in the present study. Results indicated that a maximum reducing sugar yield of 54.7g/100g dry biomass cellulose was achieved at a substrate concentration of 2%, 100rpm agitation, 0.6g/g enzyme loading, 40°C hydrolysis temperature, 50h saccharification time, and pH 5.0. Scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy were used to identify structural changes in native, pretreated biomass and hydrolyzed residues. Structural analysis revealed large part of amorphous cellulose and partial crystalline cellulose in the HPAP cotton stalk were hydrolyzed during enzymatic treatment. HPAP cotton stalk can be used as a potential feed stock for bioethanol production.

Crystal structure of phosphoglucomutase from Leishmania major at 3.5Å resolution.

The crystal structure of phosphoglucomutase (LmPGM) from the parasite Leishmania major has been solved at 3.5Å resolution. Although the active form of the enzyme is monomeric in solution, four molecules (A, B, C, D) were found in the asymmetric unit, of which the pairs (A,D) and (B,C) were of identical inter-subunit geometry. The parasitic enzyme constituted of four domains exhibited the canonical 'heart' shape of the protein, with domains I to III having a conserved α|β core, while the fourth (IV) domain being structurally distinct from the rest. Conformational variability of the IVth domain, postulated to be responsible for the catalytic function of the enzyme has been studied by normal mode analysis (NMA) and the conformational features responsible for domain movement in the 'hinge region' analyzed in detail. Although the active site of phosphoglucomutase is highly conserved from parasite to human, initial calculations show that a ligand binding pocket could exist near the hinge region, which is unique to the parasite. The enzymatic parameters of LmPGM have been determined and compared with other PGMs from orthologous species in addition to elucidating its mechanism of action by docking the substrate, intermediate onto the active site.

A continuous kinetic assay for protein and DNA methyltransferase enzymatic activities.

BackgroundMethyltransferases (MTs) catalyze the S-adenosylmethionine (SAM)-dependent methylation of a wide variety of protein and DNA substrates. Methylation of lysine, arginine or cytosine regulates a variety of biological processes including transcriptional activation and gene silencing. Despite extensive studies of the cellular roles of MTs, their quantitative kinetic characterization remains challenging. In the past decade, several assays have been developed to monitor methyl transfer activity utilizing different approaches including radiolabeling, antibodies or mass-spectrometry analysis. However, each approach suffers from different limitation and no easy continuous assay for detection of MT activity exists.ResultsWe have developed a continuous coupled assay for the general detection of MTs activity. In this assay, the formation of S-adenosylhomocysteine (SAH) product is coupled NAD(P)H oxidation through three enzyme reactions including glutamate dehydrogenase leading to absorbance changes at 340 nm. The utility and versatility of this assay is demonstrated for SET7/9 and SETD6 with peptides and full length protein substrates and for M.HaeIII with a DNA substrate.ConclusionsThis study shows a simple and robust assay for the continuous monitoring of MT enzymatic activity. This assay can be used for the determination of steady-state kinetic enzymatic parameters (e.g., kcat and KM) for a wide variety of MTs and can be easily adapted for high-throughput detection of MT activity for various applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0048-y) contains supplementary material, which is available to authorized users.

Human Intestinal Raf Kinase Inhibitor Protein (RKIP) Catalyzes Prasugrel as a Bioactivation Hydrolase

Prasugrel is a thienopyridine antiplatelet prodrug that undergoes rapid hydrolysis in vivo to a thiolactone metabolite by human carboxylesterase-2 (hCE2) during gastrointestinal absorption. The thiolactone metabolite is further converted to a pharmacologically active metabolite by cytochrome P450 isoforms. The aim of the current study was to elucidate hydrolases other than hCE2 involved in the bioactivation step of prasugrel in human intestine. Using size-exclusion column chromatography of a human small intestinal S9 fraction, another peak besides the hCE2 peak was observed to have prasugrel hydrolyzing activity, and this protein was found to have a molecular weight of about 20 kDa. This prasugrel hydrolyzing protein was successfully purified from a monkey small intestinal cytosolic fraction by successive four-step column chromatography and identified as Raf-1 kinase inhibitor protein (RKIP) by liquid chromatography-tandem mass spectrometry. Second, we evaluated the enzymatic kinetic parameters for prasugrel hydrolysis using recombinant human RKIP and hCE2 and estimated the contributions of these two hydrolyzing enzymes to the prasugrel hydrolysis reaction in human intestine, which were approximately 40% for hRKIP and 60% for hCE2. Moreover, prasugrel hydrolysis was inhibited by anti-hRKIP antibody and carboxylesterase-specific chemical inhibitor (bis p-nitrophenyl phosphate) by 30% and 60%, respectively. In conclusion, another protein capable of hydrolyzing prasugrel to its thiolactone metabolite was identified as RKIP, and this protein may play a significant role with hCE2 in prasugrel bioactivation in human intestine. RKIP is known to have diverse functions in many intracellular signaling cascades, but this is the first report describing RKIP as a hydrolase involved in drug metabolism.

