Separation and purification of pyrroloquinoline quinone from Gluconobacter oxydans fermentation broth using supramolecular solvent complex extraction.

In this paper, new supramolecular extractants, which contained surfactant, alkane and alkanol, were designed and used to separate PQQ. After a series of tests, the optimal extractant composition was determined as benzalkalonium (C8-C16) chloride (BC): n-hexane:n-pentanol, and the highest extraction rate could reach 98%. The extraction equilibrium could be reached in five minutes. The mechanism of the extraction selectivity was inferred as an ion-pair and π-π complexation interaction between PQQ and BC, which was indicated by UV and fluorescence quenching experiments. To recycle the organic extractant, the extract was back-extracted with sodium chloride solution. After extraction, back extraction and crystallization, an isolated product with a purity of 97.5% was obtained from G. oxydans fermentation broth. The product was identified as PQQ by HPLC analysis and MS. Above all, the present research developed a simple and efficient method for the separation of PQQ from fermentation broth.

Investigating the Product Profiles and Structural Relationships of New Levansucrases with Conventional and Non-Conventional Substrates.

The synthesis of complex oligosaccharides is desired for their potential as prebiotics, and their role in the pharmaceutical and food industry. Levansucrase (LS, EC 2.4.1.10), a fructosyl-transferase, can catalyze the synthesis of these compounds. LS acquires a fructosyl residue from a donor molecule and performs a non-Lenoir transfer to an acceptor molecule, via ß-(2→6)-glycosidic linkages. Genome mining was used to uncover new LS enzymes with increased transfructosylating activity and wider acceptor promiscuity, with an initial screening revealing five LS enzymes. The product profiles and activities of these enzymes were examined after their incubation with sucrose. Alternate acceptor molecules were also incubated with the enzymes to study their consumption. LSs from Gluconobacter oxydans and Novosphingobium aromaticivorans synthesized fructooligosaccharides (FOSs) with up to 13 units in length. Alignment of their amino acid sequences and substrate docking with homology models identified structural elements causing differences in their product spectra. Raffinose, over sucrose, was the preferred donor molecule for the LS from Vibrio natriegens, N. aromaticivorans, and Paraburkolderia graminis. The LSs examined were found to have wide acceptor promiscuity, utilizing monosaccharides, disaccharides, and two alcohols to a high degree.

Pyrroloquinoline quinone from Gluconobacter oxydans fermentation broth enhances superoxide anion-scavenging capacity of Cu/Zn-SOD.

A bioassay-guided fractionation of extract from Gluconobacter oxydans fermentation broth afforded Compound 1, which was identified as pyrroloquinoline quinone (PQQ) by spectroscopic methods. PQQ has been shown to enhance the superoxide anion-scavenging capacity significantly for Cu/Zn-SOD. To illustrate the mechanism, the interaction between PQQ and Cu/Zn-SOD was investigated. The multiple binding sites involving hydrogen bonds and van der Waals force between PQQ and Cu/Zn-SOD were revealed by isothermal titration calorimetry. The α-helix content was increased in the Cu/Zn-SOD structure with the addition of PQQ into the solution through ultraviolet (UV) spectroscopy. These results indicated that PQQ could change the conformation of Cu/Zn-SOD through interaction, which could enhance its superoxide anion-scavenging capacity. Therefore, PQQ is a potential natural antioxidant.

Improving the production yield and productivity of 1,3-dihydroxyacetone from glycerol fermentation using Gluconobacter oxydans NL71 in a compressed oxygen supply-sealed and stirred tank reactor (COS-SSTR).

In this study, a compressed oxygen gas supply was connected to a sealed aerated stirred tank reactor (COS-SSTR) bio-system, leading to a high-oxygen pressure bioreactor used to improve the bio-transformative performance in the production of 1,3-dihydroxyacetone (DHA) from glycerol using Gluconobacter oxydans NL71. A concentration of 301.2 ± 8.2 g L(-1) DHA was obtained from glycerol after 32 h of fed-batch fermentation in the COS-SSTR system. The volumetric productivity for this process was 9.41 ± 0.23 g L(-1) h(-1), which is presently the highest obtained level of glycerol bioconversion into DHA. These results show that the application of this bioreactor would enable microbial production of DHA from glycerol at the industrial scale.

