Emiko Shinagawa

NEW DEVELOPMENTS IN OXIDATIVE FERMENTATION

Abstract. Oxidative fermentations have been well established for a long time, especially in vinegar and in L-sorbose production. Recently, information on the enzyme systems involved in these oxidative fermentations has accumulated and new developments are possible based on these findings. We have recently isolated several thermotolerant acetic acid bacteria, which also seem to be useful for new developments in oxidative fermentation. Two different types of membrane-bound enzymes, quinoproteins and flavoproteins, are involved in oxidative fermentation, and sometimes work with the same substrate but produce different oxidation products. Recently, there have been new developments in two different oxidative fermentations, D-gluconate and D-sorbitol oxidations. Flavoproteins, D-gluconate dehydrogenase, and D-sorbitol dehydrogenase were isolated almost 2 decades ago, while the enzyme involved in the same oxidation reaction for D-gluconate and D-sorbitol has been recently isolated and shown to be a quinoprotein. Thus, these flavoproteins and a quinoprotein have been re-assessed for the oxidation reaction. Flavoprotein D-gluconate dehydrogenase and D-sorbitol dehydrogenase were shown to produce 2-keto-D-gluconate and D-fructose, respectively, whereas the quinoprotein was shown to produce 5-keto-D-gluconate and L-sorbose from D-gluconate and D-sorbitol, respectively. In addition to the quinoproteins described above, a new quinoprotein for quinate oxidation has been recently isolated from Gluconobacter strains. The quinate dehydrogenase is also a membrane-bound quinoprotein that produces 3-dehydroquinate. This enzyme can be useful for the production of shikimate, which is a convenient salvage synthesis system for many antibiotics, herbicides, and aromatic amino acids synthesis. In order to reduce energy costs of oxidative fermentation in industry, several thermotolerant acetic acid bacteria that can grow up to 40°C have been isolated. Of such isolated strains, some thermotolerant Acetobacter species were found to be useful for vinegar fermentation at a high temperature such 38–40°C, where mesophilic strains showed no growth. They oxidized higher concentrations of ethanol up to 9% without any appreciable lag time, while alcohol oxidation with mesophilic strains was delayed or became almost impossible under such conditions. Several useful Gluconobacter species of thermotolerant acetic acid bacteria are also found, especially L-erythrulose-producing strains and cyclic alcohol-oxidizing strains. Gluconobacter frateurii CHM 43 is able to rapidly oxidize meso-erythritol at 37°C leading to the accumulation of L-erythrulose, which may replace dihydroxyacetone in cosmetics. G. frateurii CHM 9 is able to oxidize cyclic alcohols to their corresponding cyclic ketones or aliphatic ketones, which are known to be useful for preparing many different physiologically active compounds such as oxidized steroids or oxidized bicyclic ketones. The enzymes involved in these meso-erythritol and cyclic alcohol oxidations have been purified and shown to be a similar type of membrane-bound quinoproteins, consisting of a high molecular weight single peptide. This is completely different from another quinoprotein, alcohol dehydrogenase of acetic acid bacteria, which consists of three subunits including hemoproteins.

Existence of a novel prosthetic group, PQQ, in mebrane-bound, electron transport chain-linked, primary dehydrogenases of oxidative bacteria

We have already purified membrane-bound, electron transport-linked, D-glucose dehydrogenase from Pseudomonas jluorescens [I], and found that the enzyme had an unknown prosthetic group differing from NAD(P) and flavin. Furthermore, it appears that alcohol [2], aldehyde [3] and D-glucose [4] dehydrogenases isolated in our laboratory from Gluconobacter suboxydans also have the same prosthetic group. This group’s characteristics closely resembled those of glucose dehydrogenase purified from Acinetobacter [S] and of methanol dehydrogenases from Pseudomonas sp. M27 [6], Rhodopseudomonas acidophila [7] and Hyphomicrobium X [8]. Duine et al. [9] showed recently, that the novel prosthetic group was a pyrrolo-quinoline quinone (PQQ), the structure of which was determined using an X-ray analysis by Salisbury et al. [lo]. To obtain more information on the prosthetic group, we isolated a mutant of P. aeruginosa having less glucose dehydrogenase activity than normal. This activity was shown to be restored by adding the prosthetic group extracted from the purified glucose dehydrogenase of P. fluorescens. Thus, the membrane of the mutant has an apo-glucose dehydrogenase and may be available for the identification of PQQ. Using the mutant membrane, we showed that alcohol, aldehyde and glucose dehydrogenases from G. suboxydans also had the same prosthetic group, PQQ.

