Glucose Dehydrogenase Activity Research Articles

Enhancing the Imine Reductase Activity of a Promiscuous Glucose Dehydrogenase for Scalable Manufacturing of a Chiral Neprilysin Inhibitor Precursor

Enhancing the Imine Reductase Activity of a Promiscuous Glucose Dehydrogenase for Scalable Manufacturing of a Chiral Neprilysin Inhibitor Precursor

A Novel Chinese Medicine Formula Inhibits Non-small Cell Lung Cancer by Triggering Oxidative Stress Dependent on Pentose Phosphate Pathway

Non-small cell lung cancer (NSCLC) is one of the most lethal malignancies worldwide. A novel Chinese medicine formula-01 (NCHF-01) has shown significant clinical efficacy in the treatment of NSCLC, but the mechanism of this formula in the treatment of NSCLC is not fully understood. The aim of this study is to investigate the molecular mechanism of NCHF-01 in inhibiting NSCLC. Lewis lung cells (LLC) tumor bearing mice were established to detect the tumor inhibitory effect of NCHF-01. The morphological changes of tissues and organs in LLC tumor-bearing mice were detected by hematoxylin-eosin (HE) staining. NSCLC cells were treated by NCHF-01. The effects of cell viability and proliferation were detected by MTT and crystal violet staining experiment. Flow cytometry was used to detect cell cycle, apoptosis and reactive oxygen species (ROS). Network pharmacology was used to predict the mechanism of its inhibitory effect of NSCLC. Western blot and immunohistochemistry (IHC) were used to detect the expression of related proteins. NCHF-01 can inhibit tumor growth in LLC tumor-bearing mice, and has no obvious side effects on other tissues and organs. NCHF-01 could inhibit cell viability and proliferation, induce G2/M phase arrest and apoptosis, and promote the increase of ROS level. Network pharmacological analysis showed that NCHF-01 exerts anti-NSCLC effects through various biological processes such as oxidative stress and central carbon metabolism. NCHF-01 can reduce the protein expression and enzyme activity of the key enzymes 6-phosphate glucose dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) in the pentose phosphate pathway (PPP). NCHF-01 can inhibit NSCLC through oxidative stress dependent on the PPP.

Notch signaling generates the “cut here line” on the cuticle of the puparium in Drosophila melanogaster

During a molt or eclosion, insects shed their cuticle, an extracellular matrix made by underlying epidermal cells, by cleavage along a defined line. This means that the "cut here line" is pre-formed on the cuticle, and its formation is indispensable for insect life. Here, we show that the proper formation of the operculum ridge (OR), which is the "cut here line" on the puparium (pupal case) of Drosophila melanogaster, involves Notch signaling activation in the epidermal cells just beneath the future OR region (OR-forming cells). The inhibition of Notch signaling causes defects in eclosion due to failure in OR cleavage, the chitin organization and several cuticular proteins localization, glucose dehydrogenase (Gld) activity, and OR-forming cell shape. Our findings provide the first insight into the molecular basis of the structure and formation of the "cut here line" on the cuticle.

Recombinant Protein Expression Chassis Library of Vibrio natriegens by Fine-Tuning the Expression of T7 RNA Polymerase.

Vibrio natriegens is the fastest-growing bacteria, and its doubling time is less than 10 min. At present, the T7 expression system has been introduced into V. natriegens for heterologous protein expression, including the commercial strain Vmax1 and the variant VnDX,2 which is a backup expression chassis of Escherichia coli BL21(DE3). However, the strength of the existing T7 expression system is not optimal for every recombinant protein. The different expression strengths of T7 RNA polymerase (T7 RNAP) can be obtained by changing the promoter and ribosome binding site (RBS) sequences of T7 RNAP at different transcription and translation levels. In this work, we obtained a robust VnDX variant library with the fine-tuning T7 RNAP using the industrially used enzyme glucose dehydrogenase (GDH) as the reporter protein. Among this library, the variant VnDX-tet, whose promoter of T7 RNAP was changed from PlacUV5 to Ptet, showed that the reporter enzyme GDH activity was increased by 109% by the T7 expression system. Similarly, variants with different T7 RNAP translation levels were obtained by changing RBS sequences upstream of T7 RNAP, and the results showed that the variant VnDX-RBS12/pGDH had the highest GDH activity, which increased by 12.6%. The VnDX variant library constructed in this study with different T7 expression strengths provides a choice for expressing various recombinant proteins, greatly expanding the application of V. natriegens.