Identification of mannitol as compatible solute in Gluconobacter oxydans.

Gluconobacter oxydans is an industrially important bacterium owing to its regio- and enantio-selective incomplete oxidation of various sugars, alcohols, and polyols. The complete genome sequence is available, but it is still unknown how the organism adapts to highly osmotic sugar-rich environments. Therefore, the mechanisms of osmoprotection in G. oxydans were investigated. The accumulation and transport of solutes are hallmarks of osmoadaptation. To identify potential osmoprotectants, G. oxydans was grown on a yeast glucose medium in the presence of 100 mM potassium phosphate (pH 7.0) along with various concentrations of sucrose (0-600 mM final concentration), which was not metabolized. Intracellular metabolites were analyzed by HPLC and (13)C NMR spectroscopy under stress conditions. Both of these analytical techniques highlighted the accumulation of mannitol as a potent osmoprotectant inside the stressed cells. This intracellular mannitol accumulation correlated with increased extracellular osmolarity of the medium. For further confirmation, the growth behavior of G. oxydans was analyzed in the presence of small amounts of mannitol (2.5-10 mM) and 300 mM sucrose. Growth under sucrose-induced osmotic stress conditions was almost identical to control growth when exogenous mannitol was added in low amounts. Thus, mannitol alleviates the osmotic stress of sucrose on cellular growth. Moreover, the positive effect of exogenous mannitol on the rate of glucose consumption and gluconate formation was also monitored. These results may be helpful to optimize the processes of industrial product formation in highly concentrated sugar solutions.

Chemoenzymatic synthesis of carbohydrates as antidiabetic and anticancer drugs.

Different chemoenzymatic strategies for the preparation of carbohydrates and analogues possessing antidiabetic or anticancer activity are summarized. In this sense, some examples illustrating the use of enzymes such as aldolases, lipases or glycosidases (in some cases improved by genetic engineering techniques) are presented, showing the advantages of the implementation of chemoenzymatic protocols, which combine the flexibility of chemical synthesis with the efficiency, selectivity and sustainability of biotransformations to obtain diverse complex carbohydrates, glycoconjugates and glycomimetics.

A novel functional conducting polymer as an immobilization platform.

Here, we present the fabrication of conducting polymer based enzymatic and microbial biosensors. To obtain immobilization platforms for both pyranose oxidase (PyOx) and Gluconobacter oxydans, the graphite electrode surface was modified with the polymer of 4-amino-N-(2,5-di(thiophen-2-yl)-1H-pyrrol-1-yl)benzamide (HKCN) which has free amino groups on the surface for further bioconjugation reactions with the biomolecules. Initially, the electrode surface was covered with HKCN via electropolymerization. Then, either PyOx or G. oxydans cell was stabilized using glutaraldehyde as a cross-linker. After optimization of biosensors, analytical characterization and surface imaging studies were investigated. The change of current depends on glucose concentration between 0.05-1.0mM and 0.25-2.5mM with HKCN/PyOx and HKCN/G. oxydans biosensors in batch systems. Also, the calibration graphs were obtained for glucose in FIA mode, and in this case, linear ranges were found to be 0.01-1.0mM and 0.1-7.5mM for HKCN/PyOx and HKCN/G. oxydans, respectively.

Structural insights into substrate and coenzyme preference by SDR family protein Gox2253 from Gluconobater oxydans.

