Asymmetric oxidation by Gluconobacter oxydans

Asymmetric oxidation is of great value and a major interest in both research and application. This review focuses on asymmetric oxidation of organic compounds by Gluconobacter oxydans. The microbe can be used for bioproduction of several kinds of important chiral compounds, such as vitamin C, 6-(2-hydroxyethyl)amino-6-deoxy-α-l-sorbofuranose, (S)-2-methylbutanoic acid, (R)-2-hydroxy-propionic acid and 5-keto-d-gluconic acid. Characteristics of the bacteria and research progress on the enantioselective biotransformation process are introduced.     

Kinetics of polyol oxidation with Gluconobacter oxydans

Summary The influence of culture pH on the metabolism of Gluconobacter oxydans was determined. An acidic milieu during growth of the organism enhances the oxidation rate. The CO₂ evolution rate representing the assimilation of the product is inhibited by a low pH value. Growth of the bacteria is possible both on glycerol and DHA in separate phases, which is not a controlled as diauxic growth. Product formation follows Luedeking-Piret kinetics.now: Institut für Biotechnologie, TU Graz, Petersgasse 12, 8010 Graz, Austria     

Aldehyde production by alcohol oxidation with Gluconobacter oxydans

The microbial oxidation of various primary alcohols to the corresponding aldehydes has been investigated. A focused screening performed amongst some acetic acid bacteria showed that a newly isolated strain of Gluconobacter oxydans oxodizes various short-chain aliphatic alcohols to the corresponding aldehydes with negligible acid production. 3-Methyl-1-butanol (isoamyl alcohol) proved to be the better substrate with high yields (more than 90%) without by-product formation. This biotransformation also occurs with continuous or semicontinuous addition of substrate since the volatile product is removed from the medium under vigorous aeration conditions. Product recovery is attained either by the use of cold traps or by reversible complex formation.     

Kinetic aspects of glucose oxidation by Gluconobacter oxydans cells immobilized in calcium alginate

Summary Gluconobacter oxydans subspecies suboxydans (ATCC 621 H), when growing at high glucose concentrations, oxidizes this substrate incompletely and gluconic acid accumulates in the medium in almost stoichiometric amounts. Such cells were harvested and entrapped in various alginate gels. The preparation with the highest retention of glucose oxidizing activity was used in further studies with the aim of developing an efficient process for continuous gluconic acid production.The retention of activity increases (up to 95%) as the alginate concentration in the gel decreases or the cell/alginate weight ratio is enhanced. In the latter case, however, transport of oxygen to and inside the biocatalyst beads rapidly becomes rate-limiting and thus lowers the efficiency of the biocatalyst. Similarly, the efficiency decreases as the size of the biocatalyst beads increases. In no case rate-limitation by transport of glucose was found. Thus, biocatalyst activity per unit volume of support, diameter of the biocatalyst beads, and aeration efficiency are important parameters for reactor design.     

Glucose oxidation and PQQ-dependent dehydrogenases in Gluconobacter oxydans

Gluconobacter oxydans is famous for its rapid and incomplete oxidation of a wide range of sugars and sugar alcohols. The organism is known for its efficient oxidation of D-glucose to D-gluconate, which can be further oxidized to two different keto-D-gluconates, 2-keto-D-gluconate and 5-keto-D-gluconate, as well as 2,5-di-keto-D-gluconate. For this oxidation chain and for further oxidation reactions, G. oxydans possesses a high number of membrane-bound dehydrogenases. In this review, we focus on the dehydrogenases involved in D-glucose oxidation and the products formed during this process. As some of the involved dehydrogenases contain pyrroloquinoline quinone (PQQ) as a cofactor, also PQQ synthesis is reviewed. Finally, we will give an overview of further PQQ-dependent dehydrogenases and discuss their functions in G. oxydans ATCC 621H (DSM 2343).     

Analysis of growth of Gluconobacter oxydans in glucose containing media

Gluconobacter oxydans oxidizes glucose via alternative pathways: one involves the non-phosphorylative, direct oxidation route to gluconic acid and ketogluconic acids, and the second requires an initial phosphorylation and then oxidation via the pentose phosphate pathway enzymes. During growth of G. oxydans in glucose-containing media, the activity of this pathway is strongly influenced by (1) the pH value of the environment and (2) the actual concentration of glucose present in the culture. At pH values below 3.5 the activity of the pentose phosphate pathway was completely inhibited resulting in an increased requirement of the organism for nutrient substances, and a poor cell yield. At pH 5.5 a triphasic growth response was observed when G. oxydans was grown in a defined medium. Above a threshold value of 5–15 mM glucose, oxidation of both glucose and gluconate by the pentose phosphate pathway enzymes was repressed, causing a rapid accumulation of gluconic acid in the culture medium. When growing under these conditions, a low affinity for the oxidation of glucose was found (K _s=13 mM). Below this threshold glucose concentration, pentose phosphate pathway enzymes were synthesized and glucose was actively assimilated via this pathway. It was shown that de novo enzyme synthesis was necessary for increased pentose phosphate pathway activity and that assimilation of gluconate by washed cell suspensions was inhibited by glucose.     

