Glucose Dehydrogenase Gene Research Articles

Azotobacters as biofertilizer.

Azotobacters have been used as biofertilizer since more than a century. Azotobacters fix nitrogen aerobically, elaborate plant hormones, solubilize phosphates and also suppress phytopathogens or reduce their deleterious effect. Application of wild type Azotobacters results in better yield of cereals like corn, wheat, oat, barley, rice, pearl millet and sorghum, of oil seeds like mustard and sunflower, of vegetable crops like tomato, eggplant, carrot, chillies, onion, potato, beans and sugar beet, of fruits like mango and sugar cane, of fiber crops like jute and cotton and of tree like oak. In addition to the structural genes of the enzyme nitrogenase and of other accessory proteins, A. vinelandii chromosomes contain the regulatory genes nifL and nifA. NifA must bind upstream of the promoters of all nif operons for enabling their expression. NifL on activation by oxygen or ammonium, interacts with NifA and neutralizes it. Nitrogen fixation has been enhanced by deletion of nifL and by bringing nifA under the control of a constitutive promoter, resulting in a strain that continues to fix nitrogen in presence of urea fertilizer. Additional copies of nifH (the gene for the Fe-protein of nitrogenase) have been introduced into A. vinelandii, thereby augmenting nitrogen fixation. The urease gene complex ureABC has been deleted, the ammonia transport gene amtB has been disrupted and the expression of the glutamine synthase gene has been regulated to enhance urea and ammonia excretion. Gluconic acid has been produced by introducing the glucose dehydrogenase gene, resulting in enhanced solubilization of phosphate.

Phosphate solubilizing bacteria with glucose dehydrogenase gene for phosphorus uptake and beneficial effects on wheat.

The aim of this study was to isolate, characterize and use phosphate solubilizing bacteria to enhance the bioavailability of insoluble Ca-phosphate for wheat plants. For this purpose, 15 phosphorus solubilizing bacteria (PSB) were isolated from wheat rhizospheric soils of Peshawar and southern Punjab region, Pakistan. These isolates were identified using light microscopy and 16S rRNA gene. Among the isolated bacteria, two strains (Pseudomonas sp. MS16 and Enterobacter sp. MS32) were the efficient P solubilizers based on their P solubilization activity determined qualitatively (solubilization index 3.2–5.8) as well as quantitatively (136–280 μg mL-1). These two strains produced indole-3-acetic acid (25.6–28.1 μg mL-1), gibberellic acid (2.5–11.8), solubilized zinc compounds (SI 2.8–3.3) and showed nitrogenase and 1-Aminocyclopropane-1-carboxylic acid deaminase activity in vitro. Phosphate solubilization activity of Pseudomonas sp. MS16 was further validated by amplification, sequencing and phylogenetic analysis of glucose dehydrogenase (gcd) gene (LT908484) responsible for P solubilization. Response Surface Methodology for large-scale production was used to find optimal conditions (Temperature 22.5°C, pH 7) for P solubilization. Glucose was found to support higher P solubilization in vitro. In an in vitro experiment, PSB treated wheat seedlings improved germination and seedling vigor (11% increases) as compared to un-inoculated control. Rhizoscanning of these seedlings showed an increase in various root growth parameters. Wheat inoculation with selected strain MS16 showed pronounced effect on grain yield in pot (38.5% increase) and field (17–18% increase) experiments compared to non-inoculated control. Root colonization by PSB through Florescent in situ Hybridization and Confocal Laser Scanning Microscopy confirmed their rhizosphere competence in soil. BOX-PCR confirmed the re-isolated colonies of Pseudomonas sp. MS16. The results indicated that gluconic acid producing Pseudomonas sp. MS16 from un-explored soils may be cost effective and environment friendly candidate to improve plant growth and phosphorous uptake by wheat plants.

