Agric. Biol. Chem., 55 (3), 837-844, 1991 837 Polyethylene Glycol Dehydrogenating Activity Demonstrated by Dye-linked Alcohol Dehydrogenase of Rhodopseudomonas acidophila M402 Kei Yamanaka Department of Applied Chemistry, Faculty of Engineering, OkayamaUniversity of Science, Ridai-cho, Okayama 700, Japan Received October 18, 1990 Ethylene glycols and polyethylene glycols inhibited the growth of a photosynthetic bacterium, Rhodopseudomonasacidophila, strain M402 under aerobic-dark and anaerobic-light culture conditions. However, polyethylene glycol dehydrogenating activity was demonstrated with phenazine methosulfate (PMS) as an electron acceptor in parallel with the PMS-linked aromatic alcohol dehydrogenase activity. Addition of ethylene glycols or polyethylene glycols to the culture neither induced aromatic alcohol dehydrogenase, nor accelerated polyethylene glycol dehydrogenating activity. Purified PMS-linked aromatic alcohol dehydrogenase had high affinities toward diethylene glycol and polyethylene glycols. This indicated that the dye-linked aromatic alcohol dehydrogenase is capable of oxidizing these xenobiotic synthetic polymer alcohols, polyethylene glycols. There is no evidence on the existence of a specific polyethylene glycol dehydrogenase in this bacterium. Broad substrate specificity is a well-known vanillyl alcohol in R. acidophila under property of alcohol and aldehyde dehydroge- aerobic-dark conditions can be postulated as nases, either dye-linked or NAD+-dependent, follows: of methylotrophs and some non-methylo- Vanillyl alcohol->vanillin-åºvanillic acid trophic bacteria.1~7) Their broad substrate specificities, however, vary depending on the The first oxidation step of vanillyl alcohol organism. The range of width of specificity was to vanillin is catalyzed by the dye-linked usually decided by the researchers' interests, vanillyl alcohol dehydrogenase, and vanillin is mainly in compounds structurally related to further dehydrogenated by the dye-linked the main substrate molecule. For instance, aldehyde dehydrogenase. Both of the purified activity of primary alcohol (methanol) dehy- dehydrogenases, interestingly, showed very drogenase was assayed on methanol.1-6) broad substrate specificities. Aromatic alcohol Therefore, a series of primary alcohols was dehydrogenase showed higher activity on selected as possible substrates. In a few cases, aliphatic alcohols than on aromatic alcohols, secondary alcohols were included.6) Wefound though it was obtained from the vanillyl a new type of dye-linked aromatic alcohol alcohol-grown cells. Aldehyde dehydrogenase dehydrogenase in the vanillyl alcohol-induced obtained from the cells induced by benzyl cells of Rhodopseudomonasacidophila, strain alcohol also showed higher activity on a series M402, under aerobic-dark conditions.8) Puri- of straight chain aldehydes than on aromatic fied enzyme was active for oxidation of vanillyl aldehydes. It is of particular interest since it is alcohol, but inert on vanillin. In connection expected to broaden the substrate specificity. with this enzyme, dye-linked aldehyde-specific Kawai et al have done an extensive study dehydrogenase was also demonstrated in the on the biodegradation of polyethylene glycol same culture.9) The metabolic pathway of (PEG)* by microorganisms.1(M1) They postu- * Abbreviations: EG, ethylene glycol; TEG, tetraethylene glycol; PEG, polyethylene glycol; PMS, phenazine methosulfate. 838 K. Yamanaka lated the oxidative metabolic pathway of PEG periment. The basal mediumand cultivation of bacteria by symbiotic mixed culture as follows: were described in a previous paper.8) For growth on polyethylene glycol was first oxidized to ethylene glycols and PEG, bacteria were inoculated in the basal medium (5ml) with ethylene glycol or PEG as sole polyethylene glycol aldehyde, which thereafter carbon sources at 3g/1 in test tubes, and were cultured received further oxidation to carboxylate.10) aerobically (shaking) or anaerobically, illuminated with Two reactions were catalyzed by respective tungsten lamps. In parallel experiments, bacteria were also dehydrogenases which were named PEG cultured on the PEGmedia supplemented with DL-malate dehydrogenase and PEG-aldehyde dehydro- (0.3%) as a dissimilative carbon source. Changes of ll,12) absorbance at 650 nmwere measuredand expressed as an genase. indication of growth. To detect PEG-dehydrogenating This metabolic pathway closely resembled activity during the culture, two media were prepared. As those of primary alcohol oxidation by carbon sources, malate (0.3%) and vanillyl