Glucose Dehydrogenase Apoenzyme Research Articles

Novel and efficient screening of PQQ high-yielding strains and subsequent cultivation optimization.

A novel and efficient screening method for pyrroloquinoline quinone (PQQ) high-yielding methylotrophic strains was developed by using glucose dehydrogenase apoenzyme (GDHA) which depended on PQQ as the cofactor. Using this high-throughput method, PQQ high-yielding strains were rapidly screened out from thousands of methylotrophic colonies at a time. The comprehensive phylogenetic analysis revealed that the highest PQQ-producing strain zju323 (CCTCC M 2016079) could be assigned to a novel species in the genus Methylobacillus of the Betaproteobacteria. After systematic optimization of different medium components and cultivation conditions, about 33.4mg/L of PQQ was obtained after 48h of cultivation with Methylobacillus sp. zju323 at the shake flask scale. Further cultivations of Methylobacillus sp. zju323 were carried out to investigate the biosynthesis of PQQ in 10-L bench-top fermenters. In the batch operation, the PQQ accumulation reached 78mg/L in the broth after 53h of cultivation. By adopting methanol feeding strategy, the highest PQQ concentration was improved up to 162.2mg/L after 75h of cultivation. This work developed a high-throughput strategy of screening PQQ-producing strains from soil samples and also demonstrated one potential bioprocess for large-scale PQQ production with the isolated PQQ strain.

Kinetics and thermodynamics of activation of quinoprotein glucose dehydrogenase apoenzyme in vivo and catalytic activity of the activated enzyme in Escherichia coli cells

Apo-glucose dehydrogenase existing in Escherichia coli is converted to the holoenzyme with exogenous pyrroloquinoline quinone (PQQ) and Mg2+. Catalytic behaviour of the E. coli cells with the holoenzyme is characterized by a Michaelis–Menten-type equation with a catalytic constant of the cell and apparent Michaelis constants for d-glucose and an artificial electron acceptor added to the E. coli suspension. The catalytic constant is expressed as the product of the number of molecules of the enzyme contained in an E. coli cell (z) and the catalytic constant of the enzyme (kcat), which were determined to be 2.2×103 and 6.8±0.8×103s-1 (phenazine methosulphate as an electron acceptor) respectively. Kinetics of the in vivo holoenzyme formation can be followed by an enzyme-electrochemical method developed by us. The rate constants for the reactions of apoenzyme with PQQ (kf,PQQ) and with Mg2+ (kf,Mg) were determined to be 3.8±0.4×104 M-1·s-1 and 4.1±0.9M-1·s-1 respectively. Equilibrium constants for the binding of apoenzyme to PQQ and Mg2+ were determined as the dissociation constants Kd,PQQ(Mg) and Kd,Mg to be 1.0±0.1nM and 0.14±0.01mM respectively. The dissociation constants for Ca2+ were also determined. The holoenzyme, once formed in E. coli, returns gradually to the apoenzyme in the absence of PQQ and/or Mg2+ in solution. EDTA was effective to remove Mg2+ from the enzyme in the cells to deactivate the enzyme completely, while PQQ remained in the E. coli cells.

Kinetics and thermodynamics of activation of quinoprotein glucose dehydrogenase apoenzyme in vivo and catalytic activity of the activated enzyme in Escherichia coli cells