Fungal FAD-dependent glucose dehydrogenases concerning high activity, affinity, and thermostability for maltose-insensitive blood glucose sensor

Six FAD-dependent glucose dehydrogenase (FAD-GDH) genes, for blood glucose sensors, from the genomic DNAs of Aspergillus species were identified by degenerated PCR screening. The enzymatic properties of the gene products expressed by Pichia pastoris were characterized. Investigation of substrate selectivity to fourteen saccharides revealed that these enzymes were maltose-insensitive FAD-GDHs. A.bisporus, A.terreus var. aureus, and A. phoenicis FAD-GDHs showed high substrate selectivity; they exhibited reactivity to only one or two saccharides. A. phoenicis FAD-GDH specifically exhibited insensitivity to d-xylose. Furthermore, six FAD-GDHs exhibited superior parameter values, such as maximum velocity (Vmax), Michaelis-Menten constant (Km), and apparent transition midpoint temperature (Tm). A. phoenicis and A. terreus var. aureus FAD-GDHs exhibited high activity, with Vmax values of (8.66 ± 0.17) × 104 U/μmol and (1.14 ± 0.03) × 105 U/μmol, respectively. A. brunneo-uniseriatus, A.carneus, and A. malignus FAD-GDHs exhibited very high affinity for glucose, with Km values between 0.41 and 0.96 mM. A. bisporus FAD-GDH exhibited high thermostability, with a Tm value of 72.5 °C. Therefore, we propose that the FAD-GDHs will be practical in blood glucose sensors and in other bioelectrochemical devices exhibiting high substrate selectivity, high current output, high sensitivity, and/or high stability to storage and use.

Identification of a l-Lactate dehydrogenase with 3,4-dihydroxyphenylpyruvic reduction activity for l-Danshensu production

Abstract Danshensu (DSS), also known as 3,4-dihydroxyphenyllactate, is an herbal preparation with prominent pharmacological activities. It has lactic acid structure and may be obtained by reducing 3,4-dihydroxyphenylpyruvate with lactate dehydrogenase. We screened a coenzyme-aspecific L-lactate dehydrogenase (LF-L-LDH0845) from lactobacillus fermentum for the bioconversion of 3,4-dihydroxyphenylpyruvate to optically pure (ee ≥ 99.99%) L-DSS. LF-L-LDH0845 has an approximate molecular weight of 33.65 kDa, exhibits wide substrate scope for 2-keto-carboxylic acids. Values of Km, Kcat, and Kcat/Km for LF-L-LDH0845 with 3,4-dihydroxy-phenylpyruvate substrate were 11.37 mM, 0.2931 s−1, and 0.0258 mM−1 s−1, respectively. LF-L-LDH0845 was most active and stable at pH 6.0, the optimum temperature was 25 °C, stability decreased with increasing temperature, and activity was lost completely at 50 °C. K+ stimulated while Fe2+ and Cu2+ inhibited the enzyme activity significantly. Glucose dehydrogenase gene was coexpressed with lf-l-ldh0845 in E. coli to regenerate cofactors by oxidising glucose, which efficiently reduced 3,4-dihydroxyphenylpyruvate to L-DSS with 95.45% isolation yield.

Tricalcium phosphate solubilization and nitrogen fixation by newly isolated Aneurinibacillus aneurinilyticus CKMV1 from rhizosphere of Valeriana jatamansi and its growth promotional effect.

