Improving Gluconobacter oxydans performance in the in situ removal of the inhibitor for asymmetric resolution of racemic 1-phenyl-1,2-ethanediol.

Gluconobacter oxydans DSM2003 was used to catalyze the oxidation of racemic 1-phenyl-1,2-ethanediol (PED) for the production of (S)-enantiomer. The oxidative product mandelic acid produced strong inhibition to this reaction and largely reduced the activity of biocatalyst, which was the key problem in the reaction. In order to overcome this bottleneck, an anion exchange resin was selected and introduced as adsorbent for the in situ removal of the inhibitor from the reaction system. This method increased the substrate concentration from 12 to 60 g/L and the yield of (S)-PED by approximately five times from 4.9 g/L, on the premise that the enantiomeric excess (ee) value of (S)-PED remained above 96% and the reaction time was no more than 20 h. Moreover, the final space-time yield was over 1.2g/L/h, which was higher than that reported from previous studies.

Pentose oxidation by acetic acid bacteria led to a finding of membrane-bound purine nucleosidase.

D-Ribose and 2-deoxy-D-ribose were oxidized to 4-keto-D-ribonate and 2-deoxy-4-keto-D-ribonate respectively by oxidative fermentation, and the chemical structures of the oxidation products were confirmed to be as expected. Both pentoses are important sugar components of nucleic acids. When examined, purine nucleosidase activity predominated in the membrane fraction of acetic acid bacteria. This is perhaps the first finding of membrane-bound purine nucleosidase.

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.

Efficient conversion of 1,2-butanediol to (R)-2-hydroxybutyric acid using whole cells of Gluconobacter oxydans.

(R)-2-Hydroxybutyric acid ((R)-2-HBA) is an important building block for azinothricin family of antitumour antibiotics and biodegradable poly(2-hydroxybutyric acid). However, optically active (R)-2-HBA could not be produced through microbial fermentation or chemical synthesis. Several biocatalytic methods have been reported for the production of (R)-2-HBA. Those processes used expensive and scarce substrates and would not be suitable for practical application. In this work, Gluconobacter oxydans DSM 2003 was confirmed to have the ability to produce (R)-2-HBA from 1,2-butanediol, a non-toxic and inexpensive compound that had a great potential for biotechnological processes. Under the optimal conditions, the biocatalytic process produced (R)-2-HBA at a high concentration (18.5 g l(-1)) and a high enantiomeric excess (99.7%). The biocatalysis process introduced in this study may provide a technically and economically interesting route for production of (R)-2-HBA.

Use of glycerol for producing 1,3-dihydroxyacetone by Gluconobacter oxydans in an airlift bioreactor.

1,3-Dihydroxyacetone can be produced by biotransformation of glycerol with glycerol dehydrogenase from Gluconobacter oxydans cells. Firstly, improvement the activity of glycerol dehydrogenase was carried out by medium optimization. The optimal medium for cell cultivation was composed of 5.6g/l yeast extract, 4.7 g/l glycerol, 42.1g/l mannitol, 0.5 g/l K(2)HPO(4), 0.5 g/l KH(2)PO(4), 0.1g/l MgSO(4)·7H(2)O, and 2.0 g/l CaCO(3) with the initial pH of 4.9. Secondly, an internal loop airlift bioreactor was applied for DHA production from glycerol by resting cells of G. oxydans ZJB09113. Furthermore, the effects of pH, aeration rate and cell content on DHA production and glycerol feeding strategy were investigated. 156.3 ± 7.8 g/l of maximal DHA concentration with 89.8±2.4% of conversion rate of glycerol to DHA was achieved after 72h of biotransformation using 10g/l resting cells at 30°C, pH 5.0 and 1.5vvm of aeration rate.

Conversion of quinate to 3-dehydroshikimate by Ca-alginate-immobilized membrane of Gluconobacter oxydans IFO 3244 and subsequent asymmetric reduction of 3-dehydroshikimate to shikimate by immobilized cytoplasmic NADP-shikimate dehydrogenase.

