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The production of conjugate vaccines within an E. coli (Escherichia coli) host provides an inexhaustible supply without the need for culture of pathogenic organisms. The machinery for expression of glycan and acceptor protein, as well as the coupling enzyme, are all housed within the E. coli chassis, meaning that there are no additional steps required for individual purification and chemical conjugation of components. In addition, there are far fewer purification steps necessary to obtain a purified glycoconjugate for use in vaccine testing. Here we describe production and purification of a HIS-tagged Campylobacter jejuni AcrA protein conjugated to Streptococcus pneumoniae serotype 4 capsule.

One-pot process for the production of ginsenoside Rd by coupling enzyme-assisted extraction with selective enzymolysis was explored in this paper. Several detection methods including HPLC-MS were used to identify and quantify the products in the enzymolysis solution of pectinase. Results showed that ginsenoside Rd was the main component in enzymolysis solution, pectinase specifically hydrolyzes protopanaxadiol (PPD)-type ginsenoside and was a selective enzyme to convert ginsenoside Rb1 to Rd in a way. In addition the influencing factors on the yield of ginsenoside Rb1 and Rd were optimized using L9(34) orthogonal design data. The enzymolysis conditions for the higher yield of Rd were 52.5 °C, pH 6 and 1 h with a yield of 0.8314 from 50 mg drug material. The controllable transformation hypothesis of the PPD-type ginsenoside was also explored from the perspective of the molecular steric hindrance. Pectinase could be used as an efficient enzyme for one-pot producing ginsenoside Rd.

UDP-glucose dehydrogenase from Capra hircus has been purified to homogeneity by salt fractionations, heat treatment and chromatographic steps. It is a homohexamer of about 300 kDa. Though the basic physical and enzymatic properties of the caprine enzyme are comparable to those of the beef liver enzyme, it has lower energy of activation and different entropy and enthalpy for the transition state during catalysis. The caprine enzyme can act suitably as an auxiliary enzyme in the coupled assay system for UDP-galactose 4-epimerase. Enzymes: UDP-Glc DH, UDP-glucose dehydrogenase (EC 1.1.1.22); Epimerase, UDP-galactose 4-epimerase (EC 5.1.3.2).

Existing assays monitoring ENPP1 activity are either not physiologically relevant or not suitable for high-throughput screening (HTS). Here, we describe the development and implementation of two new ENPP1 activity assays that address these drawbacks. These assays employ physiological substrates of ENPP1, ATP and ADP. They rely on detection of inorganic phosphate using a special modification of the malachite green-molybdate colorimetric procedure that ensures stability of acid-labile compounds, such as the ones containing phosphodiester bonds. The pyrophosphate generated in ENPP1 reaction is converted to inorganic phosphate in the presence of inorganic phosphatase; whereas, omission of this coupling enzyme enables detection of the inorganic phosphate generated by ENPP1. These new ENPP1 assays were miniaturized into high-density microplate formats. With minimal requirement for ENPP1 enzyme, low micromolar phosphate detection sensitivity, and simple protocol involving three to four simple liquid handling steps, these robust assays are suitable for HTS.

High throughput screening and the subsequent hit evaluation phase are critical components of target based drug discovery. Identification of suitable lead material remains a key milestone in any antibacterial strategy, and while target-based drug discovery has proven effective for a number of disease areas, the success rate of this approach has been challenged for antibacterial targets [23]. While there is always a component of luck in matching the right compound file with the chosen target, it is clear that careful and thoughtful assay design, execution, and hit evaluation can tilt the odds in favor of finding quality inhibitors that enable late stage drug discovery. In contrast, poorly designed assays and inadequate hit evaluation processes invariably result in the identification of questionable leads that require extensive resources and time to evaluate and usually have low probabilities for success. This chapter outlines the generic process from building a suitable assay for high throughput screening to the critical factors in selection of lead material for focused drug discovery prosecution.