Aneurinibacillus aneurinilyticus strain CKMV1 was isolated from rhizosphere of Valeriana jatamansi and possessed multiple plant growth promoting traits like production of phosphate solubilization (260 mg/L), nitrogen fixation (202.91 nmol ethylene mL−1 h−1), indole-3-acetic acid (IAA) (8.1 μg/mL), siderophores (61.60%), HCN (hydrogen cyanide) production and antifungal activity. We investigated the ability of isolate CKMV1 to solubilize insoluble P via mechanism of organic acid production. High-performance liquid chromatography (HPLC) study showed that isolate CKMV1 produced mainly gluconic (1.34%) and oxalic acids. However, genetic evidences for nitrogen fixation and phosphate solubilization by organic acid production have been reported first time for A. aneurinilyticus strain CKMV1. A unique combination of glucose dehydrogenase (gdh) gene and pyrroloquinoline quinone synthase (pqq) gene, a cofactor of gdh involved in phosphate solubilization has been elucidated. Nitrogenase (nif H) gene for nitrogen fixation was reported from A. aneurinilyticus. It was notable that isolate CKMV1 exhibited highest antifungal against Sclerotium rolfsii (93.58%) followed by Fusarium oxysporum (64.3%), Dematophora necatrix (52.71%), Rhizoctonia solani (91.58%), Alternaria sp. (71.08%) and Phytophthora sp. (71.37%). Remarkable increase was observed in seed germination (27.07%), shoot length (42.33%), root length (52.6%), shoot dry weight (62.01%) and root dry weight (45.7%) along with NPK (0.74, 0.36, 1.82%) content of tomato under net house condition. Isolate CKMV1 possessed traits related to plant growth promotion, therefore, could be a potential candidate for the development of biofertiliser or biocontrol agent and this is the first study to include the Aneurinibacillus as PGPR.

Efficient biosynthesis of ethyl (R)-4-chloro-3-hydroxybutyrate using a stereoselective carbonyl reductase from Burkholderia gladioli.

BackgroundEthyl (R)-4-chloro-3-hydroxybutyrate ((R)-CHBE) is a versatile chiral precursor for many pharmaceuticals. Although several biosynthesis strategies have been documented to convert ethyl 4-chloro-3-oxobutanoate (COBE) to (R)-CHBE, the catalytic efficiency and stereoselectivity are still too low to be scaled up for industrial applications. Due to the increasing demand of (R)-CHBE, it is essential to explore more robust biocatalyst capable of preparing (R)-CHBE efficiently.ResultsA stereoselective carbonyl reductase toolbox was constructed and employed into the asymmetric reduction of COBE to (R)-CHBE. A robust enzyme designed as BgADH3 from Burkholderia gladioli CCTCC M 2012379 exhibited excellent activity and enantioselectivity, and was further characterized and investigated in the asymmetric synthesis of (R)-CHBE. An economical and satisfactory enzyme-coupled cofactor recycling system was created using recombinant Escherichia coli cells co-expressing BgADH3 and glucose dehydrogenase genes to regenerate NADPH in situ. In an aqueous/octanol biphasic system, as much as 1200 mmol COBE was completely converted by using substrate fed-batch strategy to afford (R)-CHBE with 99.9 % ee at a space-time yield per gram of biomass of 4.47 mmol∙L−1∙h−1∙g DCW−1.ConclusionsThese data demonstrate the promising of BgADH3 in practical synthesis of (R)-CHBE as a valuable chiral synthon. This study allows for the further application of BgADH3 in the biosynthesis of chiral alcohols, and establishes a preparative scale process for producing (R)-CHBE with excellent enantiopurity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0301-x) contains supplementary material, which is available to authorized users.

Organic Acid Secretion and Phosphate Solubilizing Efficiency of Pseudomonas sp. PSB12: Effects of Phosphorus Forms and Carbon Sources

ABSTRACTSecretion of organic acids is an important mechanism for phosphate solubilizing bacteria (PSB) to dissolve insoluble phosphorus in soil. However, the composition of organic acids produced by PSB in the presence of different phosphorus compounds is poorly known, and little is known about the ability of PSB to degrade pollutants in sediment. In this study, we isolated a strain Pseudomonas sp. PSB12 from the sediment of the Qihe River. PSB12 had maximum phosphate solubilization index (SI) of 3.86 on Pikovskaya's agar medium. The phosphate solubilizing activity was associated with the release of organic acids produced from glucose, while the composition of organic acids produced by PSB12 was dependent on the phosphorus forms. When initial soluble phosphorus was insufficient (in MP1 and MP2 media), gluconic acid was the predominant organic acid. In contrast, formic acid, butyrate, and propanedioic acid were the main organic acids produced when only soluble phosphorus (MP3) was supplied. RT-PCR indicated that the expression of glucose dehydrogenase gene (gcd) of PSB12 was two- to four-fold higher in MP1 than in MP3. PSB12 also possessed the phenol hydroxylase gene (phe) suggesting that phenol could be used as the carbon source to dissolve insoluble phosphorus. PSB12 is a potential candidate for in situ bioremediation and for promoting plant growth in soil contaminated by phenol with low levels of soluble phosphorus.