The membrane fraction of Gluconobacter oxydans IFO 3244, involving membrane-bound quinoprotein quinate dehydrogenase and 3-dehydroquinate dehydratase, was immobilized into Ca-alginate beads. The Ca-alginate-immobilized bacterial membrane catalyzed a sequential reaction of quinate oxidation to 3-dehydroquinate and its spontaneous conversion to 3-dehydroshikimate under neutral pH. An almost 100% conversion rate from quinate to 3-dehydroshikimate was observed. NADP-Dependent cytoplasmic enzymes from the same organism, shikimate dehydrogenase and D-glucose dehydrogenase, were immobilized together with different carriers as an asymmetric reduction system forming shikimate from 3-dehydroshikimate. Blue Dextran 2000, Blue Dextran-Sepharose-4B, DEAE-Sephadex A-50, DEAE-cellulose, and hydroxyapatite were effective carriers of the two cytoplasmic enzymes, and the 3-dehydroshikimate initially added was converted to shikimate at 100% yield. The two cytoplasmic enzymes showed strong affinity to Blue Dextran 2000 and formed a soluble form of immobilized catalyst having the same catalytic efficiency as that of the free enzymes. This paper may be the first one on successful immobilization of NAD(P)-dependent dehydrogenases.

Optimization of immobilization for selective oxidation of benzyl alcohol by Gluconobacter oxydans using response surface methodology.

This study used the Box-Behnken design and response surface methodology to optimize immobilization of Gluconobacter oxydans in Ca-alginate gel for the production of benzaldehyde in a biphasic system. Immobilization parameters, such as Na-alginate concentration, cell load, and bead diameter, were optimized. The mathematical model developed was validated and proven to be statistically adequate and accurate in predicting the response. For both activity and stability responses, the best results were achieved at alginate concentration of 2.55% (w/v), cell load of 49.26 mg/ml, and 2.2 mm bead diameter. Under these conditions, retention activity of 87.6% could be attained for the immobilized cell. In addition, the oxidative activity of immobilized cells was retained at 53.2% compared with that of free cells after 10 repeated batch reactions, while only 15.7% of activity remained in free cells.

Enhanced production of dihydroxyacetone from glycerol by overexpression of glycerol dehydrogenase in an alcohol dehydrogenase-deficient mutant of Gluconobacter oxydans.

Gluconobacter oxydans can rapidly and incompletely oxidize glycerol to dihydroxyacetone (DHA), a versatile product extensively used in cosmetic, chemical and pharmaceutical industries. To improve DHA production, the glycerol dehydrogenase (GDH) responsible for DHA formation was overexpressed in G. oxydans M5AM, in which the gene coding for the membrane-bound alcohol dehydrogenase (ADH) was interrupted. Real-time PCR and enzyme activity assay revealed that the absence of ADH together with the overexpression of GDH gene resulted in an increased GDH activity in the resulting strain M5AM/GDH, which led to a substantially enhanced production of DHA in a resting cell system. In a batch biotransformation process, M5AM/GDH exhibited a 2.4-fold increased DHA productivity of 2.4g/g CDW/h from 1.0g/g CDW/h, yielding 96g/L DHA from 100g/L glycerol. When 140g/L glycerol was supplied, a final DHA concentration of 134g/L was accumulated within 14h. In four repeated batch runs, 385g DHA over a time period of 34h was achieved from 400g glycerol with an average productivity of 2.2g/g CDW/h. These results indicated that this newly developed strain G. oxydans M5AM/GDH with high productivity and increased tolerance against product inhibition has potential for DHA production in an industrial bioconversion process.

Purification and characterization of membrane-bound 3-dehydroshikimate dehydratase from Gluconobacter oxydans IFO 3244, a new enzyme catalyzing extracellular protocatechuate formation.

3-Dehydroshikimate dehydratase (DSD) is the first known enzyme catalyzing aromatization from 3-dehydroshikimate (DSA) to protocatechuate (PCA). Differently from cytosolic DSD (sDSD), a membrane-bound 3-dehydroshikimate dehydratase (mDSD) was found for the first time in the membrane fraction of Gluconobacter oxydans IFO 3244, and DSA was confirmed to be the direct precursor of PCA. In contrast to weak and instable sDSD, the abundance of mDSD in the membrane fraction suggested the metabolic significance of mDSD as the initial step in aromatization. mDSD was solubilized only by a detergent and was readily purified to high homogeneity. Its molecular weight was estimated to be 76,000. Purified mDSD showed a sole peak at 280 nm in the absorption spectrum and no critical cofactor requirements. The Km of DSA was measured at 0.5 mM, and the optimum pH was observed at pH 6-8. mDSD appeared to react only with DSA, and was inert to other compounds, such as 3-dehydroquinate, quinate, and shikimate.