Membrane-bound Quinoprotein D-Arabitol Dehydrogenase of Gluconobacter suboxydans IFO 3257: A Versatile Enzyme for the Oxidative Fermentation of Various Ketoses

Solubilization of membrane-bound quinoprotein D-arabitol dehydrogenase (ARDH) was done successfully with the membrane fraction of Gluconobacter suboxydans IFO 3257. In enzyme solubilization and subsequent enzyme purification steps, special care was taken to purifiy ARDH as active as it was in the native membrane, after many disappointing trials. Selection of the best detergent, keeping ARDH as the holoenzyme by the addition of PQQ and Ca2+, and of a buffer system involving acetate buffer supplemented with Ca2+, were essential to treat the highly hydrophobic and thus labile enzyme. Purification of the enzyme was done by two steps of column chromatography on DEAE-Toyopearl and CM-Toyopearl in the presence of detergent and Ca2+. ARDH was homogenous and showed a single sedimentation peak in analytical ultracentrifugation. ARDH was dissociated into two different subunits upon SDS-PAGE with molecular masses of 82kDa (subunit I) and 14kDa (subunit II), forming a heterodimeric structure. ARDH was proven to be a quinoprotein by detecting a liberated PQQ from SDS-treated ARDH in HPLC chromatography. More preliminarily, an EDTA-treated membrane fraction lost the enzyme activity and ARDH activity was restored to the original level by the addition of PQQ and Ca2+. The most predominant unique character of ARDH, the substrate specificity, was highly versatile and many kinds of substrates were oxidized irreversibly by ARDH, not only pentitols but also other polyhydroxy alcohols including D-sorbitol, D-mannitol, glycerol, meso-erythritol, and 2,3-butanediol. ARDH may have its primary function in the oxidative fermentation of ketose production by acetic acid bacteria. ARDH contained no heme component, unlike the type II or type III quinoprotein alcohol dehydrogenase (ADH) and did not react with primary alcohols.

o-Type cytochrome oxidase in the membrane of aerobically grown Pseudomonas aeruginosa

A variety of cytochrome oxidases, including the aa3-type, al-type, d-type and o-type, have been identified in bacteria. Many aa,type bacterial cytochrome oxidases have been purified and characterized [l-7]. These studies have contributed much to the analysis of cytochrome oxidase; but, except for the soluble Vitreoscilla cytochrome o, no o-type cytochrome oxidase has been characterized completely in spite of its extensive distribution in bacteria [8-lo]. Membrane-bound o-type cytochrome oxidases have been studied in Azotobacter vinelandii [ 1 l131 and Rhodopseudomonas palustris [ 141. In the case where these oxidases show high activity, they contain a c-type cytochrome the role of which is not known. In Pseudomonas aeruginosa grown aerobically, o-type cytochrome may have function as the only terminal oxidase in the membrane-bound, electron-transport system [ 151. Thus, we solubilized and purified the o-type cytochrome oxidase from the membrane of aerobically grown R aeruginosa. This oxidase consists of 4 polypeptides that include cytochrome o and cytochrome c, both of which react with carbon monoxide. branes were treated with cholate plus deoxycholate [16], then washed with 1% cholate plus 1 M KCI. The residual precipitate (600-800 mg protein) was extracted with 2% Triton X-100 overnight in 0.1 M Tris-HCI buffer (pH 7.5 at 2O’C) at 10 mg protein/ml. The extract was diluted with the same volume of cold distilled water and immediately applied to a DEAEcellulose column (2.4 X 15 cm) pre-equilibrated with 0.05 M Tris-HCl buffer (pH 7.5). The column was washed with 150 ml the same buffer containing 0.05% Brij 58, after which the oxidase was eluted with 150 ml of the same buffer containing 0.15 M KCl. Fractions which were eluted first that had high specific activity were combined, then diluted 4-fold with cold water after which they were applied to a DEAEcellulose column (2.4 X 7 cm) equilibrated with 0.05 M Tris-HCl buffer (pH 7.5) containing 0.05% Brij 58. After washing the column with 150 ml of the same buffer, the oxidase was eluted with a linear gradient of 150 ml each of the above buffer and of the same buffer containing 0.3 M KCl. At -0.15 M KCl, the oxidase activity was eluted as a single symmetrical peak that coincided with the peaks of protein and cytochrome. The orange-yellow active fraction was concentrated with an ultrafilter then used as the purified oxidase.

