A MEMBRANE SEPARATOR/BIOREACTOR COUPLED WITH ABSORBANCE MEASUREMENT FOR DETECTION OF Escherichia coli O157:H7

A membrane separator/bioreactor system was developed for rapid detection of Escherichia coli O157:H7. The system consisted of a membrane separator/bioreactor (0.45 μm of the pore size) to separate the-complexes of E. coli O157:H7 and alkaline phosphatase-conjugated anti-E. coli O157:H7 antibodies from the sample and to produce p-nitrophenol through the enzymatic reaction (p-nitrophenyl phosphate hydrolysis), and an optical detector for measuring the p-nitrophenol absorbance at 400 nm. The membrane material and the flow rate of the substrate for the enzymatic hydrolysis had great effects on the absorbance of p-nitrophenol. The optimum conditions for the enzymatic reaction were determined as 1.0 M Tris buffer, pH 8.0, and 0.1 M MgCl₂ for this system. The detection range was 10⁴± 10⁷ CFU/mL with a relative standard deviation of 4.3 ± 14.2%, and whole procedure could be completed in 50 min without any enrichment and culture. Other bacteria such as Salmonella typhimurium, Campylobacter jejuni and Listeria monocytogenes had no significant interference with the detection of E. coli O157:H7.     

A spectrophotometric assay for the transphosphatidylation activity of phospholipase D enzyme

We developed a specific spectrophotometric assay for the quantitative determination of phospholipase D-catalyzed transphosphatidylation activity. The assay measures p-nitrophenol liberated by phospholipase D-catalyzed reaction of phosphatidyl-p-nitrophenol and ethanol in an aqueous-organic emulsion system. The release of p-nitrophenol was linear to reaction time at an early stage of the reaction with phospholipase D from Streptomyces sp. In the spectrophotometric assay for the reaction with phospholipase D from Streptomyces chromofuscus, which has higher hydrolytic activity than transphosphatidylation activity, p-nitrophenol was not found. The advantages of this novel method for measuring the transphosphatidylation activity of phospholipase D are that (i) it does not use radioactive compounds, (ii) it can measure the initial velocity of the reaction, and (iii) it is rapid, easy, and accurate to perform.     

A novel assay of glucuronidation of C- and N-hydroxylated metabolites of the carcinogen N-2-fluorenylacetamide

A method for the assay of glucuronidation of C- and N-hydroxylated metabolites of the carcinogen N-2-fluorenylacetamide is described. The method employs UDP-[U-14C ))glucuronic acid and Baker C18 extraction columns for separation of the glucuronides from their aglycones and from the glucuronic acid. The 14C-labeled glucuronides, generated by rat liver microsomes, are eluted from the columns with 30% (v/v) methanol after prewashing the columns and elution of the radioactivity of 14C-glucuronic acid with 1 mM ammonium acetate, pH 6.9. The radioactivity of the eluates is measured by scintillation counting. The method is modified for assays of glucuronidation of alpha-naphthol and p-nitrophenol in that 1 mM phosphoric acid is used instead of 1 mM ammonium acetate, and the method is potentially adaptable to other aglycones. By monitoring radioactivity or uv absorbance of the column eluates, it is shown that all aglycones, except p-nitrophenol, are retained on the columns during elution of their glucuronides with 30% (v/v) methanol and are eluted only when absolute methanol is used. The identity of the glucuronides is shown by their response to hydrolysis by beta-glucuronidase in the presence and absence of D-saccharic acid-1,4-lactone and, in some instances, by chromatographic and spectral analyses of the released aglycones.     

Product inhibition study on carbonic anhydrase using spectroscopy and calorimetry

Kinetic and thermodynamic studies have been made on the effect of the p-nitrophenol product on the activity of bovine carbonic anhydrase in 50 mM Tris buffer pH 7.5, at 300K using UV spectrophotometry and isothermal titration calorimetry (ITC). A competitive inhibition was observed for p-nitrophenol as a product of the enzymatic reaction. A graphical fitting method was used for determination of the binding constant and enthalpy of inhibitor binding using ITC data. The dissociation binding constant was 0.10mM by the microcalorimetric method, which is in good agreement with the value of 0.11mM for the inhibition constant obtained from the spectrophotometric method.     