Respostas antioxidativas, fisiológicas e produtiva de arroz cultivado sob deficiência hídrica e adubação silicatada

<p>O silício é um nutriente que pode proporcionar melhor desenvolvimento às plantas de arroz cultivadas em ambientes com deficiência hídrica. Este elemento diminui as perdas de água por transpiração, aumenta a taxa fotossintética e atua como ativador do sistema de defesa enzimático. O objetivo deste trabalho foi analisar o efeito do silício em dois cultivares de arroz (Curinga e o IAC 202) submetidos à deficiência hídrica, sobre os parâmetros enzimáticos, fisiológicos e produtivo. Foram utilizados quatro tensões de água no solo (-10, –30, –50 e –70 kPa) combinadas com duas formas de correção da saturação do solo (ausência e presença de silício). Foram avaliados a atividade enzimática de Superóxido dismutase, Catalase e Ascorbato Peroxidase, a taxa de assimilação de CO2 e a produção de grãos. O delineamento experimental foi em blocos casualizado em esquema fatorial 4 x 2, com quatro repetições, sendo as médias comparadas pelo teste de Tukey (p < 0,05). A atividade enzimática aumentou com a aplicação de silício mesmo em condições de deficiência hídrica mais intensa. A taxa de assimilação de CO2 e a produção de grãos também aumentaram com a adição de silício. A aplicação de silício em ambos cultivares de arroz ameniza os efeitos do estresse ocasionado pela deficiência hídrica.</p>

Effects of Sodium Arsenite and Arsenate in Testicular Histomorphometry and Antioxidants Enzymes Activities in Rats.

The main source of environmental arsenic exposure in most countries of the world is drinking water in which inorganic forms of arsenic predominate. The present study was aimed to test the impact of two different compounds of inorganic arsenic in histomorphometric and enzymatic parameters in the testes by oral exposition. Adult Wistar male rats were exposed to sodium arsenite and arsenate in drinking water, testing for each chemical form the concentrations of 0.01 and 10mg/L per 56days. The animals intoxicated with arsenic, mainly sodium arsenite, showed reduction in the percentage of seminiferous epithelium and in proportion and volume of Leydig cells. Moreover, there was an increase in the percentage of tunica propria, lumen, lymphatic space, blood vessels, and macrophages. The activity of superoxide dismutase (SOD) did not change among the groups. However, the activity of catalase (CAT) decreased in animals exposed to both arsenic compounds. In addition, the higher concentration of arsenic, mainly as sodium arsenite, caused vacuolization in the seminiferous epithelium. The body and testes weight as well as testosterone concentration remained unchanged among the groups. In conclusion, exposition to arsenic, mainly as sodium arsenite, caused alteration in histomorphometric parameters and antioxidant defense system in the testes.

Phosphatidylinositol kinase activities in Trypanosoma cruzi epimastigotes

Phosphatidylinositol (PtdIns) metabolism through phosphatidylinositol kinase (PIKs) activities plays a central role in different signaling pathways. In Trypanosoma cruzi, causative agent of Chagas disease, PIKs have been proposed as target for drug design in order to combat this pathogen. In this work, we studied the classes of PI4K, PIPK and PI3K that could participate in signaling pathways in T. cruzi epimastigote forms. For this reason, we analyzed their enzymatic parameters and detailed responses to avowed kinase inhibitors (adenosine, sodium deoxycholate, wortmannin and LY294002) and activators (Ca2+, phosphatidic acid, spermine and heparin). Our results suggest the presence and activity of a class III PI4K, a class I PIPK, a class III PI3K previously described (TcVps34) and a class I PI3K. Class I PI3K enzyme, here named TcPI3K, was cloned and expressed in a bacterial system, and their product was tested for kinase activity. The possible participation of TcPI3K in central cellular events of the parasite is also discussed.