Directed evolution engineering to improve activity of glucose dehydrogenase by increasing pocket hydrophobicity.

Glucose dehydrogenase (GDH) is a NAD(P)+ dependent oxidoreductase, which is useful in glucose determination kits, glucose biosensors, cofactor regeneration, and biofuel cells. However, the low efficiency of the catalysis hinders the use of GDH in industrial applications. In this study, an analysis of interactions between eight GDH mutants and NADP+ is powered by AlphaFold2 and Discovery Studio 3.0. The docking results showed that more hydrogen bonds formed between mutants, such as P45A and NADP+, which indicated that these mutants had the potential for high catalytic efficiency. Subsequently, we verified all the mutants by site-directed mutagenesis. It was notable that the enzyme activity of mutant P45A was 1829 U/mg, an improvement of 28-fold compared to wild-type GDH. We predicted the hydrophobicity of the protein-ligand complexes, which was confirmed by an 8-anilino-1-naphthalenesulphonic acid fluorescent probe. The following order of increasing hydrophobicity index was deduced: GDH < N46E < F155Y < P45A, which suggested that the enzyme activity of GDH is positively related to its pocket hydrophobicity. Furthermore, P45A still showed better catalytic ability in organic solvents, reaching 692 U/mg in 10% isopropanol, which was 19-fold that of the wild-type GDH. However, its substrate affinity was affected by organic solvents. This study provides a good theoretical foundation for further improving the catalytic efficiency of GDH.

Highly sensitive and stable self-powered biosensing for exosomes based on dual metal-organic frameworks nanocarriers

Biofuel cells (BFCs)-based self-powered biosensors suffer from the limited stability of bioenzymes. Meanwhile, the poor performance of self-powered biosensors affects the sensitivity of biosensing, thus, it is significant and challenging to improve their stability and sensitivity. In our work, a BFC-based self-powered biosensor, with simultaneously enhanced stability and sensitivity, was constructed utilizing dual metal-organic frameworks (MOFs) as the carriers of the bioenzyme and the electroactive probe, respectively. Anodic enzyme, glucose dehydrogenase (GDH), was encapsulated in zeolitic imidazolate framework-8 (ZIF-8) to form GDH@ZIF-8 composites, enhancing the catalytic activity and stability of GDH. Meanwhile, another zirconium metal-organic frameworks (UiO-66-NH2) loaded with electroactive molecules (K3[Fe(CN)6]) served as nano-enrichment carriers and improved the capability of the cathode to accept electrons from the anode, further improving the sensitivity of the as-proposed biosensor. Herein, the “signal-on” BFC-based self-powered biosensing of exosomes, the model analyte, with excellent stability and outstanding sensitivity was realized with the assistance of dual MOFs, and the detection limit was down to 300 particles mL−1 (based on 3s/k), which was superior to those previously reported in literatures. Furthermore, the developed protocol was capable of detecting exosomes derived from cancer cells in complex biological samples. Overall, in this work the enhancement of both stability and sensitivity has been achieved by utilizing two types of MOFs, which laid the foundation for expanding the applications of BFC-based self-powered biosensors.

Electrochemical Aptasensor Employing Enzyme-Aptamer One-to-One Complex Showing Quasi-Direct Electron Transfer Ability

