HOMOGENEOUS ENZYME IMMUNOASSAY OF SERUM AFP BY FLUORIMETRIC KINETIC METHOD

The activity of the Ab-bound enzyme(HRP)changed after immunochemical reaction,and can be indicated by a sensitive fluorimetric kinetic method.The finding set the stage for developing sensitive homogeneous immunoassay.AFP was measured as an example.

银杏外种皮对钉螺的杀灭机理

To study the toxicity of extracts of Ginkgo biloba sarcotesta to Oncomelania hupensis,snails were exposed to 40% and 80% of 24 h LC 50 of the extract of Ginkgo bilba for 24 h,choline esterase(ChE),alanine aminotransferase(ALT),alkaline phosphatase(ALP),lactate dehydrogenase(LDH),succino dehydrogenase(SDH),malic dehydrogenase(MDH)activities in cephalopodium and liver were determined by enzyme kinetic assay.Arecoline and niclosamide were used as reference molluscicides.The results showed that sarcotesta of Ginkgo biloba could inhibit ChE,ALT,ALP and MDH activities both in cephalopodium and liver;arecoline could inhibit ChE,ALP,SDH and MDH activities in cephalopodium and ChE,ALT,ALP,SDH and MDH activities in liver.Niclosamide had inhibitory effects upon ChE,ALT,ALP,SDH and MDH activities in cephalopodium,and ChE,ALT,ALP and SDH activities in liver.All three molluscicides did not inhibite LDH activity in cephalopodium and liver.These results indicate that lethal effects of extracts of sarcotesta of Ginkgo biloba are mediated via inhibition of MDH activitiy,and interference with the NADH respiratory chains.Inhibition of vital enzymic mechanisms causes snails to death.

Characterization of Carboxylesterase Associated with Malathion Insensitivity in the Field Population of the Oriental Migratory Locust

Carboxylesterases （CarEs） from two field populations of the oriental migratory locust, Locusta migratoria manilensis （Meyen）, were examined to try to understand their contribution to malathion insensitivity. The CarEs activities in Wudi population （WD） were 1.75- and 1.50-fold significantly higher than those in Huangliu population （HL） when a-naphthyl acetate （a-NA） and [3-naphthyl acetate were used as substrates, respectively. Such elevated CarEs activities presented in the WD could be because of an increased staining intensity of the a-NA-hydrolyzing CarEs as shown on the nondenaturing polyacrylamide gel electrophoresis. Inhibition studies of CarEs using paraoxon and malaoxon indicated that CarE activities in the HL were more strongly inhibited than those in the WD. Furthermore, a 449-bp DNA fragment of CarE was obtained from L. migratoria manilensis. Hemiquantity reverse transcription-polymerase chain reaction analysis showed that CarE gene expression level in the WD was higher than that in the HL. The higher CarE activities and the increased CarE mRNA level in the WD appeared to be associated with decreased susceptibility to malathion in the WD due to the application of organophosphorus insecticides.

Comparisons of Properties of Acetylcholinesterase from Two Field-Collected Populations of Oxya chinensis Thunberg(Orthoptera:Acrididae)and the Role of Acetylcholinesterase in the Susceptibility to Malathion

In this study, acetylcholinesterase (AChE) was extracted from two field-collected populations of Oxya chinensis (XinxiangCity, Henan Province and Changzhi City, Shanxi Province). AChE activities were decreased when concentrations of ATCincreased, showing a characteristic phenomenon of substrate inhibition at high concentration in both populations. Suchinhibition occurred at relatively low concentration for AChE from Xinxiang population but relatively high for AChE fromChangzhi population. The kinetic study showed that there were no significant differences between the two populations inthe Km values. The Km value in Changzhi population was only 1.09-fold higher than that in Xinxiang population. However,significant differences were observed between the two populations in Vmax values. The Vmax value in Changzhi populationwas 1.32-fold higher than that in Xinxiang population. The inhibition study in vitro showed that the AChE from bothpopulations exhibited similar rank order in sensitivity to inhibition by three OPs, as determined by comparison of theirbimolecular rate constants (ki), from the most potent inhibition to the least was chlopyrifos-oxon > paraoxon >demeton-s-methyl for AChE from the two populations and that the ki values in Xinxiang population were lower than those in Changzhipopulation. The I50 values of AChE from Xinxiang population were 4.84-, 2.66-, and 1.92-fold less sensitive to inhibition byparaoxon, chlopyrifos-oxon, and demeton-s-methyl. These results were consistent with the results in bioassay. It isinferred that AChE insensitivity to OP insecticides plays an important role in the differences of insusceptibility of Oxyachinensis to malathion between the two populations.