Use of a 96-well microplate reader for measuring routine enzyme activities

A method is described for the routine determination of the rate of colorimetric enzyme reactions using a 96-well microtiter plate reader commonly used in immunoassay. This approach is illustrated by monitoring esterase activity using three common products: release of thiol, release of ethanol, and release of p-nitrophenylate ion. Examples include monitorings of the rate of hydrolysis of acetylthiocholine iodide by eel acetylcholinesterase and the rate of hydrolysis of malathion and nonconventional esters such as O-methyl, O-ethyl, and O-isobutyl carbonates of p-nitrophenol by commercial porcine liver carboxylesterase. Data obtained from the plate reader were compared to those obtained, under similar conditions, in a conventional spectrophotometer. Absorbance measurements made in both machines on the same solution, as well as absorbance changes measured over time, were similar. The use of the 96-well plate format tremendously increased the number of enzyme assays carried out per person and the interface with a personal computer allowed rapid manipulation of the absorbance values to calculate the desired rate data. This approach should be generally applicable to many routine colorimetric assays in the research laboratory.     

FTIR spectroscopic kinetic analysis of alkaline phosphatase under hyperbaric manipulation.

For the first time, high-pressure infrared spectroscopy has been used in an enzyme kinetics study. This technique allows not only the investigation of kinetics under very high pressure, but it also allows simultaneous determinion of changes in the secondary structure of enzymes at the corresponding pressures. In the present study, a classical enzyme reaction, the conversion of p-nitrophenol phosphate into p-nitrophenol by alkaline phosphatase was selected to demonstrate the potential of infrared spectroscopy as an alternative physical method in the high-pressure study of enzyme kinetics. The rate constants of this enzyme reaction have been determined as a function of pressure in the pressure range 0.001-14 kbar. The first-order rate constants thus obtained increases with increasing pressure up to 8.3 kbar. At this pressure, the reaction rate decreases abruptly due to the denaturation of the enzyme arising from the conformational changes of some alpha-helical segments in the enzyme molecules into beta-sheet structure. The present results suggest that the pressure-enhanced overall hydrogen-bond strength in the amide groups of the enzyme is one of the factors which stimulate the enzyme activity. Moreover, the dissociation of the dimeric enzyme into its subunits does not inhibit the enzyme activity but only attributes to a slight change in activation volume.     

Colorimetric assay for cellular transglutaminase

A colorimetric assay for cellular transglutaminase using 5-(biotinamido)pentylamine and polyvinylidine difluoride membranes for crude cellular preparations and purified enzyme has been developed. The biotinpentylamine substrate was incorporated into N,N-dimethylcasein by transglutaminase, the biotinylated products were adsorbed onto the membrane disks and conjugated with streptavidin-beta-galactosidase, and the absorbance resulting from the formation of p-nitrophenol from hydrolysis of p-nitrophenyl-beta-D-galactopyranoside was measured at 405 nm. The validity of the assay was established by showing a good correlation, gamma = 0.922, between the colorimetric procedure and the commonly used radiometric filter paper method for the enzyme. The procedure offers a rapid, sensitive, and nonisotopic method for the estimation of cellular transglutaminase activity in as low as 20 ng of purified guinea pig liver transglutaminase and 10 micrograms of crude fibroblast cytosol protein.     

An approach to assay calcineurin activity and the inhibitory effect of zinc ion

In this study, an electrochemical method to assay calcineurin activity is proposed. Although the enzyme could not exhibit electron transfer reactivity and the catalytic reaction of the substrate could not give any electrochemical wave, p-nitrophenol as the catalytic reaction production could be oxidized at the calcineurin/Triton X-100 film modified electrode to exhibit useful wave that might be employed to assay the enzyme activity. The effect of Ni(2+) and Zn(2+) on calcineurin was also investigated. Whereas Ni(2+) was confirmed to be able to enhance the enzymatic activity, Zn(2+) was found to be an inhibitor to calcineurin.     

