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Aysu Yarman Christopher Schulz Cristoph Sygmund Roland Ludwig Lo Gorton Ulla Wollenberger Frieder W. Scheller 《Electroanalysis》2014,26(9):2043-2048
For the first time the direct electron transfer of an enzyme ‐ cellobiose dehydrogenase, CDH ‐ has been coupled with the hexokinase catalyzed competition for glucose in a sensor for ATP. To enhance the signal output for ATP, pyruvate kinase was coimmobilized to recycle ADP by the phosphoenolpyruvate driven reaction. The new sensor overcomes the limit of 1 : 1 stoichiometry of the sequential or competitive conversion of ATP by effective enzymatic recycling of the analyte. The anodic oxidation of the glucose converting CDH proceeds at electrode potentials below 0 mV vs. Ag|AgCl thus potentially interfering substances like ascorbic acid or catecholamines do not influence the measuring signal. The combination of direct electron transfer of CDH with the enzymatic recycling results in an interference‐free and oxygen‐independent measurement of ATP in the lower µmolar concentration range with a lower limit of detection of 63.3 nM (S/N=3). 相似文献
2.
Silva Daniel Pereira Pessoa Adalberto Roberto Inês-Conceição Vitolo Michele 《Applied biochemistry and biotechnology》2001,91(1-9):605-613
A batch culture of Saccharomyces cerevisiae for the production of hexokinase was carried out in a 5-L fermentor containing 3 L of culture medium, which was in oculated
with cell suspension (about 0.7 g/L), and left ferm entingat 35°C and pH 4.0. The aeration and agitation were adjusted to
attain k
La values of 15, 60, 135, and 230 h−1. The highest hexokinase productivity (754.6 U/[L h]) and substrate-cell conversion yield (0.21 g/g) occurred for a k
La of 60 h−1. Moreover, the formation of hexokinase and cell growth are coupled events, which is in accordance with the constitutive character
of this enzyme. Hexokinase formation for k
La>60 h−1 was not enhanced probably owing to saturation of the respiratory pathway by oxygen. 相似文献
3.
E. A. Zaitzeva E. S. Chukria O. M. Poltorak 《Applied biochemistry and biotechnology》1996,61(1-2):67-74
Kinetic study of the mechanism of the temperature-induced loss of the catalytic activity by yeast hexokinase (HK) and yeast
glucose-6phosphate dehydrogenase (G-6-PDG) has shown the dissociative nature of the processes. In the temperature range 40–47°C,
they are satisfactorily described in terms of consecutive reactions in which steps of irreversible denaturation of the monomeric
units follow the reversible dissociation of inactive oligomeric forms into the active units, resulting in an increase in catalytic
activity. The experimental data have been analyzed in the framework of the dissociative mechanism, and a semiquantitative
method has been developed for calculating the individual rate constants. 相似文献
4.
A hydrogen peroxide based enzyme electrode for the determination of ATP has been developed by the immobilization of glucose oxidase and hexokinase. Competition between the enzymes for the substrate glucose allowed the measurement of ATP. Different immobilization procedures and different types of hexokinase have been tested. Using a BSA-glutaraldehyde procedure and hexokinase from an overproducing strain of bakers' yeast, ATP was measured in the 0.05–0.5 mmol l−1 range with a detection limit of 0.01 mmol l−1. ATP concentrations comparable to those reported in the literature and a good recovery were obtained when the enzyme electrode was used with human erythrocyte hemolysate. 相似文献
5.
George G. G. Oliveira Daniel P. Silva Ines Conceição Roberto Michele Vitolo Adalberto Pessoa Jr. 《Applied biochemistry and biotechnology》2003,108(1-3):787-797
This study dealt with the partition behavior and partial purification of hexokinase (HK) from baker’s yeast by liquid-liquid
extraction using aqueous two-phase polyethylene glycol (PEG)/citrate systems. First, we investigated the effect of agitation
type (vortex and 8 rpm rotation) on the stability of the system, and then the effects of sodium citrate concentration, PEG
concentration, and molar mass of PEG on the partition coefficient of this enzyme by using a 25 factorial experimental design. The results of this factorial experiment showed the possibility of a partial purification
of HK by using two extraction steps, since the enzyme preferentially migrated to the top phase and the total proteins (mainly
contaminants) remained in the bottom phase. The purification factor (Pur
TOP) of the enzyme in the top phase was 1.87, and the partition coefficient of the total proteins (K
Prot
) was 0.47. 相似文献
6.
HEPES-based biological buffer is subject to photooxidation upon exposure to fluorescent illumination. Thereby hydrogen peroxide
is generated, which interferes with amperometric oxidoreductase-based biosensors for glucose or adenosine triphosphate (ATP).
These biosensors operate at an oxidation potential above 500 mV vs. the standard calomel electrode (SCE) and involve hydrogen
peroxide as the electroactive molecule detected at the electrode surface. False-positive detection of ATP was observed in
HEPES buffer utilizing an amperometric microbiosensor based on the co-immobilization of glucose oxidase and hexokinase for
detection of ATP in biological specimens. Electrochemical, mass spectrometric, 31P NMR, and 1H NMR studies indicate that complexation of ATP and HEPES induced by the presence of Ca2+ in HEPES buffer decreases the photooxidation of HEPES. Consequently, the hydrogen peroxide background concentration is reduced,
thereby leading to erroneous ATP detection at the dual-enzyme microbiosensor, which determines an increase in ATP via a reduced
hydrogen peroxide signal. 相似文献
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