[31] d-Gluconate dehydrogenase from bacteria, 2-keto-d-gluconate-yielding, membrane-bound

Publisher Summary This chapter describes an assay method for the isolation of D-gluconate dehydrogenase from bacteria. D-gluconate dehydrogenase occurs on the outer surface of cytoplasmic membrane of oxidative bacteria, such as Pseudomonas, Klebsiella, Serratia , and acetic acid bacteria. The enzyme activity is linked to the electron transport chain in the cytoplasmic membrane constituting a D-gluconate oxidase system. The assay is performed spectrophotometrically at 25°C by measuring the decrease of absorbance at 600 nm of 2,6-dichlorophenolindophenol (DCIP) mediated with phenazine methosulfate (PMS). The activity is also measured with PMS, DCIP, ferricyanide, or coenzyme Q (CoQ) as an electron acceptor. The purification procedures of the enzyme from P. fluorescens and K. pneumoniae are described in the chapter. Potassium phosphate buffer, pH 6.0, containing 5 m M MgCl 2 is used throughout the purification of P. fluorescens . The steps involved in the purification procedure are (1) the preparation of membrane fraction, (2) the solubilization of enzyme, (3) ammonium sulfate fractionation, and (4) hydroxyapatite column chromatography. Purified D-gluconate dehydrogenase shows a visible absorption spectrum of the cytochrome c type having an asymmetrical α-peak.

Membrane-bound Sugar Alcohol Dehydrogenase in Acetic Acid Bacteria catalyzes L-Ribulose Formation and NAD-Dependent Ribitol Dehydrogenase is Independent of the Oxidative Fermentation

To identify the enzyme responsible for pentitol oxidation by acetic acid bacteria, two different ribitol oxidizing enzymes, one in the cytosolic fraction of NAD(P)-dependent and the other in the membrane fraction of NAD(P)-independent enzymes, were examined with respect to oxidative fermentation. The cytoplasmic NAD-dependent ribitol dehydrogenase (EC 1.1.1.56) was crystallized from Gluconobacter suboxydans IFO 12528 and found to be an enzyme having 100kDa of molecular mass and 5 s as the sedimentation constant, composed of four identical subunits of 25 kDa. The enzyme catalyzed a shuttle reversible oxidoreduction between ribitol and D-ribulose in the presence of NAD and NADH, respectively. Xylitol and L-arabitol were well oxidized by the enzyme with reaction rates comparable to ribitol oxidation. D-Ribulose, L-ribulose, and L-xylulose were well reduced by the enzyme in the presence of NADH as cosubstrates. The optimum pH of pentitol oxidation was found at alkaline pH such as 9.5-10.5 and ketopentose reduction was found at pH 6.0. NAD-Dependent ribitol dehydrogenase seemed to be specific to oxidoreduction between pentitols and ketopentoses and D-sorbitol and D-mannitol were not oxidized by this enzyme. However, no D-ribulose accumulation was observed outside the cells during the growth of the organism on ribitol. L-Ribulose was accumulated in the culture medium instead, as the direct oxidation product catalyzed by a membrane-bound NAD(P)-independent ribitol dehydrogenase. Thus, the physiological role of NAD-dependent ribitol dehydrogenase was accounted to catalyze ribitol oxidation to D-ribulose in cytoplasm, taking D-ribulose to the pentose phosphate pathway after being phosphorylated. L-Ribulose outside the cells would be incorporated into the cytoplasm in several ways when need for carbon and energy sources made it necessary to use L-ribulose for their survival. From a series of simple experiments, membrane-bound sugar alcohol dehydrogenase was concluded to be the enzyme responsible for L-ribulose production in oxidative fermentation by acetic acid bacteria.

