Biosci. Biotechnol. Biochem., 66 (2), 307–318, 2002 L-Erythrulose Production by Oxidative Fermentation is Catalyzed by PQQ-Containing Membrane-bound Dehydrogenase Duangtip MOONMANGMEE,† Osao ADACHI,‡ Emiko SHINAGAWA,* Hirohide TOYAMA, Gunjana THEERAGOOL,** Napha LOTONG,** and Kazunobu MATSUSHITA Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan *Department of Chemical and Biological Engineering, Ube National College of Technology, Tokiwadai, Ube 755-8555, Japan **Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand Received August 10, 2001; Accepted September 20, 2001 Thermotolerant Gluconobacter frateurii CHM 43 was alkaline pHs such as 9.0–10.5. L-Erythrulose reduction selected for L-erythrulose production from meso- was found at pH 6.0 with NADH as coenzyme. Judging erythritol at higher temperatures. Growing cells and the from the catalytic properties, the NAD-dependent en- membrane fraction of the strain rapidly oxidized meso- zyme in the cytosolic fraction was regarded as a typical erythritol to L-erythrulose irreversibly with almost pentitol dehydrogenase of NAD-dependent and the en- 100% of recovery at 379C. L-Erythrulose was also zyme was independent of the oxidative fermentation of produced e‹ciently by the resting cells at 379Cwith L-erythrulose production. 85% recovery. The enzyme responsible for meso- erythritol oxidation was found to be located in the Key words: acetic acid bacteria; membrane-bound cytoplasmic membrane of the organism. The EDTA- meso-erythritol dehydrogenase; NAD- resolved enzyme required PQQ and Ca 2＋ for L- dependent meso-erythritol de- erythrulose formation, suggesting that the enzyme hydrogenase; L-erythrulose reductase; catalyzing meso-erythritol oxidation was a quino- oxidative fermentation protein. Quinoprotein membrane-bound meso- erythritol dehydrogenase (QMEDH) was solubilized In relation to sugar and sugar alcohol metabolism and puriˆed to homogeneity. The puriˆed enzyme in acetic acid bacteria, we have characterized many showedasinglebandinSDS-PAGEofwhichthe membrane-bound dehydrogenases localized in the molecular mass corresponded to 80 kDa. The optimum cytoplasmic membranes.1) Membrane-bound de- pH of QMEDH was found at pH 5.0. The Michaelis hydrogenases catalyze substrate oxidation, yielding constant of the enzyme was found to be 25 mM for an oxidation product by which the oxidative fermen- meso-erythritol as the substrate. QMEDH showed a tations have the actual function such as acetate fer- broad substrate speciˆcity toward C3-C6 sugar alcohols mentation, D-gluconate fermentation, L-sorbose in which the erythro form of two hydroxy groups exist- fermentation, and so on. In the cytosolic fraction of ed adjacent to a primary alcohol group. On the other the same organisms, diŠerent kinds of NAD(P)- hand, the cytosolic NAD-denpendent meso-erythritol dependent dehydrogenases have been indicated.1) The dehydrogenase (CMEDH) of the same organism was NAD(P)-dependent dehydrogenases characteristical- puriˆed to a crystalline state. CMEDH showed a ly catalyze reduction of the oxidized product after it molecular mass of 60 kDa composed of two identical is incorporated into the cytoplasm. In our previous subunits, and an apparent sedimentation constant was study,2) membrane-bound dehydrogenses related to 3.6 s.CMEDH catalyzed oxidoreduction between meso- pentitol oxidation in acetic acid bacteria were report- erythritol and L-erythrulose. The oxidation reaction was ed, indicating that they are responsible for the oxida- observed to be reversible in the presence of NAD at tive fermentation and for which NAD-dependent ‡ To whom correspondence should be addressed. Laboratory of Applied Microbiology, Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan; Tel: +81-83-933-5857; Fax: +81-83-933-5820; E-mail: osao＠ agr.yamaguchi-u.ac.jp † On leave from the Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand. A part of this paper was presented in the annual meeting of the Japan Society for Bioscience, Biotechnology, and Agrochemistry held in Kyoto from March 24 to 27, 2001. The abstract paper appears in the Nippon Nogeikagaku Kaishi, 75, 215 (2001). 308 D. MOONMANGMEE et al. cytosolic enzymes have no use. Regarding oxidative vation was done aerobically at 309Cor379Cina fermentations of C4 sugar alcohols, no information 500-ml side-armed Erlenmeyer ‰ask. Bacterial has been accumulated, though some earlier reports growth was monitored by measuring the turbidity by on meso-erythritol oxidation by aerobic bacteria a Klett-Summerson photoelectric colorimeter with a were made.3–4) Since L-erythrulose is not available red ˆlter. readily from commercial sources, it is important to investigate the fermentation proˆle of L-erythrulose Preparation of L-erythrulose. Areactionmixture production, identiˆcation of the enzyme responsible obtained from incubation of meso-erythritol with for meso-erythritol oxidation, and puriˆcation and resting cells of thermotolerant Gluconobacter CHM characterization of the responsible enzyme, provid- 43 at 379C for 6 h was centrifuged at 9,000×g for ing basic information for L-erythrulose production. 