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Immobilization of enzymes is valuably important as it improves the stability and hence increases the reusability of enzymes. The present investigation is an attempt for immobilization of purified glucose-6-phosphate dehydrogenase from pigeon pea on different matrix. Maximum immobilization was achieved when alginate was used as immobilization matrix. As compared to soluble enzyme the alginate immobilized enzyme exhibited enhanced optimum pH and temperature. The alginate immobilized enzyme displayed more than 80% activity up to 7 continuous reactions and more than 50% activity up to 11 continuous reactions.

Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP(+) (k cat/K m = 2618 mM(-1) s(-1), k cat = 249 s(-1), K m = 0.10 ± 0.01 mM) as cofactor, although NAD(+) (k cat/K m = 138 mM(-1) s(-1), k cat = 604 s(-1), K m = 4.37 ± 0.56 mM) could also be accepted. The K m values of glucose-6-phosphate were 0.27 ± 0.07 mM and 5.08 ± 0.68 mM with NADP(+) and NAD(+) as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8-9.0 for NADP(+) and at pH 7.0-8.6 for NAD(+) while the optimal temperature was 80 °C for NADP(+) and 70 °C for NAD(+). This was the first observation that the NADP(+)-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD(+)-linked and growth (75 °C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase.

Glucose-6-phosphate dehydrogenase (G6PD) is a key regulatory enzyme involved in the pentose phosphate pathway. This works represents purification of two buffalo liver glucose-6-phosphate dehydrogenases (BLG6PD1 and BLG6PD2) using combination of ammonium sulfate precipitation and several chromatographic columns. Both enzymes (BLG6PD1 and BLG6PD2) were homogenous on both native PAGE as well as 12% SDS PAGE with molecular weights of 28 and 66 kDa. The molecular weight of BLG6PD1 and BLG6PD2 native forms were determined to be 28 and 66 kDa by gel filtration; indicating monomeric proteins. The K(m) values for BLG6PD1 and BLG6PD2 estimated to be 0.059 and 0.06 mM of β-nicotinamide adenine dinucleotide phosphate. The optimum activity of BLG6PD1 and BLG6PD2 were displayed at pH 8.0 and 8.2 with an isoelectric point (pI) of pH 7.7-7.9 and 5.7-5.9. The divalent cations MgCl2, and CoCl2 act as activators, on the other hand, FeCl2, CuCl2 and ZnCl2 are potent inhibitors of BLG6PD1 and BLG6PD2 activity. NADPH inhibited both isoenzymes competitively with Ki values of 0.012 and 0.030 mM. This study describes a reproducible purification scheme of G6PD from the liver of buffalo as a rich source.

Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first step of the pentose phosphate pathway. In erythrocytes, the functionality of the pathway is crucial to protect these cells against oxidative damage. G6PD deficiency is the most frequent enzymopathy in humans with a global prevalence of 4.9 %. The clinical picture is characterized by chronic or acute hemolysis in response to oxidative stress, which is related to the low cellular activity of G6PD in red blood cells. The disease is heterogeneous at genetic level with around 160 mutations described, mostly point mutations causing single amino acid substitutions. The biochemical studies aimed to describe the detrimental effects of mutations on the functional and structural properties of human G6PD are indispensable to understand the molecular physiopathology of this disease. Therefore, reliable systems for efficient expression and purification of the protein are highly desirable. In this work, human G6PD was heterologously expressed in Escherichia coli and purified by immobilized metal affinity chromatography in a single chromatographic step. The structural and functional characterization indicates that His-tagged G6PD resembles previous preparations of recombinant G6PD. In contrast with previous protein yield systems, our method is based on commonly available resources and fully accessible laboratory equipment; therefore, it can be readily implemented.

Psychrophilic organisms have evolved a number of modifications of cellular structures to survive in the cold environment; among them it is worth noting an increased efficiency of enzymes at lower temperatures. Glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) was purified and characterised from the psychrophilic green alga Koliella antarctica (Trebouxiophyceae, Chlorophyta) from the Ross Sea (Antarctica). It was possible to isolate a single G6PDH using biochemical strategies; its maximum activity was measured at 35 °C, and the enzyme showed an E (a) of 39.6 kJ mol(-1). This protein reacted with antibodies raised against higher plants plastidic isoforms. KaG6PDH showed peculiar kinetic properties, with a K (iNADPH) value lower than [Formula: see text]. Notably, catalytic activity was inactivated in vitro by DTT and chloroplastic thioredoxin f. These biochemical properties of G6PDH are discussed with respect to higher plant G6PDHs and the adaptation of K. antarctica to polar low-temperature environment.

Proteins from thermophilic microorganisms are stabilized by various mechanisms to preserve their native folded states at higher temperatures. A thermostable glucose-6-phosphate dehydrogenase (tG6PDH) from the hyperthermophilic bacterium Aquifex aeolicus was expressed as a recombinant protein in Escherichia coli. The A. aeolicus G6PDH is a homodimer exhibiting remarkable thermostability (t1/2 = 24 hr at 90 degrees C). Based on homology modeling and upon comparison of its structure with human G6PDH, it was predicted that cysteine 184 of one subunit could form a disulfide bond with cysteine 352 of the other subunit resulting in reinforced intersubunit interactions that hold the dimer together. Site-directed mutagenesis was performed on tG6PDH to convert C184 and C352 to serines. The tG6PDH double mutant exhibited a dramatic decrease in the half-life from 24 hr to 3 hr at 90 degrees C. The same decrease in half-life was also found when either C184 or C352 was mutated to serine. The result indicates that C184 and C352 may play a crucial role in strengthening the dimer interface through disulfide bond formation, thereby contributing to the thermal stability of the enzyme.