Antioxidative and immunological responses in the haemolymph of wolf spider Xerolycosa nemoralis (Lycosidae) exposed to starvation and dimethoate

The aim of this study was to assess the intensity of enzymatic antioxidative parameters [catalase (CAT), glutathione peroxidase (GSTPx), glutathione reductase (GR), total antioxidant capacity (TAC)] and percentage of high granularity cells as well as low to medium granularity cells in haemolymph of wolf spiders Xerolycosa nemoralis exposed to starvation and dimethoate under laboratory conditions. Only in starved males, haemolymph included a lower percentage of high granularity cells, accompanied by high activity of CAT and GSTPx, than in the control. Exposure of males to dimethoate increased CAT activity, after single application, and significantly enhanced GR activity, after five-time application. In females, five-time contact with dimethoate elevated the percentage of high granularity cells. As in comparison to females, male X. nemoralis were more sensitive to the applied stressing factors, it may be concluded that in natural conditions both food deficiency and chemical stress may diminish the immune response of their organisms.

Azinphos-methyl and chlorpyrifos, alone or in a binary mixture, produce oxidative stress and lipid peroxidation in the freshwater gastropod Planorbarius corneus

Azinphos-methyl (AZM) and chlorpyrifos (CPF) are broad-spectrum organophosphate insecticides used for pest control on a number of food crops in many parts of the world that have been shown to inhibit cholinesterase activity in the non-target freshwater gastropod Planorbarius corneus. The present study was undertaken to determine: (a) whether AZM and CPF induce oxidative stress in P. corneus, and (b) whether a mixture of both organophosphates that causes a higher neurotoxicity than single pesticides also causes an enhanced oxidative stress. To this end, non-enzymatic and enzymatic parameters were measured in the soft tissues of snails acutely exposed to the insecticides in single-chemical (2.5mg AZML−1 and 7.5μgCPFL−1) and a binary-mixture (1.25mgAZML−1 plus 3.75μgCPFL−1) studies. At 24h, all pesticide-exposed groups showed significantly decreased glutathione (GSH) and glutathione disulfide (GSSG) levels when compared to control animals. At 48h, all exposed groups showed an alteration of the redox status (GSH/GSSG ratio) and a significant increase in malondialdehyde levels. The exposure for 48h to AZM and CPF, alone or in the binary mixture, also resulted in a significant decrease of the antioxidant superoxide dismutase activity. The greatest decrease was observed with CPF exposure (59% of decrease relative to the control group). A significant increase in catalase and glutathione S-transferase activities was observed in CPF group and in CPF and AZM+CPF groups, respectively. The activities of glutathione reductase and glucose 6-phosphate dehydrogenase did not show significant changes with respect to controls in any treatment group. In conclusion, the data shown in the present study provide evidence that AZM, CPF and a mixture of both organophosphates are able to induce oxidative stress and oxidative damage in P. corneus tissues. However, no similarities between the degree of neurotoxicity and the degree of alterations of the measured oxidative stress parameters were found.

Molecular and biochemical characterization of the UDP-glucose: Anthocyanin 5-O-glucosyltransferase from Vitis amurensis

Generally, red Vitis vinifera grapes only contain monoglucosidic anthocyanins, whereas most non-vinifera red grapes of the Vitis genus have both monoglucosidic and bis-glucosidic anthocyanins, the latter of which are believed to be more hydrophilic and more stable. Although previous studies have established the biosynthetic mechanism for formation of monoglucosidic anthocyanins, less attention has been paid to that of bis-glucosidic anthocyanins. In the present research, the full-length cDNA of UDP-glucose: anthocyanin 5-O-glucosyltransferase from Vitis amurensis Rupr. cv. ‘Zuoshanyi’ grape (Va5GT) was cloned. After acquisition and purification of recombinant Va5GT, its enzymatic parameters were systematically analyzed in vitro. Recombinant Va5GT used malvidin-3-O-glucoside as its optimum glycosidic acceptor when UDP-glucose was used as the glycosidic donor. Va5GT-GFP was found to be located in the cytoplasm by analyzing its subcellular localization with a laser-scanning confocal fluorescence microscope, and this result was coincident with its metabolic function of modifying anthocyanins in grape cells. Furthermore, the relationship between the transcriptional expression of Va5GT and the accumulation of anthocyanidin bis-glucosides during berry development suggested that Va5GT is a key enzyme in the biosynthesis of bis-glucosidic anthocyanins in V. amurensis grape berries.