Active-site titration of serine proteinases acting selectively on cationic substrates by N-alpha-carbobenzoxy-L-arginine p-nitrophenyl ester and N-alpha-carbobenzoxy-L-lysine p-nitrophenyl ester; determination of active enzyme concentration

A titration method for determination of trypsin-like serine proteinase concentration has been developed by using ZArgONp and ZLysONp, two specific chromogenic amino-acid derivatives which show the characteristics of optimal active-site titrants. Active proteinase concentration has been estimated from the effect of titrant concentration on the amplitude, at time zero, of the time-course for the instantaneous release of p-nitrophenol, preceding the steady-state reaction (burst phase).     

Estimation of Cellobiohydrolase Ⅰ Activity by Numerical Differentiation of Dynamic Ultraviolet Spectroscopy

1,4-β-D-glucan cellobiohydrolase Ⅰ （CBH Ⅰ）, p-nitrophenyl β-D-cellobioside, p-nitrophenol and cellobiose show distinct ultraviolet spectra, allowing the design of an assay to track the dynamic process of p-nitrophenyl β-D-cellobioside hydrolysis by CBH Ⅰ. Based on the linear relationship between p-nitrophenol formation in the hydrolysate and its first derivative absorption curve of AUC340-400 m （area under the curve）, a new sensitive assay for the determination of CBH Ⅰ activity was developed. The dynamic parameters of catalysis reaction, such as Vm and kcat, can all be derived from this result. The influence of β-glucosidase and endoglucanase in crude enzyme sample on the assay was discussed in detail. This approach is useful for accurate determination of the activity of CBHs.     

A high-throughput and generic assay method for the determination of substrate specificities of thermophilic alpha-aminotransferases

For the determination of substrate specificities of thermophilic alpha-aminotransferases (AATs), a novel high-throughput assay method was developed. In this method, a thermophilic omega-aminotransferase (OAT) and a thermophilic aldehyde dehydrogenase (ALDH) are coupled to the AAT reaction. Glutamic acid is used as an amino group donor for the AAT reaction yielding 2-oxoglutalic acid. 2-Oxoglutalic acid produced by the AAT reaction is used as an amino group acceptor in the OAT reaction regenerating glutamic acid. The amino group donor of the OAT reaction is 5-aminopentanoic acid yielding pentanedioic acid semialdehyde which is oxidized by ALDH to pentanedioic acid with concomitant reduction of NADP(+) to NADPH. NADPH thus produced then reduces colorless tetrazolium salt into colored formazan. To construct such a reaction system, the genes for a thermophilic AAT, a thermophilic OAT and a thermophilic ALDH were cloned and expressed in Escherichia coli. These enzymes were subsequently purified and used to determine the activities of AAT for the synthesis of unnatural amino acids. This method allowed the clear detection of the AAT activities as it measures the increase in the absorbance on a low background absorbance reading.     

对硝基苯酚冲击对UASB反应器中污泥活性和微生物种群的影响

研究了在稳定运行的上流式厌氧污泥床(Upflow Anaerobic Sludge Blanket,UASB)反应器中,对硝基苯酚(p-NP)冲击对反应器活性的影响。采用PCR-DGGE技术监测了反应器受对硝基苯酚冲击后微生物种群多样性的变化。实验结果表明,p-NP冲击对污泥的产甲烷活性和COD去除活性均有严重的抑制,污泥活性的恢复需要较长时间;高浓度冲击比低浓度冲击产生更严重的影响,20mg/L和40mg/Lp-NP冲击后污泥活性的恢复期分别为16d和27d。p-NP冲击后,真细菌和古菌的多样性均发生了显著的变化,而且p-NP冲击对真细菌的影响大于对古菌的影响。p-NP冲击后甲烷产量下降的主要与Methanosaetasp.的活性下降以及Methanomicrobia sp.丰度的下降有关。而冲击后真细菌的主要变化表现为Chloroflexisp.、Bacteroidesp.和Anaerovibrio sp.的丰度均下降;Rheinheimera sp.在受到20mg/Lp-NP冲击时丰度下降,继续受到40mg/Lp-NP冲击时该种群消失。Flavobacteria sp.是p-NP冲击后新出现的细菌种群,可能与p-NP的降解有关。     

A model for formulation of protein assay.