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.

The genus Gluconobacter oxydans: comprehensive overview of biochemistry and biotechnological applications.

The genus Gluconobacter comprises some of the most frequently used microorganisms when it comes to biotechnological applications. Not only has it been involved in "historical" production processes, such as vinegar production, but in the last decades many bioconversion routes for special and rare sugars involving Gluconobacter have been developed. Among the most recent are the biotransformations involved in the production of L-ribose and miglitol, both very promising pharmaceutical lead molecules. Most of these processes make use of Gluconobacter's membrane-bound polyol dehydrogenases. However, recently other enzymes have also caught the eye of industrial biotechnology. Among them are dextran dextrinase, capable of transglucosylating substrate molecules, and intracellular NAD-dependent polyol dehydrogenases, of interest for co-enzyme regeneration. As such, Gluconobacter is an important industrial microbial strain, but it also finds use in other fields of biotechnology, such as biosensor-technology. This review aims to give an overview of the myriad of applications for Gluconobacter, with a special focus on some recent developments.

Production of Gluconobacter oxydans cells from low-cost culture medium for conversion of glycerol to dihydroxyacetone.

Gluconobacter oxydans could be immobilized as a biocatalyst for the conversion of glycerol to dihydroxyacetone. To reduce the production cost, the cells were produced from agricultural byproducts. Corn meal hydrolysate and corn steep liquor were employed to replace of sorbitol and yeast extract as medium for G. oxydans cell production. The optimal medium contained 80 g/L reducing sugar, 25 g/L corn steep liquor, and 10 g/L glycerol. The cell mass was about 4.22 g/L and the glycerol dehydrogenase activity was about 5.23 U/mL. For comparison, the cell mass was about 4.0 g/L and the glycerol dehydrogenase activity was about 5.35 U/mL cultured in sorbitol and yeast extract medium. These studies shown the corn meal hydrolysate and corn steep liquor medium was similar in performance to a nutrient-rich medium, but the cost of production was only 15% of that cultured in sorbitol and yeast extract medium. It was an economical process for the production of G. oxydans cells as biocatalyst for the conversion of glycerol to dihydroxyacetone in industry.

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.

Optimization of the microbial synthesis of dihydroxyacetone from glycerol with Gluconobacter oxydans.

An optimized repeated-fed-batch fermentation process for the synthesis of dihydroxyacetone (DHA) from glycerol utilizing Gluconobacter oxydans is presented. Cleaning, sterilization, and inoculation procedures could be reduced significantly compared to the conventional fed-batch process. A stringent requirement was that the product concentration was kept below a critical threshold level at all times in order to avoid irreversible product inhibition of the cells. On the basis of experimentally validated model calculations, a threshold value of about 60 kg x m(-3) DHA was obtained. The innovative bioreactor system consisted of a stirred tank reactor combined with a packed trickle-bed column. In the packed column, active cells could be retained by in situ immobilization on a hydrophilized Ralu-ring carrier material. Within 17 days, the productivity of the process could be increased by 75% to about 2.8 kg x m(-3) h(-1). However, it was observed that the maximum achievable productivity had not been reached yet.

Novel enzymatic method for the production of xylitol from D-arabitol by Gluconobacter oxydans.

Microorganisms capable of producing xylitol from D-arabitol were screened for. Of the 420 strains tested, three bacteria, belonging to the genera Acetobacter and Gluconobacter, produced xylitol from D-arabitol when intact cells were used as the enzyme source. Among them, Gluconobacter oxydans ATCC 621 produced 29.2 g/l xylitol from 52.4 g/l D-arabitol after incubation for 27 h. The production of xylitol was increased by the addition of 5% (v/v) ethanol and 5 g/l D-glucose to the reaction mixture. Under these conditions, 51.4 g/l xylitol was obtained from 52.4 g/l D-arabitol, a yield of 98%, after incubation for 27 h. This conversion consisted of two successive reactions, conversion of D-arabitol to D-xylulose by a membrane-bound D-arabitol dehydrogenase, and conversion of D-xylulose to xylitol by a soluble NAD-dependent xylitol dehydrogenase. Use of disruptants of the membrane-bound alcohol dehydrogenase genes suggested that NADH was generated via NAD-dependent soluble alcohol dehydrogenase.