A coupled enzyme assay for aldose 1-epimerase

Aldose 1-epimerase (mutarotase) EC 5.1.3.3. catalyzes the mutarotation of selected pyranose sugars (1). The enzyme has been implicated as a component of the sugar transport system in kidney and intestine (2,3,4). Conventional analytical methods for monitoring catalytic activity involve relatively long, finite-interval polarimetric or spectrophotometric measurements of mutarotation rates employing α-d-glucose as the substrate (5). We have found this method to be somewhat cumbersome and time consuming, as α-d-glucose solutions spontaneously epimerize at rates requiring their individual preparation for each experiment. We report here a kinetic assay method for aldose 1-epimerase based upon fastin situ generation of α-d-glucose employing hydrolysis of sucrose by β-fructofuranosidase and a subsequent reporter reaction involving the aerobic oxidation of β-d-glucose via glucose oxidase. Analytical monitoring of the rate limiting epimerization step in the three-enzyme system is achieved by measurement of oxygen depletion in solution employing a conventional Clark electrode assembly.     

Rapid polarographic mutarotase assay with -D-glucose oxidase

This rapid mutarotase assay consists of polarographic measurements of rates of oxygen consumptions due to the enzymic oxidation with β-d-glucose oxidase of β-d-glucose formed from α-d-glucose in the absence and presence of mutarotase. This method requires only 5 min for each sample, and is applicable to aqueous solutions or suspensions with any turbidity and also to mutarotase samples containing large quantities of d-glucose. The mutarotase contents in rat tissues were determined by this method.     

Optimization of an Aspergillus niger glucose oxidase production process

The purpose of the present study was to ascertain the optimal concentration of dissolved oxygen in order to maximize the intracellular glucose oxidase formation in Aspergillus niger. Cultivations performed in a 3.5 l laboratory reactor showed that a dissolved oxygen concentration at 3% of saturation at a total pressure of 1.2 bar was optimal for maximizing intracellular glucose oxidase activity. Cultivations performed at higher dissolved oxygen concentrations did not produce as much glucose oxidase as those performed at 3%, although the formation rate was high. Experiments revealed that maximal intracellular glucose oxidase formation for the A. niger strain used, is accomplished by limiting the gluconic acid production rate by means of maintaining a low dissolved oxygen concentration. Several attempts to achieve higher intracellular glucose oxidase activity were also made by manipulating the glucose concentration at a 3% dissolved oxygen concentration. However, no enhancement in glucose oxidase activity was observed.     

Enhancement of glucose oxidase production in batch cultivation of recombinant Saccharomyces cerevisiae: optimization of oxygen transfer condition

AIMS: To obtain an optimal combination of agitation speed and aeration rate for maximization of specific glucose oxidase (GOD) production in recombinant Saccharomyces cerevisiae, and to establish a correlation between kLa vis-à-vis oxygen transfer condition and specific glucose oxidase production. METHODS AND RESULTS: The oxygen transfer condition was manifested indirectly by manipulating the impeller speed and aeration rate in accordance with a Central Composite Rotatory Design (CCRD). The dissolved oxygen concentration and the volumetric oxygen transfer coefficient (kLa) were determined at corresponding combinations of impeller speed and aeration rate. The maximal specific extracellular glucose oxidase production (3.17 U mg-1 dry cell mass) was achieved when the initial dissolved oxygen concentration was 6.83 mg l-1 at the impeller speed of 420 rev min-1 and at the rate of aeration of 0.25 vvm. It was found out that while impeller speed had a direct effect on the production of enzyme, a correlation between kLa and specific GOD production could not be established. CONCLUSION: At the agitation speed of 420 rev min-1 and at 0.25 vvm aeration rate, the degree of turbulence and the dissolved oxygen concentration were thought to be optimal both for cellular growth and production of enzyme. SIGNIFICANCE AND IMPACT OF THE STUDY: The combined effect of agitation and aeration on recombinant glucose oxidase production in batch cultivation has not yet been reported in the literature. Therefore, this study gives an insight into the effect of these two important physical parameters on recombinant protein production. It also suggests that since there is no correlation between kLa and specific production of GOD, kLa should not be used as one of the scale-up parameters.     