The role of gluconate production by Pseudomonas spp. in the mineralization and bioavailability of calcium–phytate to Nicotiana tabacum

Organic phosphorus (P) is abundant in most soils but is largely unavailable to plants. Pseudomonas spp. can improve the availability of P to plants through the production of phytases and organic anions. Gluconate is a major component of Pseudomonas organic anion production and may therefore play an important role in the mineralization of insoluble organic P forms such as calcium-phytate (CaIHP). Organic anion and phytase production was characterized in 2 Pseudomonas spp. soil isolates (CCAR59, Ha200) and an isogenic mutant of strain Ha200, which lacked a functional glucose dehydrogenase (Gcd) gene (strain Ha200 gcd::Tn5B8). Wild-type and mutant strains of Pseudomonas spp. were evaluated for their ability to solubilize and hydrolyze CaIHP and to promote the growth and assimilation of P by tobacco plants. Gluconate, 2-keto-gluconate, pyruvate, ascorbate, acetate, and formate were detected in Pseudomonas spp. supernatants. Wild-type pseudomonads containing a functional gcd could produce gluconate and mineralize CaIHP, whereas the isogenic mutant could not. Inoculation with Pseudomonas improved the bioavailability of CaIHP to tobacco plants, but there was no difference in plant growth response due to Gcd function. Gcd function is required for the mineralization of CaIHP in vitro; however, further studies will be needed to quantify the relative contribution of specific organic anions such as gluconate to plant growth promotion by soil pseudomonads.

Highly Efficient Synthesis of Optically Pure (S)-1-phenyl-1,2-ethanediol by a Self-Sufficient Whole Cell Biocatalyst.

Terminal vicinal diols are important chiral building blocks and intermediates in organic synthesis. Reduction of α-hydroxy ketones provides a straightforward approach to access these important compounds. In this study, it has been found that asymmetric reduction of a series of α-hydroxy aromatic ketones and 1-hydroxy-2-pentanone, catalyzed by Candida magnolia carbonyl reductase (CMCR) with glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration, afforded 1-aryl-1,2-ethanediols and pentane-1,2-diol, respectively, in up to 99 % ee. In order to evaluate the efficiency of the bioreduction, lyophilized recombinant Escherichia coli whole cells coexpressing CMCR and GDH genes were used as the biocatalyst and α-hydroxy acetophenone as the model substrate, and the reaction conditions, such as pH, cosolvent, the amount of biocatalyst and the presences of a cofactor (i.e., NADP+), were optimized. Under the optimized conditions (pH 6, 16 h), the bioreduction proceeded smoothly at 1.0 m substrate concentration without the external addition of cofactor, and the product (S)-1-phenyl-1,2-ethanediol was isolated with 90 % yield and 99 % ee. This offers a practical biocatalytic method for the preparation of these important vicinal diols.

Knock-out of glucose dehydrogenase gene in Gluconacetobacter xylinus for bacterial cellulose production enhancement

Glucose dehydrogenase (GDH) locates in the cytoplasmic membrane of Gluconacetobacter xylinus oxidizes glucose to gluconic acid that decreases the conversion of glucose to bacterial cellulose (BC). In this study, a mutant of G. xylinus was generated by knocking-out the membrane bound GDH gene via homologous recombination of a defect GDH gene. The production of BC by G. xylinus mutant (GDH-KO strain) using glucose as a carbon source was investigated. Without the membrane bound GDH activity, the mutant strain still produces BC and increases glucose utilization efficiency for cellulose biosynthesis. In contrast, the wild-type strain oxidized a large fraction of glucose to gluconic acid that decreased the conversion yield of glucose to BC. Our results showed that the BC production from GDH-KO strain was about 40 and 230% higher than that of wild-type strain in static and shaken culture, respectively.