The Susceptibilities of Oxya chinensis(Orthoptera:Acridoidea) to Malathion and Comparison of the Esterase Properties from Three Collected Populations in Tianjin Area,China

The susceptibilities of Oxya chinensis to malathion were studied in three populations collected from outskirt of Tianjin, China, using bioassays and biochemical analysis. Populations were chosen to represent different exposure to insecticides： BDG （Beidagang; low exposure）, BD （Baodi; high exposure previously but low exposure now）, and JN （Jinnan; high exposure）. The results showed that the LD50 values of BD and JN populations were 3.95- and 12.02-fold and 3.64- and 10.07- fold higher than that of BDG population in females and males, respectively. The LD50 values in females were higher than those in males. The results of biochemical analysis indicated that the esterase （EST） activities in JN population were higher than those in BD and BDG populations. They showed that when α-NA, α-NB, and α-NA were used as substrates, females＇ EST activities of JN population were 1.11-, 1.30-, and 1.14-fold and 1.39-, 1.59-, and 1.54-fold higher than those of BD and BDG populations, respectively. When α-NA, α-NB, and β-NA were used as substrates, males＇ EST activities of JN population were 1.13-, 1.12-, and 1,00-fold and 1.20-, 1.14-, and 1.07-fold higher than those of BD and BDG populations, respectively. The results also showed that the specific activities of the females were higher than those of the males in the BD and JN populations, whereas the specific activities of the males were higher than those of the females in the BDG population. The results of bioassay were consistent with those of biochemical analysis. Thus, it was inferred that the elevated ESTs activities might play an important role in conferring the differences of susceptibility of O. chinensis to malathion in the three collected populations. Enzyme kinetic studies indicated that the Km and Vmax values were different among the three collected populations and between the females and the males. The observed changes in the kinetic parameters might be explained by differential expression patterns of isozymes so that the insect esterases have different affinities and maximum velocities toward the same substrate.

Screening and Statistical Optimization of Physiochemical Parameters for the Production of Xylanases from Agro-Industrial Wastes

Xylanases are mostly produced through submerged fermentation;nonetheless solid-state fermentation has increased profound attention and consideration of scholars having high conversion level biomass to energy conservation. This study depicted the purification of xylanases and their possible utilization in industry. The present study was carried out to examine the culture influence of fungal strain Fomes fomentarius (F. fomentarius) using different agro-industrial residues (wheat straw, rice husk, sugarcane bagasse and siris pods). F. fomentarius showed maximum enzyme production after 72 h of fermentation, when grown on wheat straw in solid state fermentation process while maximum activity showed on pH 6.0 at 30°C. The other parameters optimized by statistical design (RSM) showed maximum xylanase activity (146 ± 8 IU/mL) at 65% moisture content, 4 mL inoculums size, 175 mg Ammonium sulphate, 200 mg Calcium carbonate and 1.4 grams of glucose. Xylanase was salted out at 60% ammonium sulphate concentration and enzyme was further purified by Sephadex G-100 gel filtration chromatography with 2.2 fold increase in activity. The purified xylanase from F. fomentarius had optimum pH 6.0 and 40°C. Xylanase showed higher specificity for oat spelt xylan with kinetic constants Km 1.25 mg/mL and Vmax 54 mM/min. Xylanases have an industrial important enzyme used extensively in food, feed and paper industry.

Effects of ausforming strain on bainite transformation in nanostructured bainite steel

The effects of ausforming strain on bainite transformation in high-carbon low-alloy nanobainite steel were investigated using a Gleeble 3500 thermomechanical simulator machine. The bainite transformation speed at 300°C was found to be accelerated by ausforming at 300, 600, and 700°C under applied strains ranging from 10% to 50% followed by isothermal transformation at 300°C. The ausformed bainite volume fraction varied with the ausforming strain because of the mechanical stabilization of the deformed austenite. Ausforming at low temperatures not only enhanced the bainite ferrite volume fraction but also refined the microstructure substantially. Although the amount of bainite ferrite might have been reduced with increasing strain, the microstructures were refined by ausforming. © 2017, University of Science and Technology Beijing and Springer-Verlag Berlin Heidelberg.