1. Introduction In this study, we report an innovative electrochemical aptasensor based on the labeling aptamer with quasi-direct electron transfer (quasi-DET)-type redox-enzyme, by developing a breakthrough technological platform to prepare one-to-one complex of enzyme and aptamer.The chronoamperometric-based electrochemical aptasensor is an ideal sensor principle using aptamers for biomolecular recognition considering signal amplification by the labelled redox-enzyme. However, conventional methods to label aptamers with enzymes are chemical modification using cross-linking reagents, which often result in uncontrollable multimer-complexes. Therefore, the method to prepare enzyme-aptamer one-to-one complexes which yields in reproducible and designed enzyme-aptamer complex is technologically challenging. We here report the development of a novel technological platform to prepare enzyme-aptamer one-to-one complex, based on biomolecular engineering concept. 2. Methods Enzyme-aptamer one-to-one complex was constructed utilizing monomeric streptavidin (mSA) and biotin complex formation [1] [2]. We used FAD-dependent glucose dehydrogenase (GDH) from Aspergillus flavus as an aptamer labelling enzyme, and anti-vascular endothelial growth factor (VEGF) aptamer, as to proof of this conceptional study. VEGF is a biomarker for cancer diagnosis. We created the recombinant fusion protein of GDH and mSA (GDH-mSA). The combination of GDH-mSA with biotinylated anti-VEGF aptamer results in their one-to-one complex. To evaluate the formation of the complex and detect VEGF using GDH activity, we performed enzyme-linked aptamer assay (ELAA). Next, GDH-mSA was evaluated as a label enzyme for electrochemical aptasensor, by chronoamperometry analysis using one-to-one complex of GDH-mSA and thiolated and biotinylated aptamer on the gold electrode. Before the analysis, we modified GDH-mSA covalently with phenazine ethosulfate (PES) which was a redox probe enabling redox-enzymes to possess quasi-DET ability [3]. The formation of the enzyme-aptamer one-to-one complex on the electrode and quasi-DET ability of PES-modified GDH-mSA was evaluated. 3. Results GDH-mSA showed dye-mediated GDH activity, indicating the fusion of mSA did not negatively affect the catalytic activity. As a result of ELAA, GDH-mSA showed remarkable GDH activity in the presence of VEGF and biotinylated anti-VEGF aptamer. This indicated one-to-one complex of GDH-mSA and biotinylated anti-VEGF aptamer was formed as we designed, and showed specific recognition and binding with VEGF. Chronoamperometry analysis was carried out, and the signal was increased strikingly upon the addition of glucose only with the gold electrode immobilizing PES-modified GDH-mSA with biotinylated aptamer. Unmodified GDH-mSA did not show quasi-DET. These results indicated our success in the construction of a novel technological platform to prepare enzyme-aptamer one-to-one complex, based on biomolecular engineering concept. 4. Conclusions In this study, to circumvent the formation of uncontrollable multimer-complexes of enzymes and aptamers, we created enzyme-aptamer one-to-one complex based on the mSA-biotin interaction. Thus designed enzyme-aptamer one-to-one complex was prepared without losing both characteristics and acknowledging both benefits. These results suggested that GDH-mSA and biotinylated aptamer complex can be applied for the development of quasi-DET-type aptasensor by simply modifying GDH-mSA with PES. These benefits of utilizing GDH-mSA combined with aptamer will lead to the further application of aptamer-based biosensing of various targets. 5.

Downregulation of T7 RNA polymerase transcription enhances pET-based recombinant protein production in Escherichia coli BL21 (DE3) by suppressing autolysis.

Escherichia coli BL21 (DE3) is an excellent and widely used host for recombinant protein production. Many variant hosts were developed from BL21 (DE3), but improving the expression of specific proteins remains a major challenge in biotechnology. In this study, we found that when BL21 (DE3) overexpressed glucose dehydrogenase (GDH), a significant industrial enzyme, severe cell autolysis was induced. Subsequently, we observed this phenomenon in the expression of 10 other recombinant proteins. This precludes a further increase of the produced enzyme activity by extending the fermentation time, which is not conducive to the reduction of industrial enzyme production costs. Analysis of membranestructure and messenger RNA expression analysis showed that cells could underwent a form of programmed cell death (PCD) during the autolysis period. However, blocking three known PCD pathways in BL21 (DE3) did not completely alleviate autolysis completely. Consequently, we attempted to develop a strong expression host resistant to autolysis by controlling the speed of recombinant protein expression. To find a more suitable protein expression rate, the high- and low-strength promoter lacUV5 and lac were shuffled and recombined to yield the promoter variants lacUV5-1A and lac-1G. The results showed that only one base in lac promoter needs to be changed, and the A at the+1 position was changed to a G, resulting in the improved host BL21 (DE3-lac1G), which resistant to autolysis. As a consequence, the GDH activity at 43 h was greatly increased from 37.5 to 452.0 U/ml. In scale-up fermentation, the new host was able to produce the model enzyme with a high rate of 89.55 U/ml/h at 43 h, compared to only 3 U/ml/h achieved using BL21 (DE3). Importantly, BL21 (DE3-lac1G) also successfully improved the production of 10 other enzymes. The engineered E. coli strain constructed in this study conveniently optimizes recombinant protein overexpression by suppressing cell autolysis, and shows great potential for industrial applications.