Coexpression of Lactobacillus brevis ADH with GDH or G6PDH in Arxula adeninivorans for the synthesis of 1-(R)-phenylethanol.

The yeast Arxula adeninivorans was used for the overexpression of an ADH gene of Lactobacillus brevis coding for (R)-specific alcohol dehydrogenase (LbADH) to synthesise enantiomerically pure 1-(R)-phenylethanol. Glucose dehydrogenase gene from Bacillus megaterium (BmGDH) or glucose 6-phosphate dehydrogenase of Bacillus pumilus (BpG6PDH) were coexpressed in Arxula to regenerate the cofactor NADPH by oxidising glucose or glucose 6-phosphate. The yeast strain expressing LbADH and BpG6PDH produced 5200U l(-1) ADH and 370U l(-1) G6PDH activity, whereas the strain expressing LbADH and BmGDH produced 2700U l(-1) ADH and 170U l(-1) GDH activity. However, the crude extract of both strains reduced 40mM acetophenone to pure 1-(R)-phenylethanol with an enantiomeric excess (ee) of >99% in 60min without detectable by-products. An increase in yield was achieved using immobilised crude extracts (IEs), Triton X-100 permeabilised cells (PCs) and permeabilised immobilised cells (PICs) with PICs being most stable with GDH regeneration over 52 cycles. Even though the activity and synthesis rate of 1-(R)-phenylethanol with the BpG6PDH and LbADH coexpressing strain was higher, the BmGDH-LbADH strain was more stable over successive reaction cycles. This, combined with its higher total turnover number (TTN) of 391 mol product per mole NADP(+), makes it the preferred strain for continuous reaction systems. The initial non-optimised semi-continuous reaction produced 9.74gl(-1)day(-1) or 406gkg(-1) dry cell weight (dcw) day(-1) isolated 1-(R)-phenylethanol with an ee of 100% and a TTN of 206mol product per mole NADP(+). In conclusion, A. adeninivorans is a promising host for LbADH and BpG6PDH or BmGDH production and offers a simple method for the production of enantiomerically pure alcohols.

Identification and characterization of rhizospheric microbial diversity by 16S ribosomal RNA gene sequencing.

In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization.

Production of medium chain length polyhydroxyalkanoate in metabolic flux optimized Pseudomonas putida.

BackgroundPseudomnas putida is a natural producer of medium chain length polyhydroxyalkanoates (mcl-PHA), a polymeric precursor of bioplastics. A two-fold increase of mcl-PHA production via inactivation of the glucose dehydrogenase gene gcd, limiting the metabolic flux towards side products like gluconate was achieved before. Here, we investigated the overproduction of enzymes catalyzing limiting steps of mcl-PHA precursor formation.ResultsA genome-based in silico model for P. putida KT2440 metabolism was employed to identify potential genetic targets to be engineered for the improvement of mcl-PHA production using glucose as sole carbon source. Here, overproduction of pyruvate dehydrogenase subunit AcoA in the P. putida KT2440 wild type and the Δgcd mutant strains led to an increase of PHA production. In controlled bioreactor batch fermentations PHA production was increased by 33% in the acoA overexpressing wild type and 121% in the acoA overexpressing Δgcd strain in comparison to P. putida KT2440. Overexpression of pgl-encoding 6-phosphoglucolactonase did not influence PHA production. Transcriptome analyses of engineered PHA producing P. putida in comparison to its parental strains revealed the induction of genes encoding glucose 6-phosphate dehydrogenase and pyruvate dehydrogenase. In addition, NADPH seems to be quantitatively consumed for efficient PHA synthesis, since a direct relationship between low levels of NADPH and high concentrations of the biopolymer were observed. In contrast, intracellular levels of NADH were found increased in PHA producing organisms.ConclusionProduction of mcl-PHAs was enhanced in P. putida when grown on glucose via overproduction of a pyruvate dehydrogenase subunit (AcoA) in combination with a deletion of the glucose dehydrogenase (gcd) gene as predicted by in silico elementary flux mode analysis.