Acetate production from lactate and glucose fermentation by Gluconobacter oxydans

Summary The production of acetate from the fermentation of lactate by Gluconobacter oxydans was studied. Batch experiments showed that glucose was the preferred substrate compared to lactate. A fed-batch culture was fed with a mixture of glucose and lactate followed by periodic addition of lactate. The maximum productivity of acetate was 0.16 g/l h but this value decreased during the fedbatch culture due to growth inhibition by acetate.     

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.     

Intracytoplasmic membrane formation and increased oxidation of glycerol growth of Gluconobacter oxydans.

Gluconobacter oxydans is well known for the limited oxidation of compounds and rapid excretion of industrially important oxidation products. The dehydrogenases responsible for these oxidations are reportedly bound to the cell's plasma membrane. This report demonstrates that fully viable G. oxydans differentiates at the end of exponential growth by forming dense regions at the end of each cell observed with the light microscope. When these cells were thin sectioned, their polar regions contained accumulations of intracytoplasmic membranes and ribosomes not found in undifferentiated exponentially growing cells. Both freeze-fracture-etched whole cells and thin sections through broken-cell envelopes of differentiated cells demonstrate that intracytoplasmic membranes occur as a polar accumulation of vesicles that are attached to the plasma membrane. When cells were tested for the activity of the plasma membrane-associated glycerol dehydrogenase, those containing intracytoplasmic membranes were 100% more active than cells lacking these membranes. These results suggest that intracytoplasmic membranes are formed by continued plasma membrane synthesis at the end of active cell division.     

A Gluconobacter oxydans mutant converting glucose almost quantitatively to 5-keto-d-gluconic acid

Gluconobacter oxydans converts glucose to gluconic acid and subsequently to 2-keto-d-gluconic acid (2-KGA) and 5-keto-d-gluconic acid (5-KGA) by membrane-bound periplasmic pyrroloquinoline quinone-dependent and flavin-dependent dehydrogenases. The product pattern obtained with several strains differed significantly. To increase the production of 5-KGA, which can be converted to industrially important l-(+)-tartaric acid, growth parameters were optimized. Whereas resting cells of G. oxydans ATCC 621H converted about 11% of the available glucose to 2-KGA and 6% to 5-KGA, with growing cells and improved growth under defined conditions (pH 5, 10% pO₂, 0.05% pCO₂) a conversion yield of about 45% 5-KGA from the available glucose was achieved. As the accumulation of the by-product 2-KGA is highly disadvantageous for an industrial application of G. oxydans, a mutant was generated in which the membrane-bound gluconate-2-dehydrogenase complex was inactivated. This mutant, MF1, grew in a similar way to the wild type, but formation of the undesired 2-KGA was not observed. Under improved growth conditions, mutant MF1 converted the available glucose almost completely (84%) into 5-KGA. Therefore, this newly developed recombinant strain is suitable for the industrial production of 5-KGA.     

Effects of membrane-bound glucose dehydrogenase overproduction on the respiratory chain of Gluconobacter oxydans

The acetic acid bacterium Gluconobacter oxydans incompletely oxidizes carbon sources as a natural part of its metabolism, and this feature has been exploited for many biotechnological applications. The most important enzymes used to harness the biocatalytic oxidative capacity of G. oxydans are the pyrroloquinoline quinone (PQQ)-dependent dehydrogenases. The membrane-bound PQQ-dependent glucose dehydrogenase (mGDH), encoded by gox0265, was used as model protein for homologous membrane protein production using the previously described Gluconobacter expression vector pBBR1p452. The mgdh gene had ninefold higher expression in the overproduction strain compared to the parental strain. Furthermore, membranes from the overexpression strain had a five- and threefold increase of mGDH activity and oxygen consumption rates, respectively. Oxygen consumption rate of the membrane fraction could not be increased by the addition of a substrate combination of glucose and ethanol in the overproduction strain, indicating that the terminal quinol oxidases of the respiratory chain were rate limiting. In contrast, addition of glucose and ethanol to membranes of the control strain increased oxygen consumption rates approaching the observed rates with G. oxydans overproducing mGDH. The higher glucose oxidation rates of the mGDH overproduction strain corresponded to a 70 % increase of the gluconate production rate compared to the control strain. The high rate of glucose oxidation may be useful in the industrial production of gluconates and ketogluconates, or as whole-cell biosensors. Furthermore, mGDH was purified to homogeneity by one-step strep-tactin affinity chromatography and characterized. To our knowledge, this is the first report of a membrane integral quinoprotein being purified by affinity chromatography and serves as a proof-of-principle for using G. oxydans as a host for membrane protein expression and purification.     