Purification and immobilization of engineered glucose dehydrogenase: a new approach to producing gluconic acid from breadwaste

BackgroundPlatform chemicals are essential to industrial processes. Used as starting materials for the manufacture of diverse products, their cheap availability and efficient sourcing are an industrial requirement. Increasing concerns about the depletion of natural resources and growing environmental consciousness have led to a focus on the economics and ecological viability of bio-based platform chemical production. Contemporary approaches include the use of immobilized enzymes that can be harnessed to produce high-value chemicals from waste.ResultsIn this study, an engineered glucose dehydrogenase (GDH) was optimized for gluconic acid (GA) production. Sulfolobus solfataricus GDH was expressed in Escherichia coli. The Km and Vmax values for recombinant GDH were calculated as 0.87 mM and 5.91 U/mg, respectively. Recombinant GDH was immobilized on a hierarchically porous silica support (MM-SBA-15) and its activity was compared with GDH immobilized on three commercially available supports. MM-SBA-15 showed significantly higher immobilization efficiency (> 98%) than the commercial supports. After 5 cycles, GDH activity was at least 14% greater than the remaining activity on commercial supports. Glucose in bread waste hydrolysate was converted to GA by free-state and immobilized GDH. After the 10th reuse cycle on MM-SBA-15, a 22% conversion yield was observed, generating 25 gGA/gGDH. The highest GA production efficiency was 47 gGA/gGDH using free-state GDH.ConclusionsThis study demonstrates the feasibility of enzymatically converting BWH to GA: immobilizing GDH on MM-SBA-15 renders the enzyme more stable and permits its multiple reuse.

Кортико-вегетативна регуляція деяких показників обміну вуглеводів у крові холостих свиноматок

The article is devoted to the coverage of the impact of cortical and vegetative regulation mechanisms on homeostasis of carbohydrate metabolism indicators in blood of dry sows. Clinically healthy 3-years-old dry sows of large white breed were used for the studies. Experimental groups of animals were formed depending on certain typological features of nervous activity. The tone of the autonomic nervous system in sows was examined using a trigeminal vagal test. Types of higher nervous activity in pigs were determined using the method of conditioned food reflexes. Serum glucose and lactate dehydrogenase activity were determined. Correlation and analysis of variance were performed to determine the effect of cortical and vegetative mechanisms on carbohydrate metabolism indicators. The results of studies have shown that the tone of the autonomic nervous system is important in glucose homeostasis regulation in dry sows under physiological conditions. This is confirmed by the results of analysis of variance, which showed that sympathicotonia has a probable effect (η 2 x =0.38; p&lt;0.05) on serum glucose. It was found that sows with high excitability of the sympathetic nervous system were characterized by a tendency to a higher content (by 10.36%; p&lt;0.1) of serum glucose than sows with high excitability of the parasympathetic nervous system. Correlation analysis showed that there is a relationship (r=0.50; p&lt;0.05) between the intensity of excitation and inhibition processes in the cerebral cortex and the activity of lactate dehydrogenase in the sows’ blood serum. Under these conditions, sows of strong balanced mobile type of higher nervous activity showed a tendency to higher lactate dehydrogenase activity (by 20.93%; p&lt;0.1) than sows of weak type.

Distinct biotite-weathering activities of Arthrobacter pascens F74 under different contact conditions.

Bacteria play important roles in mineral weathering and soil formation. However, little is known regarding the interactions between biotite and Arthrobacter strains. In this study, the mineral-mineral activities of the Arthrobacter pascens F74 isolated from a weathered rock surface were evaluated for its weathering behavior under direct contact and no contact with biotite. No contact was obtained by using dialysis bags. When directly in contact with biotite, Al and Fe concentrations increased by 9- to 47-fold compared with the controls in the presence of strain F74. Furthermore, strain F74 increased mobilized Al by 106% to 175% and Fe by 29% to 123% under direct contact than under no contact conditions. During biotite dissolution, significantly higher cell numbers and lower pH in the culture medium were observed in the presence of strain F74 under direct contact conditions than under no contact conditions. Significantly higher gluconic acid concentration and glucose dehydrogenase activity were found under direct contact conditions than under no contact and no biotite conditions. Scanning electron microscopy analysis showed cell adhesion on the biotite surface. These results demonstrated that strain F74 behaved differently with respect to biotite-weathering effectiveness and mechanisms under different contact conditions. The results also suggested that direct contact between biotite and strain F74 was important for the production of gluconic acid, cell adhesion on the mineral surface, and the mineral dissolution of the strain.