Gox2253 from Gluconobacter oxydans belongs to the short-chain dehydrogenases/reductases family, and catalyzes the reduction of heptanal, octanal, nonanal, and decanal with NADPH. To develop a robust working platform to engineer novel G. oxydans oxidoreductases with designed coenzyme preference, we adopted a structure based rational design strategy using computational predictions that considers the number of hydrogen bonds formed between enzyme and docked coenzyme. We report the crystal structure of Gox2253 at 2.6 Å resolution, ternary models of Gox2253 mutants in complex with NADH/short-chain aldehydes, and propose a structural mechanism of substrate selection. Molecular dynamics simulation shows that hydrogen bonds could form between 2'-hydroxyl group in the adenosine moiety of NADH and the side chain of Gox2253 mutant after arginine at position 42 is replaced with tyrosine or lysine. Consistent with the molecular dynamics prediction, Gox2253-R42Y/K mutants can use both NADH and NADPH as a coenzyme. Hence, the strategies here could provide a practical platform to engineer coenzyme selectivity for any given oxidoreductase and could serve as an additional consideration to engineer substrate-binding pockets.

Phosphorylation of HPr by HPr kinase in Gluconobacter oxydans 621H.

DNA sequencing has revealed that Gluconobacter oxydans may contain an incomplete phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS), but the function of individual members of the system remains unknown. Here we demonstrated that the predicted histidine protein HPr, an essential component of PTS, can be phosphorylated by a predicted HPr kinase in G. oxydans, where Ser54 in HPr is the site responsible for such a phosphorylation. The discovery implies that G. oxydans may regulate PTS activity in a way similar to that identified in some Gram-positive bacteria with low GC content.

[Bioanode for a microbial fuel cell based on Gluconobacter oxydans inummobilized into a polymer matrix].

Acetic acid bacteria Gluconobacter oxydans subsp. industrius RKM V-1280 were immobilized into a synthetic matrix based on polyvinyl alcohol modified with N-vinylpyrrolidone and used as biocatalysts for the development ofbioanodes for microbial fuel cells. The immobilization method did not significantly affect bacterial substrate specificity. Bioanodes based on immobilized bacteria functioned stably for 7 days. The maximum voltage (fuel cell signal) was reached when 100-130 µM of an electron transport mediator, 2,6-dichlorophenolindophenol, was added into the anode compartment. The fuel cell signals reached a maximum at a glucose concentration higher than 6 mM. The power output of the laboratory model of a fuel cell based on the developed bioanode reached 7 mW/m2 with the use of fermentation industry wastes as fuel.

Chitosan-ferrocene film as a platform for flow injection analysis applications of glucose oxidase and Gluconobacter oxydans biosensors.

Chitosan-ferrocene (CHIT-Fc) hybrid was synthesized through covalent modification and its electrochemical properties in immobilized form were studied by using cyclic voltammetry. The hybrid film exhibited reversible electrochemistry with a formal potential of +0.35 V (vs. Ag/AgCl) at pH 5.5. The Fc in CHIT matrix retained its electrocatalytic activity and did not diffuse from the matrix. This redox-active hybrid was further employed as a support for immobilization of glucose oxidase (GOx) and whole cells of Gluconobacter oxydans using glutaraldehyde on a glassy carbon electrode (GCE). The experimental conditions were optimized and the analytical characteristics of enzyme and microbial biosensors were evaluated for glucose in flow injection analysis (FIA) system. Under optimized conditions, both enzyme and microbial biosensors exhibited wide linear ranges for glucose from 2.0 to 16.0 mM and from 1.5 to 25.0 mM, respectively. Moreover, the biosensors have the advantages of relatively fast response times, good reproducibility and stability in FI mode. It was demonstrated that CHIT-Fc provides a biocompatible microenvironment for both bioctalysts and an electron transfer pathway. Additionally, integration of the enzyme and microbial biosensors into the FIA system has several advantages including capability of automation and high throughput at low cost. This promising redox hybrid can be utilized as an immobilization matrix for biomolecules in biosensor systems.

A novel technique for in situ aggregation of Gluconobacter oxydans using bio-adhesive magnetic nanoparticles.