The principle of the protein assay using the reaction of an alkaline copper-protein complex with the Folin-Ciocalteu phenol reagent has been investigated. In contrast to the long-established Lowry method, a stable and rapid protein assay is developed without a buffering agent in alkaline copper solution. In the absence of a buffering agent, the reaction pH drops relatively rapidly and moves the reaction toward a more stable pH. When the reaction of alkaline copper-protein complex with Folin-Ciocalteu reagent is started at around pH 11.7, the reaction color absorbance reaches a plateau in approximately 10 min and remains stable to allow a reliable measurement of the absorbance. In the absence of the buffering agent sodium carbonate, the alkaline copper solution is also stable for months. The principle of the protein assay is presented as a model that can be used to formulate protein assays of desired specification.     

A simple and rapid assay of collagen-like polymer in crude lysate from Escherichia coli

An assay for the quantification of collagen-like polymer (CLP) in Escherichia coli cells utilizing the specific reaction between collagenase and CLP is presented. It involves thermal treatment to precipitate non-CLP proteins, digestion of CLP by collagenase and detection of the absorbance of the liberated amino acids and peptides from CLP by a ninhydrin-based method. CLP concentration is determined from the absorbance measurement.     

Lung microsomal p-nitrophenol hydroxylase -- characterization and reconstitution of its activity

1. Formation of catechols from benzene and nitrobenzene have been implicated in the carcinogenic activity of these chemicals. In liver, p-nitrophenol, an intermediate of p-nitrobenzene is enzymatically converted to 4-nitrocatechol. 2. For the first time in this study, the presence of a highly active enzyme catalyzing the formation of 4-nitrocatechol from p-nitrophenol was detected in lung microsomes. The average specific activity of lung p-nitrophenol hydroxylase was found to be 0.494 nmol 4-nitrocatechol formed mg prot-1 min-1. 3. The optimum conditions for sheep lung microsomal p-nitrophenol hydroxylase were established. The maximal activity was noted at pH 6.8. The rate of p-nitrophenol hydroxylation was linear up to 2 mg prot/ml of incubation mixture. The maximal rate of 4-nitrocatechol formation was observed with 0.25 mM p-nitrophenol. 4. The Lineweaver-Burk and Eadie-Hofstee plots were found to be curve-linear. Two different Km values were calculated as 11.6 and 71.4 microM from the Lineweaver-Burk plot and as 10.7 and 74.5 microM from the Eadie-Hofstee plot. This suggested that there were either two forms of enzyme or two different enzymes participating in ortho hydroxylation of p-nitrophenol in lung microsomes. 5. Lung microsomal p-nitrophenol hydroxylase activity of sheep was reconstituted in the presence of purified lung microsomal cytochrome P-450, NADPH dependent cytochrome P-450 reductase and synthetic lipid, phosphatidylcholine dilauroyl.     

Effect of carbon starvation on p-nitrophenol degradation by a Moraxella strain in buffer and river water