Quinate oxidation in Gluconobacter oxydans IFO3244: purification and characterization of quinoprotein quinate dehydrogenase

Quinoprotein quinate dehydrogenase (QDH) is a membrane-bound enzyme containing pyrroloquinoline quinone (PQQ) as the prosthetic group. QDH in Gluconobacter oxydans IFO3244 was found to be inducible by quinate and it is not constitutively expressed in the absence of quinate. The purification of holo-form of QDH to nearly homogeneity was achieved. The purified QDH appears to have two subunits of approximately 65 and 21 kDa, which could be the result of proteolysis of single polypeptide. Kinetic analysis indicated that the purified enzyme is much more specific to quinate than QDH from Acinetobacter calcoaceticus. The efficiency of the artificial electron acceptor was also determined.     

Directing cell catalysis of glucose to 2-keto-d-gluconic acid using Gluconobacter oxydans NL71

2-Keto-d-gluconic acid (2-KGA) is a high-value-added product from lignocellulosic straw-based biorefining process obtained via cell catalysis using Gluconobacter oxydans (G. oxydans); nonetheless, the economical production has some limitations because of the formation of undesirable mixture products of keto-gluconic acid from glucose. In this study, it was demonstrated that pulse addition of hydrogen peroxide (H₂O₂) to synthetic media was beneficial to improve the conversion rate of glucose to 2-KGA by 1.47-fold in G. oxydans. Moreover, some lignocellulosic degradation compounds served as substitute and played the role of H₂O₂ on corn stover hydrolysate for the production of 2-KGA. By conducting qRT-PCR analysis, eight dehydrogenase genes were identified in G. oxydans, which were responsible for regulating 2-KGA production. Moreover, most of the identified genes were found to produce membrane-bound glucose dehydrogenase and gluconate 2-dehydrogenase, and were up-regulated by adding H₂O₂ or degradation compounds. These up-regulated genes could be responsible for directing cell catalysis of glucose to 2-KGA in G. oxydans.     

Monitoring of dihydroxyacetone production during oxidation of glycerol by immobilized Gluconobacter oxydans cells with an enzyme biosensor

A bi-enzymatic biosensor for monitoring of dihydroxyacetone production during oxidation of glycerol by bacterial cells of Gluconobacter oxydans is presented. Galactose oxidase oxidizes dihydroxyacetone efficiently producing hydrogen peroxide, which reacts with co-immobilized peroxidase and ferrocene pre-adsorbed on graphite electrode. This mediator-based bi-enzymatic biosensor possesses very high sensitivity (4.7 μA/mM in phosphate buffer), low detection limit (0.8 μM, signal/noise = 3), short response time (22 s, 95% of steady-state) and broad linear range (0.002-0.55 mM in phosphate buffer). The effect of pH, temperature, type of buffer, as well as different stabilizers (combinations of a polyelectrolyte and a polyol) on the sensor performance were carefully optimized and discussed. Dihydroxyacetone produced during a batch conversion of glycerol by the pectate-immobilized bacteria in an air-lift reactor was determined by the biosensor and by reference spectrophotometric method. Both methods were compared and were in a very good correlation. The main advantage of the biosensor is a very short time needed for sample analysis (less than 1 min).     

Characterization of Gluconobacter oxydans immobilized in calcium alginate

Summary Gluconobacter oxydans cells were immobilized in calcium alginate and the preparation was used for the oxidation of glycerol to dihydroxyacetone. The characterization was done according to the guidelines given by the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology. The pH optimum of the preparation was found to be 5.0 and the temperature optimum was 40°C. However, the operational stability was better at 30°C. The glycerol concentration required to obtain half the maximal reaction rate was about 5 mM for both immobilized and free cells. At low concentrations of glycerol and high concentrations of dihydroxyacetone a slight inhibition was noted. No loss of activity of the immobilized preparation was observed after storage for 68 days at +4°C. Investigation of the operational stability revealed a half-life of 5 days. Studies of the influence of particle size and cell densities as well as that of oxygen concentration revealed that the oxygen supply was the rate limiting step.