Modulation of PQQ-dependent glucose dehydrogenase (mGDH and sGDH) activity by succinate in phosphate solubilizing plant growth promoting Acinetobacter sp. SK2.

Prospective plant growth promoting rhizobacteria isolated from the rhizosphere of Vigna radiata was identified as Acinetobacter sp. SK2 that solubilized 682μgml-1 of tricalcium phosphate (TCP) and 86μgml-1 of rock phosphate (RP) with concomitant decrease in pH up to 4 due to the production of gluconate. The biochemical basis of the P solubilization suggested that the gluconate production was mediated by mGDH and sGDH enzymes. Our results illustrate the role of succinate in repression of P solubilization via suppression of mGDH and sGDH activity which correlated with repression of expression of respective genes, gdhA and gdhB. SK2 also produced IAA (117μgml-1), siderophore (87% units), HCN, ammonia and solubilized minerals of Zn and K. Our findings imply that it is important to understand the cause of failure of several phosphate solubilizing bacteria in field conditions where catabolite repression may control the expression of several genes and pathways including that of mineral phosphate solubilization. Furthermore, Acinetobactersp. SK2 bearing two glucose dehydrogenase (gdhA and gdhB) genes was recognized as promising strain for P biofortification and enhanced plant growth promotion.

Trimethyl-ε-caprolactone synthesis with a novel immobilized glucose dehydrogenase and an immobilized thermostable cyclohexanone monooxygenase

An often associated drawback with Baeyer-Villiger monooxygenases, is its poor operational stability. Furthermore, these biocatalysts frequently suffer from substrate/product inhibition.In this work, a thermostable cyclohexanone monooxygenase (TmCHMO) was immobilized and used in the synthesis of trimethyl-ε-caprolactone (CHL). As a cofactor regeneration enzyme, a novel and highly active glucose dehydrogenase (GDH-01) was used immobilized for the first time. MANA-agarose was the carrier chosen since it presented an immobilization yield of 76.3 ± 0.7% and a retained activity of 62.6 ± 2.3%, the highest metrics among the supports tested.Both immobilized enzymes were studied either separately or together in six reaction cycles (30 mL; [substrate] =132.5 mM). A biocatalyst yield of 37.3 g g−1 of TmCHMO and 474.2 g g−1 of GDH-01 were obtained. These values represent a 3.6-fold and 1.9-fold increase respectively, compared with a model reaction where both enzymes were used in its soluble form.

Unique glucose oxidation catalysis of Gluconobacter oxydans constitutes an efficient cellulosic gluconic acid fermentation free of inhibitory compounds disturbance.

Toxic inhibitory compounds from lignocellulose pretreatment are the major obstacle to achieve high bioconversion efficiency in biorefinery fermentations. This study shows a unique glucose oxidation catalysis of Gluconobacter oxydans with its gluconic acid productivity free of inhibitor disturbance. The microbial experimentations and the transcriptome analysis revealed that both the activity of the membrane-bound glucose dehydrogenase and the transcription level of the genes in periplasmic glucose oxidation respiratory chain of G. oxydans were essentially not affected in the presence of inhibitory compounds. G. oxydans also rapidly converted furan and phenolic aldehyde inhibitors into the less toxic alcohols or acids. The synergy of the robust periplasmic glucose oxidation and the rapid inhibitor conversion of G. oxydans significantly elevated the efficiency of the oxidative fermentation in lignocellulose hydrolysate. The corresponding genes responsible for the conversion of furan and phenolic aldehyde inhibitors were also mined by DNA microarrays. The synergistic mechanism of G. oxydans provided an important option of metabolic modification for enhancing inhibitor tolerance of general fermentation strains.