This study examines the effect of carbon starvation on the ability of a Moraxella sp. strain to degrade p-nitrophenol (PNP). Carbon starvation for 24 h decreased the induction time for p-nitrophenol degradation by the bacterium in a minimal salt medium from 6 to 1 h but it did not completely eliminate the induction time. Moraxella cells with 2-day carbon starvation had an induction time of 3 h and the induction time of the 3-day starved cells was 6 h. A 100% increase in density of the non-starved cells did not affect the induction time for p-nitrophenol degradation by the bacterium, indicating that the initial increase in cell density of the carbon-starved culture did not cause the faster onset of p-nitrophenol degradation. However, the initial uptake of p-nitrophenol of the 1-day carbon-starved Moraxella cells was 3-fold higher than the non-starved cells. A green fluorescent protein gene (gfp)-labelled Moraxella (M6 strain) was constructed to examine the survival of and p-nitrophenol degradation by the bacterium in non-sterile river water samples. Similar p-nitrophenol degradation behaviour was observed in the river water samples inoculated with the M6 cells. The time needed for complete degradation of p-nitrophenol by the non-starved M6 was 19-27 and 33 h in samples spiked with 80, 200 and 360 microM p-nitrophenol, respectively. However, the 1-day carbon-starved inocula required about 16 h to degrade the p-nitrophenol completely regardless of its concentration in the water samples. Survival of the carbon-starved and non-starved M6 was not significantly different from each other in the river water regardless of the p-nitrophenol concentration. In the absence of p-nitrophenol, the inoculum density decreased continuously. At 200 and 360 microM p-nitrophenol, the cell densities of M6 increased in the first two days of incubation and declined steadily afterward.     

Determination of metabolites by observing the change in reaction rate during the early course of a first order reaction, as applied to fructose-1 phosphate.

An enzymatic method is described for the determination of fructose-1-phosphate photometrically with fructose-6-phosphate kinase. By using the reaction rate to calculate the absorbance at the end of the reaction, the precision of the method is improved and the time taken for one assay reduced from 2 to 3 hr to about 1 hr.     

Extinction coefficient of polymerized diaminobenzidine complexed with cobalt as final reaction product of histochemical oxidase reactions

The extinction coefficient is essential for the conversion of cytophotometric (mean integrated) absorbance values into absolute units of enzyme activity, for instance expressed in terms of moles of substrate converted per unit time and per unit wet weight of tissue. The extinction coefficient of polymerized diaminobenzidine (polyDAB) complexed with cobalt as the final reaction product of oxidase reactions was estimated at 575 nm by comparison of the amounts of final reaction products formed after incubation of serial unfixed cryostat sections of rat kidney to demonstrate D-amino acid oxidase activity with either the tetrazolium salt method or the cerium-DAB-cobalt-hydrogen peroxide method. Both procedures resulted in similar localization patterns of final reaction product in a granular form in epithelial cells of proximal tubules in rat kidney. The granules were peroxisomes. Linear relationships were found for both methods between the specific amounts of final reaction product generated by D-amino acid oxidase activity and incubation time. The cerium salt method gave rise to 7.4 times higher absorbance values of final reaction product generated per unit time and per unit wet weight of tissue than the tetrazolium salt procedure. The extinction coefficient of tetranitro BT-formazan is 19 000 at 557 nm. Therefore, the cytophotometric extinction coefficient of the poly DAB-cobalt complex as final reaction product of oxidase reactions was established to be 140 000.     

An ultraviolet absorbance method for determining acetylsalicylic acid hydrolase activity

An ultraviolet absorbance method for quantitation of acetylsalicylic acid esterase (hydrolase) activity has been developed and validated. The sensitivity of the method was found to be 2.8 nmol/ml-min in the assay cuvette. Linearity of the reaction with enzyme concentration and time has been demonstrated. The product of the enzymatic reaction, salicylic acid, has been identified by thin-layer chromatography using acetyl-[¹⁴C]salicylic acid. The quantities of salicylic acid produced in 5, 10, and 15 min of incubation were equal when assayed by the spectrophotometric method and by the acetyl-[¹⁴C]salicylic acid thin-layer chromatographic method. The time required for assay by ultraviolet absorbance is approximately 3 min/sample.     

有机磷农药在水生态系中生物净化机理研究——2.CTP-02降解对硝基酚的特性和动力学

从鸭儿湖氧化塘中分离出具有分解对硝基酚能力的细菌Pseudomonas sp.,代号CTP-02。在实验室条件下,细菌培养物降解对硝基酚的速度与时间之间的动力学方程为dc/dt=-K1t-K2,细菌降解对硝基酚的最适温度为35℃,最适pH为7.5。CTP-02菌降解对硝基酚过程中首先发生脱硝基作用。