Bioinformatics based structural characterization of glucose dehydrogenase (gdh) gene and growth promoting activity of Leclercia sp. QAU-66.

Glucose dehydrogenase (GDH; EC 1.1. 5.2) is the member of quinoproteins group that use the redox cofactor pyrroloquinoline quinoine, calcium ions and glucose as substrate for its activity. In present study, Leclercia sp. QAU-66, isolated from rhizosphere of Vigna mungo, was characterized for phosphate solubilization and the role of GDH in plant growth promotion of Phaseolus vulgaris. The strain QAU-66 had ability to solubilize phosphorus and significantly (p ≤ 0.05) promoted the shoot and root lengths of Phaseolus vulgaris. The structural determination of GDH protein was carried out using bioinformatics tools like Pfam, InterProScan, I-TASSER and COFACTOR. These tools predicted the structural based functional homology of pyrroloquinoline quinone domains in GDH. GDH of Leclercia sp. QAU-66 is one of the main factor that involved in plant growth promotion and provides a solid background for further research in plant growth promoting activities.

Cloning and characterization of uridine diphosphate glucose dehydrogenase gene from Ipomoea batatas

In order to study the structure and expression patterns of uridine diphosphate glucose dehydrogenase (UDPGH) genes in Ipomoea batatas, the transcriptome database of this plant constructed in our lab was first analyzed to screen UDPGH contigs. It was found that there were 23 UDPGH contigs of different sizes in the transcriptome database. Primers were designed to amplify the coding regions of UDPGH, and five UDPGH-coding genes (named IbUDPGH1-IbUDPGH5) were cloned and sequenced. Open reading frames of all the UDPGH were 1443 bp in length, and their identity was more than 97 and 99% at the nucleotide and protein level, respectively. Homology comparison among different plant UDPGH showed that the identity ranged from 73 to 95% at the nucleotide level and from 84 to 95% at the protein level. The results of digital gene expression profile analysis (DGE) displayed that IbUDPGH1 had the highest expression in the tuberous roots, lower in the young and mature leaves, and the lowest in stems and fibrous roots, while IbUDPGH2 and IbUDPGH5 had the highest transcript level in stems, lower in roots, and very low in leaves. The rest genes were expressed at a low level in different tissues. Semi-quantitative RT-PCR results were similar to above data from the DGE. These results imply that the high expression of UDPGH might make large contribution to the accumulation of cell wall polysaccharides in sweet potato stems and roots.

ADH from Rhodococcus ruber expressed in Arxula adeninivorans for the synthesis of 1-(S)-phenylethanol

Abstract The RrADH gene of Rhodococcus ruber coding for (S)-specific alcohol dehydrogenase (RrADH) was overexpressed in the yeast Arxula adeninivorans and used for the synthesis of enantiomerically pure alcohols. The substrates acetophenone, 2,5-hexandione and 2-nonanon used for this synthesis, were reduced by RrADH to produce S-configured alcohols. Regeneration of the cofactor, NADH, was required for the reaction and this was provided by using isopropanol as a second ADH substrate to reduce NAD + or by cloning the glucose dehydrogenase gene from Bacillus megaterium ( BmGDH ) into the yeast which regenerated NADH by oxidizing glucose. Expressing both RrADH and BmGDH in the yeast provided a strain that could synthesize 1-(S)-phenylethanol from acetophenone with NADH being regenerated by the oxidation of glucose. Both bioreduction systems led to the synthesis of pure (S) enantiomer of 1-phenylethanol, but only the enzyme coupled approach reduced 40 mM acetophenone completely in 150 min. 75 mg of 98% pure product could be isolated from 20 ml. In conclusion the synthesis potential of the RrADH expressed in A. adeninivorans is very promising for 1-(S)-phenylethanol synthesis.