Method of enzymatic determination of pyrroloquinoline quinone

An improved enzymatic method for the determination of pyrroloquinoline quinone, a novel prosthetic group of some important oxidoreductases, has been developed with cytoplasmic membrane of Escherichia coli K-12, in which D-glucose dehydrogenase (EC 1.1.99.17) was completely resolved to apo-enzyme by EDTA treatment. Incubation of the EDTA-treated membrane with exogenous pyrroloquinoline quinone in the presence of magnesium ions gave a quantitative determination of pyrroloquinoline quinone by assaying the restored D-glucose dehydrogenase activity. This novel enzymatic method was confirmed to be highly reproducible up to 10 ng of pyrroloquinoline quinone and could be applied to a routine assay of pyrroloquinoline quinone.

Purification, characterization and reconstitution of cytochrome o-type oxidase from Gluconobacter suboxydans

Abstract The Gluconobacter suboxydans respiratory chain has a cytochrome o as a terminal oxidase. The cytochrome o-type oxidase was solubilized with octyl glucoside after washing the membranes with Triton X-100, and was purified by one-step ion-exchange chromatography. The purified oxidase contains four polypeptides, two b-type cytochromes (b-558 and b-562), and 2 mol of heme/mol of enzyme. The oxidase was shown to be a typical cytochrome o and to have two CO-binding sites in the molecule. The enzyme catalyzes the oxidation of ubiquinol, and the activity is inhibited with KCN or quinone analogues. The purified cytochrome o can be reconstituted with phospholipids prepared from G. suboxydans into proteoliposomes by octyl glucoside dilution. The proteoliposome generates a proton electrochemical gradient (inside negative and alkaline) of about −140 mV during ubiquinol oxidation. The generation of membrane potential and pH gradient was determined by fluorometric methods using carbocyanine and dansylglycine, respectively. Thus, cytochrome o of G. suboxydans was shown to be an ubiquinol oxidase functioning as an energy-generator.

