Agric. Biol Chem., 49 (4), 1001 -1010, 1985 1001 Solubilization, Purification and Properties of Membrane-bound Glycerol Dehydrogenase from Gluconobacter industrius Minoru Ameyama,Emiko Shinagawa, Kazunobu Matsushita and Osao Adachi Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753, Japan Received July 30, 1984 Membrane-bound glycerol dehydrogenase was solubilized and purified about 100-fold from the membraneof Gluconobacter industrius IFO 3260 grown on a glycerol-glutamate medium. Solubilization of the enzyme was successfully achieved by use of 0.5% dimethyldodecylamineoxide in 0.05 m Tris-HCl, pH 8.0. Alcohol dehydrogenase and D-glucose dehydrogenase, which were abundantly formed in the same bacterial membrane, were eliminated on solubilization. Glycerol dehydrogenase was further purified through fractionation with polyethylene glycol 6000. The enzymeshowed a broad substrate specificity and various kinds of polyhydroxyl alcohols, in addition to glycerol, were rapidly oxidized in the presence of 2,6-dichlorophenolindophenoi and phenazine methosulfate as the electron acceptor but NADand NADPwere inert. The enzyme was proved to be a quinoprotein in which pyrroloquinoline quinone functioned as the prosthetic group. The first report on microbial oxidation of localization of the oxidase system in cells of G. glycerol to dihydroxyacetone was by Bertrand liquefaciens and found that the oxidation of with a strain capable of L-sorbose fermen- glycerol and raeso-erythritol was carried out by tation.1* Two types of glycerol dehydrogenase the particulate fraction. Unlike glycerol oxi- have been reported, one is pyridine nucleotide dase from fungi,13) the pyridine nucleotide dependent and the other is independent and independent glycerol dehydrogenase in the membrane-bound.As to the former type of bacterial membranedid not require oxygen as enzymeyielding either glyceraldehyde or di- an electron acceptor but was linked to the hydroxyacetone, NAD-dependent glycerol de- electron transport system of the organism. hydrogenase has been partially purified from Thus the localization and enzymatic properties the soluble fractions of Escherichia coii,2) of the membrane-bound glycerol dehydro- Acetobacter suboxydans and Klebsiella pneu- genase yielding dihydroxyacetone of acetic monia.^ Recently, Yamada et al.4'~6) have acid bacteria have remained to be elucidated. crystallized NAD-dependent glycerol dehy- On the other hand, production of dihydroxy- drogenase yielding dihydroxyacetone from acetone by growing cells or resting cells of Cellulomonas sp. and characterized its enzy- acetic acid bacteria has actually advanced. In matic and structural properties in detail. recent report by Chibata et a/.,14~16) fermen- NADP-dependent glycerol dehydrogenase tative and enzymatic production of dihydroxy- yielding dihydroxyacetone has been partially acetone by acetic acid bacteria was exam- purified from various origins.7~10) King and ined to increase the productivity in fermen- Cheldelinn) reported that an enzyme catalyz- tation processes which remained low until ing glycerol oxidation yielding dihydroxy- recently. In spite of such production and usage acetone independent of ATPor NADwas of dihydroxyacetone, characterization of the present in the cell free extract of Gluconobacter membrane-bound glycerol dehydrogenase of suboxydans. De Ley and Dochy12) studied the acetic acid bacteria has not been achieved. In 1002 M. Ameyamaet al. this paper, we describe the purification and enzyme solution and lOO'/miol of glycerol in a total properties of the membrane-bound glycerol volume of 1.0ml. The reaction was initiated at 25°C by the addition of glycerol and terminated by adding 0.5 ml dehydrogenase of acetic acid bacteria. At the of ferric-Dupanol reagent. Then, 3.5ml of water was same time, it is also reported that the mem- added to the reaction mixture. The resulting Prussian- brane-bound glycerol dehydrogenase con- blue color was measured with a photometer at 660nm tains a novel prosthetic group, pyrrolo- after standing at 25°C for 20min. One unit of enzyme quinoline quinone (PQQ), as in the cases activity was defined as the amount of enzyme catalyzing of alcohol, aldehyde, and D-glucose dehydro- the oxidation of 1 ^mol of glycerol per min under these conditions; 4.0 absorbance units equaled 1 pimol of glyc- genases of acetic acid bacteria.17) erol oxidized. Alcohol dehydrogenase in Table I was assayed under essentially the same conditions as for glyc- MATERIALS AND METHODS erol dehydrogenase, except that the substrate was changed to ethanol. Materials. The yeast extract was a kind gift from Assaying of the glycerol dehydrogenase with the solubi- Oriental Yeast Industry Co. Dimethyldodecylamineoxide, lized enzymefrom the membranefraction was performed dodecylsulfobetaine and dodecylbetaine were kind gifts spectrophotometrically at 25°C by measuring the decrease from Kao-Atlas Co. Lauroylsarcosine was a product of in absorbance at 600nmof DCIPwith PMS. Once the Wako Pure Chemicals Ind., and /?