Isolation and Biochemical Characterization of a Glucose Dehydrogenase from a Hay Infusion Metagenome

Glucose hydrolyzing enzymes are essential to determine blood glucose level. A high-throughput screening approach was established to identify NAD(P)-dependent glucose dehydrogenases for the application in test stripes and the respective blood glucose meters. In the current report a glucose hydrolyzing enzyme, derived from a metagenomic library by expressing recombinant DNA fragments isolated from hay infusion, was characterized. The recombinant clone showing activity on glucose as substrate exhibited an open reading frame of 987 bp encoding for a peptide of 328 amino acids. The isolated enzyme showed typical sequence motifs of short-chain-dehydrogenases using NAD(P) as a co-factor and had a sequence similarity between 33 and 35% to characterized glucose dehydrogenases from different Bacillus species. The identified glucose dehydrogenase gene was expressed in E. coli, purified and subsequently characterized. The enzyme, belonging to the superfamily of short-chain dehydrogenases, shows a broad substrate range with a high affinity to glucose, xylose and glucose-6-phosphate. Due to its ability to be strongly associated with its cofactor NAD(P), the enzyme is able to directly transfer electrons from glucose oxidation to external electron acceptors by regenerating the cofactor while being still associated to the protein.

Cloning and Characterization of Uridine Diphosphate Glucose Dehydrogenase Gene fromIpomoea batatas

Cloning and Characterization of Uridine Diphosphate Glucose Dehydrogenase Gene from<i>Ipomoea batatas</i>

Scalable biocatalytic synthesis of optically pure ethyl (R)-2-hydroxy-4-phenylbutyrate using a recombinant E. coli with high catalyst yield

Ethyl (R)-2-hydroxy-4-phenylbutanoate [(R)-HPBE] is a versatile and important chiral intermediate for the synthesis of angiotensin-converting enzyme (ACE) inhibitors. Recombinant E. coli strain coexpressing a novel NADPH-dependent carbonyl reductase gene iolS and glucose dehydrogenase gene gdh from Bacillus subtilis showed excellent catalytic activity in (R)-HPBE production by asymmetric reduction. IolS exhibited high stereoselectivity (>98.5% ee) toward α-ketoesters substrates, whereas fluctuant ee values (53.2–99.5%) for β-ketoesters with different halogen substitution groups. Strategies including aqueous/organic biphasic system and substrate fed-batch were adopted to improve the biocatalytic process. In a 1-L aqueous/octanol biphasic reaction system, (R)-HPBE was produced in 99.5% ee with an exceptional catalyst yield (gproduct/gcatalyst) of 31.7 via bioreduction of ethyl 2-oxo-4-phenylbutyrate (OPBE) at 330g/L.

Transcriptional control by two interacting regulatory proteins: identification of the PtxS binding site at PtxR

The PtxS and PtxR regulators control the expression of the glucose dehydrogenase genes from the Pgad promoter in Pseudomonas aeruginosa. These regulators bind to their cognate operators, that are separated by ∼50 nt, within the promoter region and interact with each other creating a DNA-loop that prevents RNA polymerase promoter access. Binding of the 2-ketogluconate effector to PtxS caused PtxS/PtxR complex dissociation and led to the dissolution of the repression loop facilitating the entry of the RNA polymerase and enabling the transcription of the gad gene. We have identified a hydrophobic surface patch on the PtxR putative surface that was hypothesized to correspond to the binding site for PtxS. Two surface-exposed residues in this patch, V173 and W269, were replaced by alanine. Isothermal titration calorimetry assays showed that PtxS does not interact with the mutant variants of PtxR. Electrophoretic mobility shift assay and DNAase I footprinting assays proved that both regulators bind to their target operators and that failure to interact with each other prevented the formation of the DNA-loop. In vitro transcription showed that PtxS per se is sufficient to inhibit transcription from the Pgad promoter, but that affinity of PtxS for its effector is modulated by PtxR.