New quinoproteins in oxidative fermentation

Several quinoproteins have been newly indicated in acetic acid bacteria, all of which can be applied to fermentative or enzymatic production of useful materials by means of oxidative fermentation. (1) D-Arabitol dehydrogenase from Gluconobacter suboxydans IFO 3257 was purified from the bacterial membrane and found to be a versatile enzyme for oxidation of various substrates to the corresponding oxidation products. It is worthy of notice that the enzyme catalyzes D-gluconate oxidation to 5-keto-D-gluconate, whereas 2-keto-D-gluconate is produced by a flavoprotein D-gluconate dehydrogenase. (2) Membrane-bound cyclic alcohol dehydrogenase was solubilized and purified for the first time from Gluconobacter frateurii CHM 9. When compared with the cytosolic NAD-dependent cyclic alcohol dehydrogenase crystallized from the same strain, the reaction rate in cyclic alcohol oxidation by the membrane enzyme was 100 times stronger than the cytosolic NAD-dependent enzyme. The NAD-dependent enzyme makes no contribution to cyclic alcohol oxidation but contributes to the reduction of cyclic ketones to cyclic alcohols. (3) Meso-erythritol dehydrogenase has been purified from the membrane fraction of G. frateurii CHM 43. The typical properties of quinoproteins were indicated in many respects with the enzyme. It was found that the enzyme, growing cells and also the resting cells of the organism are very effective in producing L-erythrulose. Dihydroxyacetone can be replaced by L-erythrulose for cosmetics for those who are sensitive to dihydroxyacetone. (4) Two different membrane-bound D-sorbitol dehydrogenases were indicated in acetic acid bacteria. One enzyme contributing to L-sorbose production has been identified to be a quinoprotein, while another FAD-containing D-sorbitol dehydrogenase catalyzes D-sorbitol oxidation to D-fructose. D-Fructose production by the oxidative fermentation would be possible by the latter enzyme and it is superior to the well-established D-glucose isomerase, because the oxidative fermentation catalyzes irreversible one-way oxidation of D-sorbitol to D-fructose without any reaction equilibrium, unlike D-glucose isomerase. (5) Quinate dehydrogenase was found in several Gluconobacter strains and other aerobic bacteria like Pseudomonas and Acinetobacter strains. It has become possible to produce dehydroquinate, dehydroshikimate, and shikimate by oxidative fermentation. Quinate dehydrogenase was readily solubilized from the membrane fraction by alkylglucoside in the presence of 0.1 M KCl. A simple purification by hydrophobic chromatography gave a highly purified quinate dehydrogenase that was monodispersed and showed sufficient purity. When quinate dehydrogenase purification was done with Acinetobacter calcoaceticus AC3, which is unable to synthesize PQQ, purified inactive apo-quinate dehydrogenase appeared to be a dimer and it was converted to the monomeric active holo-quinate dehydrogenase by the addition of PQQ.

Characterization of Quinohemoprotein Amine Dehydrogenase from Pseudomonas putida

Quinohemoprotein amine dehydrogenase (AMDH) was purified and crystallized from the soluble fraction of Pseudomonas putida IFO 15366 grown on n-butylamine medium. AMDH gave a single component in analytical ultracentrifugation showing an intrinsic sedimentation coefficient of 5.8s. AMDH showed a typical absorption spectrum of cytochrome c showing maxima at 554, 522, 420, and 320 nm in the reduced form and one peak at 410 nm, a shoulder at 350 nm, and a broad hill around 530 nm in the oxidized form. The oxidized enzyme was specifically reduced by the addition of amine substrate. AMDH was composed of three different subunits, 60, 40, and 20 kDa, with the total molecular weight of 120,000. Two moles of heme c were detected per mole of AMDH and the 60-kDa subunit was found to be the heme c-carrying subunit. By redox-cycling quinone staining, a positive reaction band corresponding to the 20-kDa subunit was detected after developed by SDS-PAGE, but the 20 kDa band was scarcely stained by conventional protein staining. Only a silver staining method was possible to detect the subunit after the protein was developed by SDS-PAGE. p-Nitrophenylhydrazine-inhibited AMDH was dissociated into subunits and the 20-kDa subunit showed an absorption maximum at 455 nm, indicating Schiff base formation between the carbonyl cofactor in AMDH and the carbonyl reagent. Thus, AMDH is different from nonheme quinoprotein methylamine dehydrogenase and aromatic amine dehydrogenase in many respects. The presence of an azurin-like blue protein was identified and purified from the same cell-free extract of P. putida as AMDH was purified. The blue protein was reduced specifically during AMDH reaction, suggesting that the blue protein is the direct electron acceptor in amine oxidation. The amine oxidation system was reconstituted successfully only by AMDH, the blue protein, and the cytoplasmic membranes of the organism. The function of the 40-kDa subunit is unknown at the moment. The properties of AMDH were compared with other bacterial amine dehydrogenases so far reported.