Apo-glucose dehydrogenase existing in Escherichia coli is converted to the holoenzyme with exogenous pyrroloquinoline quinone (PQQ) and Mg(2+). Catalytic behaviour of the E. coli cells with the holoenzyme is characterized by a Michaelis-Menten-type equation with a catalytic constant of the cell and apparent Michaelis constants for D-glucose and an artificial electron acceptor added to the E. coli suspension. The catalytic constant is expressed as the product of the number of molecules of the enzyme contained in an E. coli cell (z) and the catalytic constant of the enzyme (k(cat)), which were determined to be 2.2x10(3) and 6.8+/-0.8x10(3) s(-1) (phenazine methosulphate as an electron acceptor) respectively. Kinetics of the in vivo holoenzyme formation can be followed by an enzyme-electrochemical method developed by us. The rate constants for the reactions of apoenzyme with PQQ (k(f,PQQ)) and with Mg(2+) (k(f,Mg)) were determined to be 3.8+/-0.4x10(4) M(-1).s(-1) and 4. 1+/-0.9 M(-1).s(-1) respectively. Equilibrium constants for the binding of apoenzyme to PQQ and Mg(2+) were determined as the dissociation constants K(d,PQQ(Mg)) and K(d,Mg) to be 1.0+/-0.1 nM and 0.14+/-0.01 mM respectively. The dissociation constants for Ca(2+) were also determined. The holoenzyme, once formed in E. coli, returns gradually to the apoenzyme in the absence of PQQ and/or Mg(2+) in solution. EDTA was effective to remove Mg(2+) from the enzyme in the cells to deactivate the enzyme completely, while PQQ remained in the E. coli cells.

Reconstitution of membrane-integrated quinoprotein glucose dehydrogenase apoenzyme with PQQ and the holoenzyme's mechanism of action.

Membrane-integrated quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus was produced by heterologous expression of the gene for it in an Escherichia coli recombinant strain. The apoenzyme (lacking the cofactor pyrroloquinoline quinone, PQQ) was solubilized with Triton X-100 and purified to homogeneity. Reconstitution of the apoenzyme to full activity in the assay was achieved with a stoichiometric amount of PQQ in the presence of Mg2+. Just as for other PQQ-containing dehydrogenases where Ca2+ fulfills this role, Mg2+ anchors PQQ to the mGDH protein and activates the bound cofactor. This occurs in a precise way since high anomer specificity was found for the enzyme toward the sugars tested. Although the steady-state-type kinetics were as expected for a dye-linked dehydrogenase (ping-pong) and the PQQ in it was present in oxidized form, addition of glucose to the holoenzyme resulted in a very slow but continuous production of gluconolactone; i.e., the reaction did not stop after one turnover, with O2 apparently acting as an (albeit poor) electron acceptor by reoxidizing PQQH2 in the enzyme. The surprisingly low reactivity with glucose, in the absence of dye, as compared to the activity observed in the steady-state assay appeared to be due to formation of an anomalous enzyme form, mGDH. Formation of normal holoenzyme, mGDH, reducing added glucose immediately to gluconolactone (in one turnover), was achieved by treating mGDH with sulfite, by reconstituting apoenzyme with PQQ in the presence of sulfite, or by applying assay conditions to mGDH (addition of PMS/DCPIP). As compared to other quinoprotein dehydrogenases, mGDH appears to be unique with respect to the mode of PQQ-binding, as expressed by the special conditions for reconstitution and the absorption spectra of the bound cofactor, and the reactivity of the reduced enzyme toward O2. The primary cause for this seems not to be related to a different preference for the activating bivalent metal ion but to the special way of binding of PQQ to mGDH.

Lack of PQQ dependent glucose dehydrogenase apoenzyme in some Acinetobacter spp.

Seventy four asaccharolytic strains of Acinotobacter were tested for the presence of PQQ dependent glucose dehydrogenase apoenzyme. This apoenzyme could not be detected in any of five strains of Acinetobacter haemolyticus; it was also lacking in one strain of each A. junii and A. lwoffi.

The separate roles of PQQ and apo-enzyme syntheses in the regulation of glucose dehydrogenase activity in Klebsiella pneumoniae NCTC 418

No holoenzyme pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase and only very low apoenzyme levels could be detected in cells of Klebsiella pneumoniae, growing anaerobically, or carrying out a fumarate or nitrate respiration. Low glucose dehydrogenase activity in some aerobic glucose-excess cultures of K. pneumoniae (ammonia or sulphate limitation) was increased significantly by addition of PQQ, whereas in cells already possessing a high glucose dehydrogenase activity (phosphate or potassium limitation) extra PQQ had almost no effect. These observations indicate that the glucose dehydrogenase activity in K. pneumoniae is modulated by both PQQ synthesis and synthesis of the glucose dehydrogenase apo-enzyme.