10 min to remove the cells. The resultant supernatant As has already been reviewed,1) oxidative fermenta- was freeze-dried overnight and mixed with 1z tion is only catalyzed by the membrane-bound trichloroacetic acid to removed remaining protein in dehydrogenases localized on the outer surface of the the sample. After a brief centrifugation, the solution cytoplasmic membranes of acetic acid bacteria. With was put onto a Dowex-50 W column (2×80 cm) and respect to cytosolic NAD(P)-dependent meso- eluted with distilled water. Fractions were collected erythritol dehydrogenase (CMEDH), only one en- of every 100 drops and L-erythrulose was measured zyme has been reported as L-erythrulose reductase by the resorcinol test and phenol sulfuric method es- (EC 1.1.1.162) from beef liver and L-erythrulose sentially by the same method described previously.2) reduction to meso-erythritol predominated over The pooled L-erythrulose fraction was then freeze-d- meso-erythritol oxidation.5) It is also reported that ried overnight. Purity of L-erythrulose was measured NADPH is more reactive than NADH in L-erythru- by thin layer chromatography using a silica gel 60 lose reduction with the mammalian enzyme. plate (Merck, Darmstadt, Germany) with a solvent In this paper, Gluconobacter frateurii CHM 43 system of n-butanol:ethanol:distilled water (4:1:1, was screened among thermotolerant Gluconobacter vWv). A solution of 0.5z triphenyl tetrazolium chol- and mesophilic strains as high L-erythrulose producer ride was used as the coloring agent. from meso-erythritol when grown at 379C. Enzymes catalyzing meso-erythritol oxidation from the mem- Preparation of membrane and cytosolic fraction. brane fraction of NAD-independent and from the G. frateurii CHM 43 was used throughout this study. cytosolic fraction of NAD-dependent meso-erythritol The culture conditions were the same as described in dehydrogenases of the organism were puriˆed to our previous paper used for L-ribulose production by homogeneity. The puriˆed meso-erythritol dehydro- acetic acid bacteria.2) The bacterial strain was cul- genase from the membrane fraction was identiˆed as tured in 30 l of the medium in a 50-l jar fermentor. a quinoprotein, which is the enzyme responsible for The cultivation was done at 309C for 20 h under the oxidative fermentation, but the NAD-dependent vigorous aeration. Bacterial cells were harvested with meso-erythritol dehydrogenase was independent of a Sharples centrifuge (Carl Padberg, model GLE, the oxidative fermentation. Further characterization Germany) and suspended in 2 mM Tris-HCl (pH 7.5) of the two enzymes in catalytic and physicochemical containing 10 mMD-sorbitol. The bacterial cell sus- properties are also conducted. pension chilled in ice-cold water was passed twice through a French pressure cell press (SIM Aminco, Materials and Methods Spectronic Instruments, Inc., Rochester, NY, USA) at 16,000 lbWin2. To remove intact cells and cell Chemicals. NAD, NADP, NADH, NADPH, and debris, the suspension was centrifuged at 9,000×g at yeast extract were kind gifts from Oriental Yeast Co., 49C for 15 min. Separation of the membrane fraction Tokyo. Mydol 10 was a kind gift from Kao Co., from the cytosolic fraction was done by ultracen- Tokyo. Other chemicals used were from commercial trifugation (Hitachi model 55P-72) at 150,000×g at sources of guaranteed grade unless otherwise stated. 49C for 60 min. For the EDTA (ethylenediamine- N,N,N?,N?-tetraacetic acid, disodium salt) treatment Microorganisms and culture conditions. Meso- experiment, the membrane fraction was homo- philic Gluconobacter strains used in this study were genized with 20 mM Tris-HCl (pH 7.5) while 10 mM obtained from the Institute for Fermentation, Osaka, potassium phosphate buŠer (pH 6.0) was used for (IFO). Thermotolerant Gluconobacter strains were enzyme solubilization. isolated from Thailand and characterized as has been reported previously.6) Culture medium used for EDTA treatment and reactivation of the enzyme screening for L-erythrulose-producing strains was activity. The membrane solution of which the protein composed of 1z meso-erythritol, 0.1z yeast ex- concentration was adjusted to 10 mgWml was incu- tract, and 0.1z