Inositol-3-phosphate synthase (INO1) has previously been genetically validated as a drug target againstTrypanosoma brucei, the causative agent of African sleeping sickness. Chemical intervention of this essential enzyme could lead to new therapeutic agents. Unfortunately, no potent inhibitors of INO1 from any organism have been reported, so a screen for potential novel inhibitors ofT. bruceiINO1was undertaken. Detection of inhibition ofT. bruceiINO1 is problematic due to the nature of the reaction. Direct detection requires differentiation between glucose-6-phosphate and inositol-3-phosphate. Coupled enzyme assays could give false positives as potentially they could inhibit the coupling enzyme. Thus, an alternative approach of differential scanning fluorimetry to identify compounds that interact withT. bruceiINO1 was employed to screen ~670 compounds from the MayBridge Rule of 3 Fragment Library. This approach identified 38 compounds, which significantly altered the Tmof TbINO1. Four compounds showed trypanocidal activity with ED50s in the tens of micromolar range, with 2 having a selectivity index in excess of 250. The trypanocidal and general cytotoxicity activities of all of the compounds in the library are also reported, with the best having ED50S of ~20 μM againstT. brucei.

The theoretical aspects of enzyme activity, inhibition, and inactivation have been discussed in a separate volume [44]. More extensive treatments of both theory and practice of enzyme kinetics may be found in [27, 68, 69]. Ready-to use rate equations for most mechanisms may be found in the otherwise dated monography by Segel [372], rate equations may also be obtained by a computer program described in [438].

The study of enzymatic reaction mechanisms involves researching the elementary steps that intervene during the course of a reaction, and determining the rates of appearance and disappearance of reaction intermediates, as well as their relative energetic levels. The majority of intermediates that appear have very short lifetimes and are not generally detectable by classical techniques. Increasingly rapid methods have been progressively introduced for studying enzyme reactions as is the case more generally for all kinetic studies. The discovery of techniques that discern ever lower time constants marked great progress in the approaches taken to study enzymatic reaction mechanisms.

We present here a radiochemical enzymatic endpoint assay for the guanine nucleotides GTP and GDP that is suitable for use with cell extracts. The major coupling enzyme used is phosphoenolpyruvate carboxykinase purified from chicken liver. The ancillary coupling enzyme, aspartate aminotransferase, was used to generate a low steady-state concentration of oxalacetate. GTP was determined by the overall conversion of [U-14C]aspartate into [14C]phosphoenolpyruvate. This reaction was also scaled-up as a preparative method for [U-14C]phosphoenolpyruvate. This was used with the same coupling enzymes in reverse to measure GDP by the formation of [14C]aspartate. The assay method was applied to isolated rat hepatocytes. The total GTP and GDP concentrations found were within the range reported by others for rat liver. The advantages of this assay are its sensitivity, specificity, and applicability to large numbers of samples.

This paper has two aims. The first one is to point out the shortcomings of Food and Drug Administration's (FDA's) reference method for the measurement of glucose. We found that the quantity of enzyme used in the method recommended by the FDA was more than the exact quantity needed for accurate measurement. The use of exact quantity of enzyme is important to minimize the negative effects due to impurity and side reactions of enzymes. The second aim is to simulate the coupling enzyme reaction using computer. We have successfully established the exact quantity of enzyme needed in the assay through the computer simulation. The quantity of the enzyme was lesser than the that recommended by FDA, but the reaction ended at the same time as in the FDA method. In addition, optimum conditions and inhibitory effects of various reagents have also been successfully analyzed using computer. In conclusion, we suggest a reference method using computer simulation to determine the exact quantity of the coupling enzyme needed in the assay.

Publisher Summary This chapter discusses the nonenzymatic colorimetric assay of glutathione in the presence of other mercaptans. Direct assays of total mercaptans are based on chromogenic reactions of sulfhydryl groups with electrophilic reagents. Such assays have obvious limitations in terms of specificity. Other methods are time-consuming and are based on glutathione reductase-coupled assays, with interferences of mixed disulfides of glutathione and enzyme inhibitors or on chromatographic techniques. Reliable chromatographic methods are relatively complex and require expensive materials. Such observations underline the need for faster and easier methodology, especially when large series of biological samples must be processed on a daily basis. The chapter describes a colorimetric procedure that takes advantage of a glutathione (GSH)-specific reaction in the absence of a coupling enzyme. The chapter discusses the advantages and limitations of this methodology. The assay—which is based on the colorimetric measurement of 7-trifluoromethyl-4-thioquinolone at the 400-nm wavelength—has the unique advantage to be glutathione specific in the absence of enzyme. Another advantage is that a colorimetric estimation of total sulfhydryl groups can be obtained at the 356-nm wavelength, before the addition of sodium hydroxide to the mixture of thioethers produced at pH 7.8. The sensitivities of the two assays are very similar and given the stability of chromophoric products, large series of samples can be processed within 20–30 min for subsequent absorbance measurement.