Glucose-6-phosphate dehydrogenase (G6PD) was purified from rat small intestine with 19.2% yield and had a specific activity of 53.8 units per miligram protein. The pH optimum was determined to be 8.1. The purified rat small intestinal G6PD gave one activity, one protein band on native PAGE. The observation of one band on SDS/PAGE with an Mr of 48 kDa and a specific activity lower than expected may suggest the proteolytically affected enzyme or different form of G6PD in the rat small intestine. The activation energy, activation enthalpy, Q10, and optimum temperature from Arrhenius plot for the rat small intestinal G6PD were found to be 8.52 kcal/mol, 7.90 kcal/mol, 1.59, and 38 degrees C, respectively. The Km values for G6P and NADP+ were 70.1 +/- 20.8 and 23.2 +/- 7.6 microM, respectively. Double-reciprocal plots of 1/Vm versus 1/G6P (at constant [NADP+]) and of 1/Vm versus 1/NADP+ at constant [G6P]) intersected at the same point on the 1/Vm axis to give Vm = 53.8 U/mg protein.

The gsdA gene of the extreme thermophilic bacterium Aquifex aeolicus, encoding glucose-6-phosphate dehydrogenase (G6PDH), was cloned into a high-expression vector and overexpressed as a fusion protein in Escherichia coli. Here we report the characterization of this recombinant thermostable G6PDH. G6PDH was purified to homogeneity by heat precipitation followed by immobilized metal affinity chromatography on a nickel-chelate column. The data obtained indicate that the enzyme is a homodimer with a subunit molecular weight of 55 kDa. G6PDH followed Michaelis-Menten kinetics with a K(M) of 63 micro M for glucose-6-phosphate at 70 degrees C with NADP as the cofactor. The enzyme exhibited dual coenzyme specificity, although it showed a preference in terms of k(cat)/ K(M) of 20.4-fold for NADP over NAD at 40 degrees C and 5.7-fold at 70 degrees C. The enzyme showed optimum catalytic activity at 90 degrees C. Modeling of the dimer interface suggested the presence of cysteine residues that may form disulfide bonds between the two subunits, thereby preserving the oligomeric integrity of the enzyme. Interestingly, addition of dithiothreitol or mercaptoethanol did not affect the activity of the enzyme. With a half-life of 24 h at 90 degrees C and 12 h at 100 degrees C, this is the most thermostable G6PDH described.

NMR experiments that transfer conformational information from the bound to the uncomplexed state via exchange have been utilized for many years. It is demonstrated here that inter-ligand NOEs ('ILOEs'), which exist in ternary complexes with enzymes or other macromolecular receptors, can be transferred via exchange to pairs of uncomplexed ligands. This approach is illustrated by studies of glycolate + NAD+ in the presence of porcine heart lactate dehydrogenase, and by glucose-6-phosphate + NADPH in the presence of L. mesenteroides glucose-6-phosphate dehydrogenase. This strategy opens up a general methodology for exploring the active sites of enzymes and for the development of artificial ligands which can function as inhibitors, or more generally as modifiers of protein function.

6-Phosphogluconate dehydrogenase (6PGDH) from rat-liver and kidney-cortex cytosol has been partially purified and almost completely isolated (more than 95%) from glucose-6-phosphate dehydrogenase activity. The purification and isolation procedures included high-speed centrifugation, 60-75% ammonium-sulphate fractionation, by which both hexose-monophosphate dehydrogenases activities were separated, and finally the protein fraction was applied to a chromatographic column of Sephadex G-25 equilibrated with 10 mM Tris-EDTA-NADP buffer, pH 7.6, to eliminate any contaminating metabolites. The kinetic properties of the isolated partially purified liver and renal 6PGDH were examined. The saturation curves of this enzyme in both rat tissues showed a typical Michaelis-Menten kinetic, with no evidence of co-operativity. The optimum pH for both liver and kidney-cortex 6PGDH was 8.0. The Km values of liver 6PGDH for 6-phosphogluconate (6PG) and for NADP were 157 microM and 258 microM respectively, while the specific activity measured at optimum conditions (pH 8.0 and 37 degrees C) was 424.2 mU/mg of protein. NADPH caused a competitive inhibition against NADP with an inhibition constant (Ki) of 21 microM. The Km values for 6PG and NADP from kidney-cortex 6PGDH were 49 microM and 56 microM respectively. The specific activity at pH 8.0 and 37 degrees C was 120.7 mU/mg of protein. NADPH also competitively inhibited 6PGDH activity, with a Ki of 41 microM. This paper describes a quick, easy and reliable method for the separation of the two dehydrogenases present in the oxidative segment of the pentose-phosphate pathway in animal tissues, eliminating interference in the measurements of their activities.