-octyl-glucoside a enzymewas solubilized from the membrane,the enzyme product of Sigma Chemicals Co. All other chemicals activity was no longer linked to ferricyanide as the electron used in this study were commercial products of analyti- acceptor in the oxidation of glycerol. The enzyme activi- cal grade. Authentic PQQ was prepared according to ty was expressed in /rniol of reduced DCIP per min, the method described previously.i8) with 15-OmM"1as the extinction coefficient at pH 8.0. The reaction mixture contained lOO^umolof Tris-HCl or Microbial strains and culture conditions. The bacterial potassium phosphate buffer, pH 8.0, 0.6/xmol of DCIP, strains of the genus Gluconobacter listed in Table I were 0.6//mol of PMS, 100/imol of glycerol and the enzyme generously donated by the Institute for Fermentation, solution in a total volume of 3.0ml. One unit of enzyme activity was defined as the amount of enzyme catlyzing the described.19>Osaka (IFO). TheStock compositioncultures wereofpreparedthe cultureas previouslymedium oxidation of 1 jumol of glycerol per min. containing glycerol and the method for cultivation were Protein determination. The protein content was de- also essentially the same as described previously.19) In the case of a large scale culture, 30 liters of the glycerol termined by the method of Lowry et al.21) with bovine serum albumin as the standard. The modified method mediumwas incubated in a 50-liter jar fermentor at 30°C described by Dulley and Grieve22) was employed for for about 24hr under vigorous agitation (500rpm) and aeration (25 to 30 liters per min). Bacterial cells were samples which contained detergent. harvested at the late exponential phase as soon as the jbH of the culture medium had decreased to 5.2. Identification of reaction products. The reaction prod- uct of glycerol oxidation was identified by thm-layer chro- Preparation of the cell homogenate and membrane frac- matography with acetone as the developing solvent by tion. All operations were performed below 4°C in essen- essentially the same method as that described by tially the same way as described previously.19) The pre- Yamada et al.14) Detection of dihydroxyacetone was cipitated membranefraction on ultfacentrifugation was performed by spraying with 1% /?-anisidine-HCl in n- washedwith buffer solution to removecytoplasmic ma- butanol followed by standing for 10min at 110°C. Dihy- terials by repeating the ultracentrifugation. droxyacetone gave a dar^-brown spot on a yellow back- ground with an Rfvalue of 0.75~0.80. Authentic dihy- Assay ofglycerol dehydrogenase. As for the other mem- droxyacetone and glyceraldehyde were chromato- brane-bound dehydrogenases reported elsewhere,20' graphed on the same plate. Glyceraldehyde showed an the enzyme activity of glycerol dehydrogenase could be Rf value of 0.3 to 0.4 with relatively faint coloring assayed by colorimetry in the presence of potassium ferri- with /?-anisidine. Reaction products such as D-fruc- cyanide or spectrometry in the presence of 2,6-dichlo- tose, L-sorbose and D-xylulose were also identified by rophenolindophenol (DCIP) and phenazine methosul- thin-layer chromatography with dual solvent systems fate (PMS). Hereafter, the potassium ferricyanide assay of phenol-NH3 and «-butanol-acetic acid-water (4 : 1 : was employed for routine assaying as has already been 5). Detection of such compounds was also performed reported.20) The rate of reduction of ferricyanide to ferro- with /7-anisidine. Alternatively, D-fructose was identified cyanide correlated with the ratio of glycerol oxidation. enzymatically with D-fructose dehydrogenase for The reaction mixture contained 10/miol of potassium which only D-fructose was available as the substrate.19) ferricyanide, 0.5 ml of citrate-phosphate buffer, pH 5.0, L-Sorbose was detected by reverse reaction of NADP-de- Membrane-boundGlycerol Dehydrogenase 1003 pendent sorbitol dehydrogenase23)24) prepared from G. abundantly in most strains of Gluconobacter. suboxydans var. a. IFO 3254 in the presence of NADPH D-Glucose dehydrogenase activity was also at pH 6.5. D-Fructose was inert for the NADP-depend- predominant throughout these strains al- ent sorbitol dehydrogenase. Ketoses formed by the en- zymereaction were further detected by the cysteine- though the data are not shown. In G. in- carbazole reaction.25) dustrius, the level of alcohol dehydrogenase was exceptionally low under the culture con- Extraction and