Here, we present a novel technique to immobilize magnetic particles onto whole Gluconobacter oxydans in situ via a synthetic adhesive biomimetic material inspired by the protein glues of marine mussels. Our approach involves simple coating of a cell adherent polydopamine film onto magnetic nanoparticles, followed by conjugation of the polydopamine-coated nanoparticles to G. oxydans which resulted in cell aggregation. After optimization, 21.3 mg (wet cell weight) G. oxydans per milligram of nanoparticle was aggregated and separated with a magnet. Importantly, the G. oxydan aggregates showed high specific activity and good reusability. The facile approach offers the potential advantages of low cost, easy cell separation, low diffusion resistance, and high efficiency. Furthermore, the approach is a convenient platform technique for magnetization of cells in situ by direct mixing of nanoparticles with a cell suspension.

Determination of dihydroxyacetone and glycerol in fermentation process by GC after n-methylimidazole catalyzed acetylation.

A gas chromatographic method that accurately measures glycerol and dihydroxyacetone from a fermentation broth is described in this paper. The method incorporates a sample derivatization reaction using n-methylimidazole as catalyst in the presence of acetic anhydride. Resulting derivatives are separated on a DB-5 capillary column and flame ionization detector. Results show that 10 µL n-methylimidazole and 75 µL acetic anhydride are sufficient to complete the acetylation for glycerol and dihydroxyacetone at room temperature for 5 min. The present method exhibits good linearity at a concentration range of 1-100 g/L with excellent regression (R(2) > 0.9997). The limits of detection are 0.025 and 0.013 g/L for dihydroxyacetone and glycerol, respectively. The method has been successfully applied to the monitoring and control of the fermentation process, and recoveries are in the range of 95.5-98.8% with relative standard deviations below 1%.

Asymmetric reduction of activated alkenes using an enoate reductase from Gluconobacter oxydans.

A recombinant enoate reductase from Gluconobacter oxydans was heterologously expressed, purified, characterised and applied in the asymmetric reduction of activated alkenes. In addition to the determination of the kinetic properties, the major focus of this work was to utilise the enzyme in the biotransformation of different interesting compounds such as 3,5,5-trimethyl-2-cyclohexen-1,4-dione (ketoisophorone) and (E/Z)-3,7-dimethyl-2,6-octadienal (citral). The reaction proceeded with excellent stereoselectivities (>99% ee) as well as absolute chemo- and regioselectivity, only the activated C=C bond of citral was reduced by the enoate reductase, while non-activated C=C bond and carbonyl moiety remained untouched. The described strategy can be used for the production of enantiomerically pure building blocks, which are difficult to prepare by chemical means. In general, the results show that the investigated enoate reductase is a promising catalyst for the use in asymmetric C=C bond reductions.

Characterization of two aldo-keto reductases from Gluconobacter oxydans 621H capable of regio- and stereoselective alpha-ketocarbonyl reduction.

Two cytosolic NADPH-dependent carbonyl reductases from Gluconobacter oxydans 621H, Gox0644 and Gox1615, were heterologously produced in Escherichia coli. The recombinant proteins were purified to homogeneity and characterized. Gox0644 and Gox1615 were dimers with native molecular masses of 66.1 and 74.5 kDa, respectively. The enzymes displayed broad substrate specificities and reduced alpha-ketocarbonyls at the keto moiety most proximal to the terminus of the alkyl chain to produce alpha-hydroxy carbonyls, as demonstrated by NMR. With respect to stereoselectivity, protein Gox0644 specifically reduced 2,3-pentanedione to 2R-hydroxy-pentane-3-one, whereas Gox1615 produced 2S-hydroxy-pentane-3-one. Both enzymes also reduced 1-phenyl-1,2-propanedione to 2-hydroxy-1-phenylpropane-1-one, which is a key intermediate in the production of numerous pharmaceuticals, such as antifungal azoles and antidepressants. Gox0644 displayed highest activities with 2,3-diones, alpha-ketoaldehydes, alpha-keto esters, and 2,5-diketogluconate. Gox1615 was less active with these substrates, but displayed a broader substrate spectrum reducing a variety of alpha-diketones and aldehydes.

Glycerol conversion to D-xylulose by a two-stage microbial reaction using Candida parapsilosis and Gluconobacter oxydans.