Effect of glucose and glucitol analogues on respiration by mycelia ofPuccinia graminis

Previous work has shown that the glucose analogue 2-deoxy-d-glucose (2dG) is extensively metabolized to a series of derivatives including 2-deoxy-d-glucitol (2dGol) by mycelia ofPuccinia graminis. In the present investigation, the time course of action of 2dG, 2dGol, and eight other glucose analogues as respiratory inhibitors ofP. graminis was determined by measuring the effect of each analogue on endogenous rates of oxygen uptake, using a manometric method. At a test concentration of 10 mM, the most potent inhibition was observed with 2dG, thio-β-d-glucose, and 2-fluoro-2-deoxy-d-glucose, each achieving ∼65% inhibition after 5 h; little or no inhibition was observed with 1-O-methyl-β-d-glucose, phenyl-β-d-glucose, 6-deoxyglucose, 3-O-methyl-d-glucose,N-acetyl-d-glucosamine, orl-glucose. However, mild inhibition (∼30% after 5 h) was observed with thed-glucitol (sorbitol) analogue 2dGol. It was concluded that the three inhibitory glucose analogues probably targeted central pathways of glucose metabolism, whereas 2dGol selectively inhibited an optional pathway associated with turnover of an endogenously generated pool ofd-glucose.     

Use of a bioelectrochemical cell for the synthesis of (bio)chemicals

A bioelectrochemical cell containing either d-glucose oxidase (β-d-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) or xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) plus dichlorophenol-indophenol as electron acceptor in one half-cell, and chloroperoxidase (chloride:hydrogen-peroxide oxidoreductase, EC 1.11.1.10) in the other half-cell is described. Due to a combination of chemical, biochemical and electrochemical reactions, electricity and specific (bio)chemicals can be produced in the cell simultaneously and in both compartments. Furthermore, the oxidases in a bioelectrochemical cell are not inactivated by H₂O₂ and as a result the operational lifetimes of the oxidases were increased about five-fold.     

Glucose oxidase: natural occurrence, function, properties and industrial applications

Glucose oxidase (GOX) from Aspergillus niger is a well-characterised glycoprotein consisting of two identical 80-kDa subunits with two FAD co-enzymes bound. Both the DNA sequence and protein structure at 1.9 Ǻ have been determined and reported previously. GOX catalyses the oxidation of d-glucose (C₆H₁₂O₆) to d-gluconolactone (C₆H₁₀O₆) and hydrogen peroxide. GOX is produced naturally in some fungi and insects where its catalytic product, hydrogen peroxide, acts as an anti-bacterial and anti-fungal agent. GOX is Generally Regarded As Safe, and GOX from A. niger is the basis of many industrial applications. GOX-catalysed reaction removes oxygen and generates hydrogen peroxide, a trait utilised in food preservation. GOX has also been used in baking, dry egg powder production, wine production, gluconic acid production, etc. Its electrochemical activity makes it an important component in glucose sensors and potentially in fuel cell applications. This paper will give a brief background on the natural occurrence, functions as well as the properties of glucose oxidase. A good coverage on the diverse uses of glucose oxidase in the industry is presented with a brief outline on the working principles in the various settings. Furthermore, food grade GOX preparations are relatively affordable and widely available; the readers may be encouraged to explore other potential uses of GOX. One example is that GOX-catalysed reaction generates significant amount of heat (∼200 kJ/mol), and this property has been mostly neglected in the various applications described so far.     

Application of the theory of oxygen transfer: determination of the Michaelis constant of glucose oxidase with respect to oxygen]

The oxidation of beta-D-glucose with glucose oxidase generally requires oxygen, which, under normal conditions is present at low concentrations in the reaction medium. Experiments show that glucose oxidase is no longer saturated by oxygen at enzyme concentrations greater than 0.4 mg.ml1. This is due to the decrease in the oxygen concentration of the solution. The value of the oxygen mass transfer coefficients and dissolved oxygen concentrations are determined. These dissolved oxygen concentrations are found to correlate with direct measurements with an oxygen electrode. From this, the Michaelis constant of glucose oxidase for oxygen is calculated. These experiments also show that oxygen is a limiting factor for this reaction.     