PQQ-Dependent Production of Gluconic Acid by Acinetobacter, Agrobacterium and Rhizobium Species

SUMMARY: Acinetobacter Iwoffi, Azotobacter vinelandii, Agrobacterium and Rhizobium species contain quinoprotein glucose dehydrogenase apoenzyme (EC 1.1.99.17). Addition to whole cells of pyrrolo-quinoline quinone (PQQ), the prosthetic group of this enzyme, resulted in the production of gluconic acid from glucose. The in vivo reconstitution of apo-glucose dehydrogenase with PQQ was dependent on the presence of Ca2+ or Mg2+. Optimal conditions for reconstitution allowed maximal glucose dehydrogenase activity in the presence of 1–10 nmol PQQ 1−1. Synthesis of the apoenzyme of glucose dehydrogenase was not dependent on glucose in the growth media. The physiological significance of the synthesis of apo-glucose dehydrogenase, as found in a variety of bacteria, is discussed.

Energy transduction by electron transfer via a pyrrolo-quinoline quinone-dependent glucose dehydrogenase in Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus (var. lwoffi).

The coupling of membrane-bound glucose dehydrogenase (EC 1.1.99.17) to the respiratory chain has been studied in whole cells, cell-free extracts, and membrane vesicles of gram-negative bacteria. Several Escherichia coli strains synthesized glucose dehydrogenase apoenzyme which could be activated by the prosthetic group pyrrolo-quinoline quinone. The synthesis of the glucose dehydrogenase apoenzyme was independent of the presence of glucose in the growth medium. Membrane vesicles of E. coli, grown on glucose or succinate, oxidized glucose to gluconate in the presence of pyrrolo-quinoline quinone. This oxidation led to the generation of a proton motive force which supplied the driving force for uptake of lactose, alanine, and glutamate. Reconstitution of glucose dehydrogenase with limiting amounts of pyrrolo-quinoline quinone allowed manipulation of the rate of electron transfer in membrane vesicles and whole cells. At saturating levels of pyrrolo-quinoline quinone, glucose was the most effective electron donor in E. coli, and glucose oxidation supported secondary transport at even higher rates than oxidation of reduced phenazine methosulfate. Apoenzyme of pyrrolo-quinoline quinone-dependent glucose dehydrogenases with similar properties as the E. coli enzyme were found in Acinetobacter calcoaceticus (var. lwoffi) grown aerobically on acetate and in Pseudomonas aeruginosa grown anaerobically on glucose and nitrate.

Evidence of a quinoprotein glucose dehydrogenase apoenzyme in several strains ofEscherichia coli

When grown on glucose in K+-limited chemostat culture, or in batch culture with or without 2,4-dinitrophenol, several strains of Escherichia coli (including the type strain) were found to synthesize a quinoprotein glucose dehydrogenase apoenzyme. The pyridine nucleotides, NAD+ and NADP+, would not serve as cofactor, but activity could be demonstrated upon addition of 2,7,9-tricarboxy-1 H-pyrrolo(2,3-f)quinoline-4,5-dione (PQQ). Thus, in the presence of PQQ, but not in its absence, glucose was oxidized to gluconic acid. A mutant of E. coli PC 1000 was isolated that lacked Enzyme I of the phosphoenolpyruvate phosphotransferase system (PTS) but still synthesized the glucose dehydrogenase apoenzyme. Whereas this mutant would not grow on glucose in the absence of PQQ, it would do so in the presence of low concentrations (1 μM) of this cofactor. On the basis of these observations, it is concluded that the protein (apoenzyme) formed is a genuine glucose dehydrogenase, but that it is not functional in growing cells due to their inability to synthesize the appropriate cofactor (PQQ), at least under these conditions.

Evidence of a quinoprotein glucose dehydrogenase apoenzyme in several strains of Escherichia coli

Evidence of a quinoprotein glucose dehydrogenase apoenzyme in several strains of Escherichia coli