Enzymatic Activity of Escherichia Coil Lactate Dehydrogenase and Cytochrome c Oxidase

The catalytic activity of two common bacterial enzymes, lactate dehydrogenase （LDH） and cytochrome c oxidase （COX） from Escherichia coli was examined following bacterial exposure to microwave （MW） radiation under well-defined experimental conditions. The experiments were conducted in a specialized microwave processing apparatus, with an exposure frequency of 18 GHz, and a temperature profile that was restricted to below 40℃ to avoid thermal degradation of the bacteria. The absorbed power was calculated to be 1,500 kW/m3 and the electric field was determined to be 300 Wm. Both values were theoretically confirmed using Computer Simulation Technology （CST） Microwave Studio 3D Electromagnetic Stimulation Software. Results showed that the activity of both enzymes was increased following MW radiation compared to negative controls and thermally treated samples subjected to similar temperature profiles. It is suggested that the increase in COX and LDH enzyme activity could not be explained by conventional heating alone, but was rather a result of micro-thermal effects that incorporated ＇undetectable＇ thermal mechanisms.

Simultaneous heterotrophic nitrification and aerobic denitrification at high initial phenol concentration by isolated bacterium Diaphorobacter sp. PD-7

A strain capable of phenol degradation, hetemtrophic nitrification and aerobic denitrification was isolated from activated sludge of coking-plant wastewater ponds under aerobic condition. Based on its morphology, physiology, biochemical analysis and phylogenetic characteristics, the isolate was identified as Diaphorobacter sp. PD-7. Biodegradation tests of phenol showed that the maximum phenol degradation occurred at the late phase of exponential growth stages, with 1400 mg·L^-1 phenol completely degraded within 85 h. Diaphorobacter sp. PD-7 accumulated a vast quantity of phenol hydroxylase in this physiological phase, ensuring that the cells quickly utilize phenol as a sole carbon and energy source. The kinetic behavior ofDiaphorobacter sp. PD-7 in batch cultures was investigated over a wide range of initial phenol concentrations （0-1400mg·L^-1） by using the Haldane model, which adequately describes the dynamic behavior of phenol biodegradation by strain Diaphombacter sp. PD-7. At initial phenol concentration of 1400mg· L^-l, batch experiments （0.25 L flask） of nitrogen removal under aerobic condition gave almost entirely removal of 120.69mg· L^- 1 ammonium nitrogen within 75 h, while nitrate nitrogen removal reached 91% within 65 h. Moreover, hydroxylamine oxidase, periplasmic nitrate reductase and nitrite reductase were successfully expressed in the isolate.

Seasonal dynamics,kinetics,and effects of 2-hydroxyestradiol-17βon some steroidogenic enzymes in the ovary of the catfish Heteropneustes fossilis

The synthesis of bioactive steroids is catalyzed by an array of enzymes of diverse properties and actions.In the present study,seasonal dynamics and kinetics of key steroidogenic enzymes,17α-hydroxylase(Cyp17a),3β-hydroxysteroid dehydrogenase(Hsd3b),20α-hydroxysteroid dehydrogenase(Hsd20a)and 20β-hydroxysteroid dehydrogenase(Hsd20b)were investigated in the female catfish Heteropneustes fossilis.Further,the effects of the estrogen metabolite 2-hydroxyestradiol-17β(2-OHE2)and human chorionic gonadotropin(hCG)on activities of the above enzymes,cytochrome P450 aromatase(Cyp19a1),and steroid products including testosterone and cortisol were determined.The enzymes under investigation showed significant seasonal variations across the annual ovarian cycle with low activity in the gonad resting phase.The enzymes Hsd3b and Cyp17a showed high activity during early oogenesis but the activities of Hsd20a and Hsd20b were higher towards late oogenesis in the spawning phase.Hsd3b and Cyp17a elicited high apparent Km values(low substrate affinity)and high apparent Vmax in the vitellogenic phase compared to the postvitellogenic phase.Hsd20a did not elicit any significant differences in the kinetic parameters between the two phases.Hsd20b showed high apparent Km values(low substrate affinity)and high Vmax in the postvitellogenic phase.The incubation of ovarian slices with 2-OHE2 for 24 h increased dose-dependently Hsd3b,Cyp17a,Hsd20a and Hsd20b activities,similar to hCG.The levels of the corresponding C21 steroid products,progesterone(P4),17α-hydroxyprogesterone(17-OHP4),17,20α-dihydroxy-4-pregnen-3-one(17,20α-DP)and 17,20β-dihydoxy-4-pregnen-3-one(17,20β-DP),and cortisol were elevated.However,2-OHE2 decreased significantly the C19 and C18 steroids,testosterone and E2 levels,and Cyp19a activity.The co-incubation with hCG and 2-OHE2 produced a synergistic effect on the enzyme activities except that of CYP19a.The co-incubation reversed the inhibitory effect of 2-OHE2.The data show that 2-OHE2 exerts a dual role on steroidogenesis,stimulating the C21 pathway and inhibiting the C19-C18 pathway,resulting in the steroidogenic shift.