Metabolic engineering of Escherichia coli for quinolinic acid production by assembling L-aspartate oxidase and quinolinate synthase as an enzyme complex

Quinolinic acid (QA) is a key intermediate of nicotinic acid (Niacin) which is an essential human nutrient and widely used in food and pharmaceutical industries. In this study, a quinolinic acid producer was constructed by employing comprehensive engineering strategies. Firstly, the quinolinic acid production was improved by deactivation of NadC (to block the consumption pathway), NadR (to eliminate the repression of L-aspartate oxidase and quinolinate synthase), and PtsG (to slow the glucose utilization rate and achieve a more balanced metabolism, and also to increase the availability of the precursor phosphoenolpyruvate). Further modifications to enhance quinolinic acid production were investigated by increasing the oxaloacetate pool through overproduction of phosphoenolpyruvate carboxylase and deactivation of acetate-producing pathway enzymes. Moreover, quinolinic acid production was accelerated by assembling NadB and NadA as an enzyme complex with the help of peptide-peptide interaction peptides RIAD and RIDD, which resulted in up to 3.7 g/L quinolinic acid being produced from 40 g/L glucose in shake-flask cultures. A quinolinic acid producer was constructed in this study, and these results lay a foundation for further engineering of microbial cell factories to efficiently produce quinolinic acid and subsequently convert this product to nicotinic acid for industrial applications.     

Au-nanocluster emission based glucose sensing

Fabrication of a glucose biosensor based on Au-cluster emission quenching in the UV region is reported. The glucose biosensor is highly sensitive to β-d-glucose in 2.5-25.0mM range as confirmed from a linear calibration plot between Au-cluster colloid emission intensity as a function of β-d-glucose concentration. The interaction of β-d-glucose with l-cysteine capped Au cluster colloids has been confirmed from their Fourier transformed infrared spectroscopy (FTIR) measurements. It has been found that the biomolecules present in the serum such as ascorbic and uric acids, proteins and peptides do not interfere and affect in glucose estimation as confirmed from their absorption and fluorescence (FL) emission measurements. Practical utility of this sensor based on FL quenching method has been demonstrated by estimating the glucose level in human serum that includes diabetes and the data were found to be comparable or more accurate than those of the pathological data obtained from a local hospital. In addition, this biosensor is useful to detect glucose level over a wide range with sensor response time of the order of nano to picoseconds that is emission lifetime of Au clusters.     

Identification and Structural Analysis of Amino Acid Substitutions that Increase the Stability and Activity of Aspergillus niger Glucose Oxidase

Glucose oxidase is one of the most conspicuous commercial enzymes due to its many different applications in diverse industries such as food, chemical, energy and textile. Among these applications, the most remarkable is the manufacture of glucose biosensors and in particular sensor strips used to measure glucose levels in serum. The generation of ameliorated versions of glucose oxidase is therefore a significant biotechnological objective. We have used a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. As a result, we have identified two changes that increase significantly the enzyme''s thermal stability. One (T554M) generates a sulfur-pi interaction and the other (Q90R/Y509E) introduces a new salt bridge near the interphase of the dimeric protein structure. An additional double substitution (Q124R/L569E) has no significant effect on stability but causes a twofold increase of the enzyme''s specific activity. Our results disclose structural motifs of the protein which are critical for its stability. The combination of mutations in the Q90R/Y509E/T554M triple mutant yielded a version of A. niger glucose oxidase with higher stability than those previously described.     

Strong glucose dependence of electron current in human monocytes

Reactive oxygen species (ROS) production by human monocytes differs profoundly from that by neutrophils and eosinophils in its dependence on external media glucose. Activated granulocytes produce vast amounts of ROS, even in the absence of glucose. Human peripheral blood monocytes (PBM), in contrast, are suspected not to be able to produce any ROS if glucose is absent from the media. Here we compare ROS production by monocytes and neutrophils, measured electrophysiologically on a single-cell level. Perforated-patch-clamp measurements revealed that electron current appeared after stimulation of PBM with phorbol myristate acetate. Electron current reflects the translocation of electrons through the NADPH oxidase, the main source of ROS production. The electron current was nearly abolished by omitting glucose from the media. Furthermore, in preactivated glucose-deprived cells, electron current appeared immediately with the addition of glucose to the bath. To characterize glucose dependence of PBM further, NADPH oxidase activity was assessed as hydrogen peroxide (H(2)O(2)) production and was recorded fluorometrically. H(2)O(2) production exhibited similar glucose dependence as did electron current. We show fundamental differences in the glucose dependence of ROS in human monocytes compared with human neutrophils.     