Serological, biochemical and enzymatic alterations in rodents after experimental envenomation with Hadruroides lunatus scorpion venom.

Toxic effects of Peruvian Hadruroides lunatus scorpion venom on different biochemical and enzymatic parameters in blood serum of Wistar rats and Swiss mice were determined after experimental envenomation. An increase in enzymatic activities of Aspartate Aminotransferase (AST), Lactate Dehydrogenase (LDH) and levels of serum protein and albumin were observed while a decrease in creatinine level in serum was perceived after 30 min of envenomation. No alterations in urea levels and in kidney histology were detected in the envenomed rats. The global leukocytes count was diminished, with decrease in lymphocytes, eosinophils and neutrophils levels in the bloodstream, while no alterations were found in hematological parameters of red series in rats injected with H. lunatus venom. IL-2, IL-4, IL-6, INF-γ, TNF, IL-17A and IL-10 levels were evaluated 0.5, 3 and 6 h after experimental envenomation of mice with H. lunatus venom. From all the analyzed cytokines, only IL-6 showed an increase in serum levels. Taken together, these results point out that envenomation by H. lunatus can impair hematological and immunological parameters and therefore might be monitored in accidents involving this species.

표면반응을 이용한 넙치부산물의 높은 수압 가수분해의 최적화

High hydrostatic pressure (HHP) enzymatic hydrolysis was applied to the flatfish byproducts in order to develop the functional food materials. Proximate compositions of FFP were determined and its crude protein content, which could be converted to bioactive peptide, was 20.4%. The degree of hydrolysis (DH) of FFP hydrolysates were investigated depending on the process parameters such as pressure, temperature, substrate/enzyme (S/E) ratio, pH and type of enzyme. The results showed that temperature, S/E ratio and pH are major independent parameters to optimize a low-cost commercial process under 100 MPa fixed pressure using Protamex protease. Response surface methodology (RSM) was applied to study the effects of three major HHP enzymatic hydrolysis parameters, and the RSM statistical analysis results showed the hydrolysis parameters for optimal DH; S/E ratio of 50.65% (w/w), pH 6.898 and temperature of 56.97 o C. Under the optimal condition, the DH of FFP was predicted as 32.09%. To enhance the bioavailability, flatfish byproducts hydrolysates were processed through a series of ultrafiltration membrane with molecular weight cut-off (MWCO) 10, 5 and 3 kDa. Four types of permeates including below 3 kDa, 3-5 kDa, 5-10 kDa and over 10 kDa were obtained and a comparative analysis of amino-acid compositions was conducted. These results suggest that HHP enzymatic hydrolysis is an effective processing method for utilization of flatfish byproducts as functional food materials.

Characterisation of a Recombinant Patchoulol Synthase Variant for Biocatalytic Production of Terpenes.

The patchoulol synthase (PTS) is a multi-product sesquiterpene synthases which is the central enzyme for biosynthesis of patchouli essential oil in the patchouli plant. Sesquiterpene synthases catalyse the formation of various complex carbon backbones difficult to approach by organic synthesis. Here, we report the characterisation of a recombinant patchoulol synthase complementary DNA (cDNA) variant (PTS var. 1), exhibiting significant amino acid exchanges compared to the native PTS. The product spectrum using the natural substrate E,E-farnesyl diphosphate (FDP) as well as terpenoid products resulting from conversions employing alternative substrates was analysed by GC-MS. In respect to a potential use as a biocatalyst, important enzymatic parameters such as the optimal reaction conditions, kinetic behaviour and the product selectivity were studied as well. Adjusting the reaction conditions, an increased patchoulol ratio in the recombinant essential oil was achieved. Nevertheless, the ratio remained lower than in plant-derived patchouli oil. As alternative substrates, several prenyl diposphates were accepted and converted in numerous compounds by the PTS var. 1, revealing its great biocatalytic potential.