The coupling of enzyme immunoassay with supercritical fluid extraction (SFE) is an attractive technique for analysts faced with decreasing the use of hazardous solvents, due to their adverse impact on the environment. This chapter will describe the development of supercritical fluid extraction techniques which can be combined with enzyme immunoassays for the detection of pesticide residues and similar toxicants in food and environmental samples. The use of static versus dynamic SFE will be contrasted with respect to speed of analysis, equipment requirements, and quantitative vs. qualitative analysis. Detection of the presence of pesticides in meat matrices was accomplished using different commercial test kits. Removal of various interferences from the sample extract prior to EIA is necessary to achieve quantitative results, due to the presence of lipid coextractives in the extract. The above techniques have been successfully employed to determine pesticide residue content in meat products and other matrices below their specified tolerance limit set by regulatory agencies.

A spectrophotom etric assay for th e determination of branched-chain L-amino acid am inotransferase activ­ ity is described. It is based on the transamination of L-leucine in the presence of 2-oxoglutarate yielding 4ethyl-2-oxopentanoate. The rate of formation of the branched-chain 2-oxo acid is specifically monitored in a coupled enzymatic reaction using NADf -dependent D*2-hydroxyisocaproate dehydrogenase from Lactoba­ cillus casei sap. pseudoplantarum as coupling enzyme by m easuring the decrease in NADH absorbance at 334 nm. Optimized assay conditions are provided as evalu­ ated for the (iso)enzyme from rat heart, c 1995 Academic Press, Inc.

Presentation des connaissances acquises concernant la theorie des systemes enzymatiques couples, a l'aide des modeles mathematiques elabores depuis plusieurs annees ou plus recemment

We have developed a systematic approach to optimization of reagent concentrations for assays of alanine aminotransferase and aspartate aminotransferase: (a) Michaelis constants describing the initial-velocity kinetics of the coupled enzyme reactions were evaluated by a nonlinear least-squares fit of the appropriate equation to measured enzyme activities. Activities of more than 50 normal and pathological sera were measured at 30 °C. (b) These kinetic equations are used to calculate the set of reagent amino- and keto-acid concentrations that all yield a selected fraction of the theoretical maximum enzyme velocity. An optimal pair is determined by defining an additional criterion, such as minimal reagent cost or minimal concentration to Km ratio. (c) The optimum amounts of reagent NADH and coupling enzyme, being a function of desired pre-incubation and measurement intervals, maximum aminotransferase activity to be measured, and endogenous keto-acid concentration, are determined by computer simulation. An approximate relationship and an exact method for computing assay lag time are presented, along with experimentally measured endogenous keto-acid concentrations in serum. All procedures may be applied to other enzyme assays if appropriately modified.

2,4-Dinitrophenol fails to stimulate the breakdown of the soluble high energy intermediate of oxidative phosphorylation isolated from Alcaligenes faecalis extracts. Once the intermediate is broken down, by other reactions, the coupling enzyme is prevented from reassociation with the electron transport particles by 2,4-dinitrophenol. Phosphorylating particles washed with 2,4-dinitrophenol lose coupling enzyme and with it the ability to form the energy-rich intermediate and to couple phosphorylation to electron transport. Phosphorylating particles containing bound coupling enzyme are able to form the high energy intermediate even in the presence of 2,4-dinitrophenol. However, after this first cycle of formation, no more can be formed because 2,4-dinitrophenol prevents the reassociation of coupling enzyme with the electron transport particles. 2,4-Dinitrophenol stimulates respiration in the bacterial phosphorylating particles.