Role of microbial enzymes in flavor development in foods

There are four main sources of enzymes in foods—these being the inherent enzymes, enzymes from microbial contaminants, enzymes elaborated by microorganisms added to foods, and specific enzymes added to foods. This study primarily deals with the latter two sources of enzymes in food. Although both plants and animals serve as sources of enzymes, they are not as economical or versatile sources as are enzymes obtained from microorganisms. In the meat industry, proteases are used to tenderize muscle and to obtain flavor precursors. In the preparation of cured meat products such as sausages, lipases, and proteases from bacterial cultures are utilized. Similarly, proteases and lipases are used in the dairy industry to develop flavor compounds. Proteases and amylases also have applications in the baking and milling industries where they are used to produce precursors for the nonenzymatic browning reactions. Carbohydrases such as amylase, amyloglucosidase, and glucose isomerase have found usage in the starch and syrup industry for the production of high dextrose and high fructose syrups. Other enzymes such as glucose oxidase, pectinase, and naringinase are of value to the wine and fruit juice industries. A better understanding of the mode of action of enzymes as well as the mechanisms of development of flavor compounds will further enhance the use of microbial enzymes to develop specific and desired flavors in foods.     

Expression and Characterization of Glucose Oxidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Glucose oxidase (GOX) is currently used in clinical, pharmaceutical, food and chemical industries. The aim of this study was expression and characterization of Aspergillus niger glucose oxidase gene in the yeast Yarrowia lipolytica. For the first time, the GOX gene of A. niger was successfully expressed in Y. lipolytica using a mono-integrative vector containing strong hybrid promoter and secretion signal. The highest total glucose oxidase activity was 370 U/L after 7 days of cultivation. An innovative method was used to cell wall disruption in current study, and it could be recommended to use for efficiently cell wall disruption of Y. lipolytica. Optimum pH and temperature for recombinant GOX activity were 5.5 and 37 °C, respectively. A single band with a molecular weight of 80 kDa similar to the native and pure form of A. niger GOX was observed for the recombinant GOX in SDS-PAGE analysis. Y. lipolytica is a suitable and efficient eukaryotic expression system to production of recombinant GOX in compered with other yeast expression systems and could be used to production of pure form of GOX for industrial applications.     

DEVELOPMENT OF A NEW BIOSENSOR FOR DETERMINATION OF CATALASE ACTIVITY

Catalase is one of the major antioxidant enzymes that catalyzes the hydrolysis of H₂O₂. The aim of this study was to suggest a new method for the assay of catalase activity. For this purpose, an amperometric biosensor based on glucose oxidase for determination of catalase activity was developed. Immobilization of glucose oxidase was made by a cross-linking method with glutaraldehyde on a Clark-type electrode (dissolved oxygen probe). Optimization and characterization properties of the biosensor were studied and determination of catalase activity in defined conditions was investigated in artificial serum solution. The results were compared with a reference method.     

Modeling of spherical fluorescent glucose microsensor systems: design of enzymatic smart tattoos

A two-substrate mathematical model of microspherical optical enzymatic glucose sensors is presented. The sensors are based on the well-known oxidation of glucose by glucose oxidase, and are constructed by the encapsulation of glucose oxidase within hydrogel microspheres coated with ultrathin polyelectrolyte multilayer films. In order to measure glucose via changes in oxygen concentration, a fluorescent oxygen indicator is co-encapsulated with the enzyme. The model was used to predict the temporal and spatial distributions of glucose and oxygen within the sphere for step increases in bulk glucose concentration. In addition, the model was used to observe the effect of varying sensor parameters, namely sphere size, film thickness, enzyme concentration, and mass transport of substrate and co-substrate within the sphere and film coatings, on the response of the sensors. A major finding was that the application of {PSS/PAH} films as thin as 12 nm can drastically improve the sensor performance over uncoated sensors based on calcium alginate microspheres. The model is proposed as an important tool for a priori design of these complex sensor structures.     