Promising Strain of Acinetobacter from Soil for Utilization of Gluconic Acid Production

Gluconic acid, a food additive, is used in many foods to control acidity or binds metals such as calcium, iron. Acinetobacter sp. WR326, newly isolated from soil possesses high phosphate solubilizing activity and do not require pyrroloquinoline quinone (PQQ) for glucose dehydrogenase (GDH) activity. In this study the gluconic acid production potential of these bacteria was investigated. Firstly, Acinobacter sp. WR326 was incubated in tricalcium phosphate medium (TCP) with varying glucose concentrations (100, 250, 500 mM), at a temperature of 30 °C for 5 days (120 hours). The highest gluconic acid yield (59%) was observed at a glucose concentration of 100 mM. Then three different levels of gluconic acid addition to the medium (50, 100, 200 mM) were tested. When Acinetobacter sp. WR326 strain was cultivated with a 100 mM glucose and 100 mM gluconic acid the yield increased to 95.27 %. In any trials 2 -keto D-gluconic acid was not detected in the medium. As a conclusion, Acinetobacter sp. WR326 may be considered novel potential bacterial strain for gluconic acid production.

Cadmium chloride–induced testicular toxicity in male wistar rats; prophylactic effect of quercetin, and assessment of testicular recovery following cadmium chloride withdrawal

This study assessed the effect of quercetin (QE) on cadmium chloride (CdCl2) − induced testicular toxicity, as well as the effect of withdrawal of CdCl2 treatment on same. Thirty male Wistar rats aged 10 weeks old and weighing 270–300g were assigned into 5 groups and used for this study. Rats in groups 1–4 were administered vehicle, CdCl2 (5mg/kg bwt), CdCl2+QE (5mg/kg bwt and 20mg/kg bwt, respectively) or QE (20mg/kg bwt) orally for 4 weeks. Group 5 rats received CdCl2, with 4 weeks recovery period. Results showed that cadmium accumulated in serum, testis and epididymis, decreased body weight, testicular and epididymal weights, sperm count, motility and viability. Cadmium decreased serum concentrations of reproductive hormones, but increased testicular glucose, lactate and lactate dehydrogenase activity. Cadmium decreased testicular enzymatic (superoxide dismutase, catalase and glutathione peroxidase) and non-enzymatic (glutathione, vitamins C and E) antioxidants, and increased malondialdehyde and hydrogen peroxide. Cadmium down-regulated Bcl-2 protein, up-regulated Bax protein, increased Bax/Bcl-2 ratio and cleaved caspase-3 activity. Histopathology of the testis showed decreased Johnsen’s score and Leydig cell count. These negative effects were attenuated by QE administration, while withdrawal of CdCl2 did not appreciably reverse toxicity. We conclude that QE better protected the testis from CdCl2 toxicity than withdrawal of CdCl2 administration.

Extended Catalytic Scope of a Well-Known Enzyme: Asymmetric Reduction of Iminium Substrates by Glucose Dehydrogenase.

NADP(H)-dependent imine reductases (IREDs) are of interest in biocatalytic research due to their ability to generate chiral amines from imine/iminium substrates. In reaction protocols involving IREDs, glucose dehydrogenase (GDH) is generally used to regenerate the expensive cofactor NADPH by oxidation of d-glucose to gluconolactone. We have characterized different IREDs with regard to reduction of a set of bicyclic iminium compounds and have utilized 1 H NMR and GC analyses to determine degree of substrate conversion and product enantiomeric excess (ee). All IREDs reduced the tested iminium compounds to the corresponding chiral amines. Blank experiments without IREDs also showed substrate conversion, however, thus suggesting an iminium reductase activity of GDH. This unexpected observation was confirmed by additional experiments with GDHs of different origin. The reduction of C=N bonds with good levels of conversion (>50 %) and excellent enantioselectivity (up to >99 % ee) by GDH represents a promiscuous catalytic activity of this enzyme.

Carbohydrate uptake in Advenella mimigardefordensis strain DPN7T is mediated by periplasmic sugar oxidation and a TRAP-transport system.

In this study, we investigated an SBP (DctPAm ) of a tripartite ATP-independent periplasmic transport system (TRAP) in Advenella mimigardefordensis strain DPN7T . Deletion of dctPAm as well as of the two transmembrane compounds of the tripartite transporter, dctQ and dctM, impaired growth of A. mimigardefordensis strain DPN7T , if cultivated on mineral salt medium supplemented with d-glucose, d-galactose, l-arabinose, d-fucose, d-xylose or d-gluconic acid, respectively. The wild type phenotype was restored during complementation studies of A. mimigardefordensis ΔdctPAm using the broad host vector pBBR1MCS-5::dctPAm . Furthermore, an uptake assay with radiolabeled [14 C(U)]-d-glucose clearly showed that the deletion of dctPAm , dctQ and dctM, respectively, disabled the uptake of this aldoses in cells of either mutant strain. Determination of KD performing thermal shift assays showed a shift in the melting temperature of DctPAm in the presence of d-gluconic acid (KD 11.76 ± 1.3 µM) and the corresponding aldonic acids to the above-mentioned carbohydrates d-galactonate (KD 10.72 ± 1.4 µM), d-fuconic acid (KD 13.50 ± 1.6 µM) and d-xylonic acid (KD 8.44 ± 1.0 µM). The sugar (glucose) dehydrogenase activity (E.C.1.1.5.2) in the membrane fraction was shown for all relevant sugars, proving oxidation of the molecules in the periplasm, prior to transport.