A yeast strain, 25N-2B, that produces D-arabitol from glycerol, was identified as Candida parapsilosis based on phylogenetic, morphological, physiological, and biochemical analyses. It produced 32.2 g/L D-arabitol from 170 g/L glycerol in a jar fermentor. The D-arabitol in the reaction mixture was then completely converted to D-xylulose using Gluconobacter oxydans NBRC3293. The product was isolated from the reaction mixture and confirmed to be D-xylulose by (1)H and (13)C-NMR and optical rotation.

Electrochemical polymerization of 1-(4-nitrophenyl)-2,5-di(2-thienyl)-1 H-pyrrole as a novel immobilization platform for microbial sensing.

Two types of bacterial biosensor were constructed by immobilization of Gluconobacter oxydans and Pseudomonas fluorescens cells on graphite electrodes modified with the conducting polymer; poly(1-(4-nitrophenyl)-2,5-di(2-thienyl)-1 H-pyrrole) [SNS(NO(2))]. The measurement was based on the respiratory activity of cells estimated by the oxygen consumption at -0.7 V due to the metabolic activity in the presence of substrate. As well as analytical characterization, the linear detection ranges, effects of electropolymerization time, pH and cell amount were examined by using glucose as the substrate. The linear relationships were observed in the range of 0.25-4.0 mM and 0.2-1.0 mM for G. oxydans and P. fluorescens based sensors, respectively.

Biochemical and spectroscopic properties of cyanide-insensitive quinol oxidase from Gluconobacter oxydans.

Cyanide-insensitive quinol oxidase (CioAB), a relative of cytochrome bd, has no spectroscopic features of hemes b(595) and d in the wild-type bacteria and is difficult to purify for detailed characterization. Here we studied enzymatic and spectroscopic properties of CioAB from the acetic acid bacterium Gluconobacter oxydans. Gluconobacter oxydans CioAB showed the K(m) value for ubiquinol-1 comparable to that of Escherichia coli cytochrome bd but it was more resistant to KCN and quinone-analogue inhibitors except piericidin A and LL-Z1272gamma. We obtained the spectroscopic evidence for the presence of hemes b(595) and d. Heme b(595) showed the alpha peak at 587 nm in the reduced state and a rhombic high-spin signal at g = 6.3 and 5.5 in the air-oxidized state. Heme d showed the alpha peak at 626 and 644 nm in the reduced and air-oxidized state, respectively, and an axial high-spin signal at g = 6.0 and low-spin signals at g = 2.63, 2.37 and 2.32. We found also a broad low-spin signal at g = 3.2, attributable to heme b(558). Further, we identified the presence of heme D by mass spectrometry. In conclusion, CioAB binds all three ham species present in cytochrome bd quinol oxidase.

Repeated use of immobilized Gluconobacter oxydans cells for conversion of glycerol to dihydroxyacetone.

Dihydroxyacetone (DHA) is of great interest in the fine chemical and pharmaceutical industry; therefore, the discovery of suitable biocatalysts for the efficient production of it is very necessary. In the experiment, Gluconobacter oxydans was immobilized in polyvinyl alcohol (PVA). Various parameters of the immobilized cells were investigated. The results have shown that the optimal conversion conditions by the immobilized cells were at 30 degrees C and pH 6.0. The immobilized cells remained very active over the period of 14 days for storage and only lost 10% of its original activity. Repeated use of immobilized cells for conversion of glycerol to DHA was carried out in a 1.5 L stirred tank reactor, the average conversion rate was about 86%. Despite the high shear stress, bead shape was not affected, even after five consecutive conversion cycles. The regenerated biocatalyst could recover 90% of its initial activity.

[Use of NMR spectroscopy in studies of sorbitol and glucose transformation by Gluconobacter oxydans].

NMR spectroscopy was applied for studying the products of glucose and sorbitol oxidation by cells of Gluconobacter oxydans. An analysis of 1H NMR spectra showed that the transformation of glucose results in the formation of diketogluconic acid, and sorbitol is oxidized to sorbose. In the 32P NMR spectra, only a signal of inorganic phosphate was detected, which accumulated in the medium as a result of cell lysis.