The stimulation of energy-linked reactions by low levels of oligomycin varies greatly with the type of submitochondrial particle. Particles made by disruption of combined heavy and light mitochondria by exposure to sonic oscillation and depleted by urea treatment showed no stimulation over a wide range of oligomycin concentration. However, the mitochondrial coupling factor described previously stimulated energy-linked reactions in these particles. Similar particles from heavy mitochondria gave some stimulation with low levels of oligomycin. High stimulation was obtained with alkali-treated particles derived from heavy mitochondria. The magnitude of stimulation appeared to be related to the concentration of endogenous coupling factors. Both severely depleted and fully supplemented particles were not stimulated by oligomycin, but particles with intermediate levels of endogenous factors gave striking effects.

Exact equations which describe the kinetic patterns of enzyme/enzyme complexes, when compartmented coupling occurs between them, are presented. Compartmented coupling refers to the creation of a local environment in which the concentration of an intermediate, shared by two enzymes, is higher than its solution concentration. This results in a higher coupling enzyme activity, a condition reflected in a shorter transition time for the system. In this paper, equations are presented which allow experimenters to quantitate the effect of compartmented coupling in terms of changes in the apparent Km and Vmax values. The equations presented in this paper are more exact than those previously derived since they do not incorporate first order assumptions before derivation.

Phosphoenolpyruvate carboxylase (EC 4.1.1.31), used as a coupling enzyme in the assay of the pyruvate, orthophosphate dikinase (EC 2.7.9.1) forward reaction, is a serious limiting factor for the overall rate when added at a level of 0.2–0.3 unit/ml of assay medium. Nonlimiting assay conditions are obtained by either increasing the level of the coupling enzyme to 3 units/ml or adding 6mM glucose-6-phosphate as an activator/stabilizer of phosphoenolpyruvate carboxylase.

Most people approaching the problem of enzyme purification will be following a standard procedure for measuring enzyme activity. But in some cases, it may be useful to develop an alternative assay, for instance, a rapid spectrophotometric method to avoid queuing for the use of a scintillation counter. On rare occasions a newly discovered enzyme will need a new assay method. Ultimately the biochemist wants to know the relationship between an enzyme’s activity in vitro and its physiological situation. But the pH, ionic strength, and, particularly, the substrate concentration in vivo are likely to be quite different from the conditions used to measure the enzyme activity during a purification process. What is required here is a reliable, reproducible assay which will relate the amount of enzyme in one fraction with another, regardless of what the “activity” may mean in a physiological context.

Possible ways are discussed of reducing systematic errors in initial velocities (v), principally those caused by drift and nonlinear progress curves. Information on the nature of the random experimental error in v is also summarized. The error tends to increase with the magnitude of v, but generalizations about its distribution are less easily made.

A rapid, reproducible, and sensitive colorimetric assay for carbamyl phosphate synthetase I was presented. A four-fold increase in sensitivity and reduced assay time were afforded by this procedure. The method utilized the chemical conversion of carbamyl phosphate to hydroxyurea by the action of hydroxylamine instead of employing a coupling enzyme. The hydroxyurea was quantitated in 15 min by an improved colorimetric assay for ureido compounds by measuring the absorption of the resulting chromophore at 458 nm. Optimum conditions for both the formation and quantitation of hydroxyurea were established. Activity measurements of carbamyl phosphate synthetase I obtained by this uncoupled method were identical with those obtained by the ornithine transcarbamylase coupld assay.

As discussed in chapter 4, the Michaelis-Menten equation (4.10) predicts that a plot of v 0 against S0 is a rectangular hyperbola, i.e. one with orthogonal asymptotes, passing through the origin (figure 10.1). One limb of the hyperbola lies completely in the second quadrant in the physically meaningless region of negative substrate concentration. The arc of the curve as usually drawn is in the first quadrant but this is only a portion of the limb which extends to infinite — υ 0 in the third quadrant. The asymptotes are given by S0 = — K m and υ 0 = V. Although the S0, υ 0 relationship is simple, its hyperbolic nature gives rise to problems in presenting kinetic data and in calculating K m and V. This is because it is difficult to extrapolate the hyperbola to estimate V, the asymptotic value of υ 0. In order for υ 0 to approach even to within 5 % of V, S0 must be at least 19 times K m ; apart from possible limitations in availability or solubility of the substrate, or interference with the assay method, high substrate concentrations may result in appreciable substrate inhibition (see chapter 4). As K m is defined in terms of V an accurate estimate of V is necessary for determining K m