Identification and characterization of thermostable glucose dehydrogenases from thermophilic filamentous fungi.

FAD-dependent glucose dehydrogenase (FAD-GDH), which contains FAD as a cofactor, catalyzes the oxidation of D-glucose to D-glucono-1,5-lactone, and plays an important role in biosensors measuring blood glucose levels. In order to obtain a novel FAD-GDH gene homolog, we performed degenerate PCR screening of genomic DNAs from 17 species of thermophilic filamentous fungi. Two FAD-GDH gene homologs were identified and cloned from Talaromyces emersonii NBRC 31232 and Thermoascus crustaceus NBRC 9129. We then prepared the recombinant enzymes produced by Escherichia coli and Pichia pastoris. Absorption spectra and enzymatic assays revealed that the resulting enzymes contained oxidized FAD as a cofactor and exhibited glucose dehydrogenase activity. The transition midpoint temperatures (T m) were 66.4 and 62.5°C for glycosylated FAD-GDHs of T. emersonii and T. crustaceus prepared by using P. pastoris as a host, respectively. Therefore, both FAD-GDHs exhibited high thermostability. In conclusion, we propose that these thermostable FAD-GDHs could be ideal enzymes for use as thermotolerant glucose sensors with high accuracy.

Regulation of Pyrroloquinoline Quinone-Dependent Glucose Dehydrogenase Activity in the Model Rhizosphere-Dwelling Bacterium Pseudomonas putida KT2440.

Soil-dwelling microbes solubilize mineral phosphates by secreting gluconic acid, which is produced from glucose by a periplasmic glucose dehydrogenase (GDH) that requires pyrroloquinoline quinone (PQQ) as a redox coenzyme. While GDH-dependent phosphate solubilization has been observed in numerous bacteria, little is known concerning the mechanism by which this process is regulated. Here we use the model rhizosphere-dwelling bacterium Pseudomonas putida KT2440 to explore GDH activity and PQQ synthesis, as well as gene expression of the GDH-encoding gene (gcd) and PQQ biosynthesis genes (pqq operon) while under different growth conditions. We also use reverse transcription-PCR to identify transcripts from the pqq operon to more accurately map the operon structure. GDH specific activity and PQQ levels vary according to growth condition, with the highest levels of both occurring when glucose is used as the sole carbon source and under conditions of low soluble phosphate. Under these conditions, however, PQQ levels limit in vitro phosphate solubilization. GDH specific activity data correlate well with gcd gene expression data, and the levels of expression of the pqqF and pqqB genes mirror the levels of PQQ synthesized, suggesting that one or both of these genes may serve to modulate PQQ levels according to the growth conditions. The pqq gene cluster (pqqFABCDEG) encodes at least two independent transcripts, and expression of the pqqF gene appears to be under the control of an independent promoter and terminator. Plant growth promotion can be enhanced by soil- and rhizosphere-dwelling bacteria by a number of different methods. One method is by promoting nutrient acquisition from soil. Phosphorus is an essential nutrient that plants obtain through soil, but in many cases it is locked up in forms that are not available for plant uptake. Bacteria such as the model bacterium Pseudomonas putida KT2440 can solubilize insoluble soil phosphates by secreting gluconic acid. This chemical is produced from glucose by the activity of the bacterial enzyme glucose dehydrogenase, which requires a coenzyme called PQQ. Here we have studied how the glucose dehydrogenase enzyme and the PQQ coenzyme are regulated according to differences in bacterial growth conditions. We determined that glucose dehydrogenase activity and PQQ production are optimal under conditions when the bacterium is grown with glucose as the sole carbon source and under conditions of low soluble phosphate.