Evaluation of arsenic pollution in field-contaminated soil at the soil’s actual pH

The pollution and ecological risks posed by arsenic(As)entering the soil are the major environmental challenges faced by human beings.Soil phosphatase can serve as a useful indicator for assessing As contamination under specific soil pH conditions.However,the study of phosphatase kinetics in long-term field As-contaminated soil remains unclear,presenting a significant obstacle to the monitoring and evaluation of As pollution and toxicity.The purpose of this study was to determine phosphatase activity and explore enzyme kinetics in soils subjected to long-term field As contamination.Results revealed that the soil phosphatase activity varied among the tested soil samples,depending on the concentrations of As.The relationship between total As,As fractions and phosphatase activity was found to be significant through negative exponential function fitting.Kinetic parameters,including maximum reaction velocity(Vmax),Michaelis constant(Km)and catalytic efficiency(V_(max)/K_(m)),ranged from 3.14×10^(−2)–53.88×10^(−2) mmol/(L·hr),0.61–7.92 mmol/L,and 0.46×10^(−2)–11.20×10^(−2) hr^(−1),respectively.Vmax and Vmax/Km of phosphatase decreased with increasing As pollution,while Km was less affected.Interestingly,Vmax/Km showed a significant negative correlationwith all As fractions and total As.The ecological doses(ED10)for the complete inhibition and partial inhibition models ranged from0.22–70.33 mg/kg and 0.001–55.27 mg/kg,respectively,indicating that V_(max)/K_(m) can be used as an index for assessing As pollution in field-contaminated soil.This study demonstrated that the phosphatase kinetics parameters in the soil’s pH system were better indicators than the optimal pH for evaluating the field ecotoxicity of As.

Heterologous expression and characterization of two chitinase 5 enzymes from the migratory locust Locusta migratoria

Insect chitinases are involved in degradation of chitin from the exoskeleton or peritrophic metrix ofmidgut. In Locusta migratoria, two duplicated Cht5s （LmCht5-1 and LmCht5-2） have been shown to have distinct molecular characteristics and biological roles. To explore the protein properties of the two LmCht5s, we heterologously expressed both enzymes using baculovirus expression system in SF9 cells, and characterized kinetic and carbohydrate-binding properties of purified enzymes. LmCht5-1 and LmCht5-2 exhibited similar pH and temperature optimums. LmCht5-1 has lower Km value for the oligomeric substrate （4MU-（GlcNAe）3）, and higher Km value for the longer substrate （CM-Chitin- RBV） compared with LmCht5-2. A comparison of amino acids and homology modeling of catalytic domain presented similar TIM barrel structures and differentiated amino acids between two proteins. LmCht5-1 has a chitin-binding domain （CBD） tightly bound to colloidal chitin, but LmCht5-2 does not have a CBD for binding to colloidal chitin. Our results suggested both LmCht5- I and LmCht5-2, which have the critical glutamate residue in region II of catalytic domain, exhibited chitinolytic activity cleaving both polymeric and oligomeric substrates. LmCht5-1 had relatively higher activity against the oligomeric substrate, 4MU-（GlcNAc）3, whereas LmCht5-2 exhibited higher activity toward the longer substrate, CM-Chitin-RBE These findings are helpful for further research to clarit}g their different roles in insect growth and development.

Analysis of the Inhibitory Effect of Gypenoside on Na^+, K^+-ATPase in Rats' Heart and Brain and Its Kinetics

Objective： To study the effects of gypenoside （Gyp） on the activity of microsomalNa^＋, K^＋-ATPase in rat＇s heart and brain in vitro. Methods： The microsomal Na^＋, K^＋-ATPase was prepared from rat＇s heart and brain by differential centrifugation. The activity of microsomal Na^＋, K^＋-ATPase was assayed by colorimetric technique. Enzyme kinetic analysis method was used to analyze the effect of Gyp on the microsomal Na^＋, K^＋-ATPase of rats. Results： Gyp reversibly inhibited the brain and heart＇s microsomal Na^＋, K^＋-ATPase in a concentration-dependent manner, and showed a more potent effect on enzyme in the brain. The IC50 of Gyp for the heart and brain were 58.79± 8.05 mg/L and 52.07± 6.25 mg/L, respectively. The inhibition was enhanced by lowering the Na^＋, or K^＋-concentrations or increasing the ATP concentration. Enzyme kinetic studies indicated that the inhibitory effect of Gyp on the enzyme is like that of competitive antagonist of Na^＋, the counter-competitive inhibitor for the substrate ATP, and the mixed-type inhibitor for K^＋. Cenclusien： Gyp displays its cardiotonic and central inhibitory effects by way of inhibiting heart and brain＇s microsomal Na^＋, K^＋-ATPase activities in rats.