Glutathione regulates transforming growth factor-beta-stimulated collagen production in fibroblasts

Transforming growth factor-beta (TGF-beta) is a potent fibrogenic cytokine. The molecular mechanism underlying TGF-beta fibrogenesis, however, has not been completely elucidated. In this study, we showed that TGF beta decreased the intracellular GSH content in murine embryo fibroblasts (NIH 3T3), which was followed by an increase in collagen I mRNA content and collagen protein production. Prevention of GSH depletion with N-acetylcysteine (NAC), GSH, or GSH ester abrogated TGF-beta-stimulated collagen production, whereas a decrease in intracellular GSH content with L-buthionine-S,R-sulfoximine, an inhibitor of de novo GSH synthesis, enhanced TGF-beta-stimulated collagen production. These results suggest that GSH depletion induced by TGF-beta may mediate TGF-beta-stimulated collagen production. In addition, we showed that TGF-beta stimulated superoxide production and increased release of H2O2 from the cells, whereas GSH ester decreased basal and TGF-beta + glucose oxidase-stimulated H2O2 release. H2O2, exogenously added or continuously generated by glucose oxidase, enhanced TGF-beta-stimulated collagen production, whereas suppression of superoxide production by diphenyliodonium, an NAD(P)H oxidase inhibitor, blocked TGF-beta-stimulated collagen production. These data further suggest that reactive oxygen species are involved in TGF-beta-stimulated collagen production and that the effect of GSH depletion on TGF-beta-stimulated collagen production may be mediated by facilitating reactive oxygen species signaling.     

On-line determination of glucose and lactate concentrations in animal cell culture based on fibre optic detection of oxygen in flow-injection analysis.

A flow-injection analysis (FIA) system based on fibre optic detection of oxygen consumption using immobilized glucose oxidase (GOD) and lactate oxidase (LOD) is described for the on-line monitoring of glucose and lactate concentrations in animal cell cultures. The consumption of oxygen was determined via dynamic quenching by molecular oxygen of the fluorescence of an indicator. GOD and LOD were immobilized on controlled pore glass (CPG) in enzyme reactors which were directly linked to a specially designed fibre optic flow-through cell covering the oxygen optrode. The system is linear for 0-30 mM glucose, with an r.s.d. of 5% at 30 mM (five measurements) and for 0-30 mM lactate, with an r.s.d. of 5% at 30 mM (five measurements). The enzyme reactors used were stable for more than 4 weeks in continuous operation, and it was possible to analyse up to 20 samples per hour. The system has been successfully applied to the on-line monitoring of glucose and lactate concentrations of an animal cell culture designed for the production of recombinant human antithrombine III (AT-III). Results of the on-line measurement obtained by the FIA system were compared with the off-line results obtained by a glucose and lactate analyser from Yellow Springs Instrument Company (YSI).     

Simultaneous production of catalase, glucose oxidase and gluconic acid by Aspergillus niger mutant.

The production of gluconic acid, extracellular glucose oxidase and catalase in submerged culture by a number of biochemical mutants has been evaluated. Optimization of stirrer speed, time cultivation and buffering action of some chemicals on glucose oxidase, catalase and gluconic acid production by the most active mutant, AM-11, grown in a 3-L glass bioreactor was investigated. Three hundred rpm appeared to be optimum to ensure good growth and best glucose oxidase production, but gluconic acid or catalase activity obtained maximal value at 500 or 900 rpm, respectively. Significant increase of dissolved oxygen concentration in culture (16-21%) and extracellular catalase activity were obtained when the traditional aeration was employed together with automatic dosed hydrogen peroxide.     

Electrochemical study of d-glucose oxidase autoinactivation

The immobilization of a d-glucose oxidase (β-d-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) monolayer onto a glassy carbon rotating disc electrode allows the measurement of concentrations in the enzyme's microenvironment and, hence, gives a method of easily following its activity. As it functions, d-glucose oxidase undergoes an autoinactivation which is clearly distinct from inactivation by H₂O₂, and which is more severe as the concentration of the two substrates is increased. It appears that the number of catalytic cycles is fixed at 10⁷, irrespective of the concentrations of the two substrates. Kinetically, it is the enzyme-substrate complex which seems to be inactivated. Scavengers of toxic species of O₂ have no effect on the kinetics of autoinactivation. Identical results were found in solution.