Positive intercropping effects on biomass production are species-specific and involve rhizosphere enzyme activities: Evidence from a field study

Less attention has been given to soil enzymes that contribute to beneficial rhizosphere interactions in intercropping systems.Therefore,we performed a field experiment by growing faba bean,lupine,and maize in mono and mixed cultures in a moderately fertile soil.We measured shoot biomass and the kinetic parameters(maximal velocity(V max)and Michaelis-constant(K m))of three key enzymes in the rhizosphere:Leucine-aminopeptidase(LAP),β-1,4-N-acetylglucosaminidase(NAG),and phosphomonoesterase(PHO).Faba bean benefitted in mixed cultures by greater shoot biomass production with both maize and lupine compared to its expected biomass in monoculture.Next,LAP and NAG kinetic parameters were less responsive to mono and mixed cultures across the crop species.In contrast,both the V max and K m values of PHO increased in the faba bean rhizosphere when grown in mixed cultures with maize and lupine.A positive relative interaction index for shoot P and N uptake for faba bean showed its net facilitative interactions in the mixed cultures.Overall,these results suggest that over-productivity in intercropping is crop-specific and the positive intercropping effects could be modulated by P availability.We argue that the enzyme activities involved in nutrient cycling should be incorporated in further research.

Computation of kinetic isotope effects for enzymatic reactions

We describe a computational approach,incorporating quantum mechanics into enzyme kinetics modeling with a special emphasis on computation of kinetic isotope effects.Two aspects are highlighted:(1) the potential energy surface is represented by a combined quantum mechanical and molecular mechanical(QM/MM) potential in which the bond forming and breaking processes are modeled by electronic structure theory,and(2) a free energy perturbation method in path integral simulation is used to determine both kinetic isotope effects(KIEs).In this approach,which is called the PI-FEP/UM method,a light(heavy) isotope is mutated into a heavy(light) counterpart in centroid path integral simulations.The method is illustrated in the study of primary and secondary KIEs in two enzyme systems.In the case of nitroalkane oxidase,the enzymatic reaction exhibits enhanced quantum tunneling over that of the uncatalyzed process in water.In the dopa delarboxylase reaction,there appears to be distinguishable primary carbon-13 and secondary deuterium KIEs when the internal proton tautomerism is in the N-protonated or in the O-protonated positions.These examples show that the incorporation of quantum mechanical effects in enzyme kinetics modeling offers an opportunity to accurately and reliably model the mechanisms and free energies of enzymatic reactions.

Insights into the catalytic mechanism of a type I sulfide quinone oxidoreductase(SQR)from Acidithiobacillus caldus

Acidithiobacillus caldus,a typical sulfur oxidizer,derives the majority of its energy from sulfur oxidation.And the essential enzyme for sulfide oxidation catalysis is sulfide quinone oxidoreductase(SQR),an ancient flavoprotein.Here,the catalytic mechanism of SQR generated from A.caldus was investigated(SQR^(Ac)).According to phylogenetic study,SQR^(Ac)(ACAty RS11315)is closely related to SQR(BAD99305)of Acidithiobacillus ferrooxidans NASF-1 and is classified as a type I Sqr enzyme.SQR^(Ac)heterologously produced in Escherichia coli exhibits the distinctive absorption peaks(375,450 nm)of the flavoproteins family of proteins in its absorption spectrum.Utilizing site-directed mutagenesis,the function of conserved cysteines in the catalytic pathway was determined.Based on the sulfide quinone redox reactions in vitro of SQR^(Ac)and variations,Cys160 and Cys356 have been identified as enzyme-active residues.Mutation of another cysteine present in all type I SQRs(Cys128)decreased enzyme activity by 56%,indicating that this residue plays an important but non-essential role in enzyme function.In addition,the binding affinities of SQR^(Ac),the visualization of its 3D structure,and the interaction between receptors and ligands were investigated.Finally,a suitable sulfide quinone redox catalytic mechanism for A.caldus was proposed.