alcohol (5 mM) methylotrophs13) and of vanillyl alcohol by were added to one medium. Tetraethylene glycol was replaced with vanillyl alcohol in the other medium. Rhodopseudomonas acidophila, strain M402.8) Cultures (100ml medium in a 500-ml Erlenmeyer flask) Furthermore, purified so-called PEGdehydro- were done under aerobic-dark conditions for 180 hr on the genase also showed a very wide substrate vanillyl alcohol medium, and for 240hr on the TEG specificity including various PEGs, straight medium. Small portions of culture was taken every 24hr, chain alcohols, and aromatic alcohols, and and dehydrogenase activities on vanillyl alcohol and TEG some straight chain aldehydes.14) This wide were assayed. specificity is very similar to the PMS-linked Enzymeactivity. Dye-linked dehydrogenase activities aromatic alcohol dehydrogenase in R. acidoph- were assayed as described in a previous paper.8) Aromatic ila. These studies were done rather in- alcohol dehydrogenase activity was measured on vanillyl dependently by each of us. However, this strong alcohol as substrate and polyethylene glycol dehydroge- resemblance encouraged us to work again on nating activity on TEG as substrate. For both cases, the substrate specificity of our aromatic alcohol PMSwas used as an artificial electron acceptor and ac- dehydrogenase, because we hadn't paid any tivities were assayed at pH 7.5. attention to the structural relation between Protein measurement.Protein was measured by the aromatic alcohols and polyethylene glycols. method ofBradford as described in a previous paper8) with Moreover, the metabolic possibilities of bovine serum albumin as the standard. polyethylene glycols in photosynthetic bac- Electrophoresis. Gel electrophoreses on polyacrylamide terium, Rhodopseudomonas acidophila, strain were done by the procedure described previously.8) Active M402 were not clear. bands were stained by activity staining. In this paper, we describe the induction of PEGdehydrogenating activity accompanying Purification. Purified preparations of aromatic alcohol with aromatic alcohol dehydrogenase. Finally, dehydrogenase were prepared as described in previous dehydrogenating activities on all of ethylene papers.8) Cells (185 g in wet weight) were collected from 70 liter's culture. After sonic treatment (20 kHz), cell debris glycols and PEGs were confirmed to be was removed by centrifugation at 10,000 x g for 20min. catalyzed by the aromatic alcohol dehydroge- Theresultant cell-free extracts weretreated by ammonium nase of R. acidophila, strain M402, not by the sulfate fractionation. Precipitate from 30 to 80%saturation was collected by centrifugation. After being dissolved in putative specific PEGdehydrogenase. Tris buffer, pH 7.5, it was put onto a column of DEAE-cellulose (4.8 x 22cm) at pH 7.5. Passed fractions Materials and Methods were collected and put onto a second DEAE-cellulose column (3.5 x36cm) at pH 8.0. The enzyme was eluted Materials. Ethylene glycols and polyethylene glycols with 30mMbuffer, pH 8.0 at a flow rate of40ml/hr. Active were kindly donated by Dr. F. Kawai, Kobe University fractions were collected and passed to a column of Bio-Gel of Commerce.Other chemicals were purchased from a HPT (3.0x37cm). This preparation was free from commercial source. aldehyde dehydrogenase and homogenous by poly- acrylamide gel electrophoresis. Asingle band was detected Microorganism used and cultivation. Rhodopseudomonas at an Rmof0.22 by protein staining, and 0.23 by activity acidophila, strain M402was used throughout this ex- staining with vanillyl alcohol as substrate. PEG Dehydrogenating Activity by PMS-Alcohol Dehydrogenase 839 Table I. Bacterial Growth on Ethylene Results Glycols, Polyethylene Glycols and Alcohols Effects of polyethylene glycols on bacterial growth Bacterial growth (A6S0) in All of the ethylene glycols and PEGs of 400 Carbon source to 20000 were unable to support the bacterial (0.3%) Aerobic-dark Anaerobic-light growth as a sole carbon and energy source + Malate + Malate under aerobic-dark and anaerobic-light condi- (0.3%) (0.3%) tions (Table I). Addition ofEG or PEG (0.3%) Malate, no PEG 0.25 1.16 to malate (0.3%) medium inhibited the Ethylene glycol 0.07 0.29 0.31 0.54 bacterial growth to some extent under both Diethylene glycol 0.06 0.30 0.21 0.96 culture conditions. In contrast, addition of Triethylene glycol 0.06 0.30 0.23 0.59 Tetraethylene glycol 0.06 0.20 0.22 1.26 benzyl alcohol, vanillyl alcohol, ethanol, or PEG 400 0.05 0.09 0.19 0.66 1 -butanol to malate mediumclearly accelerated PEG 600 0.05 0.08 0.29 0.67 the bacterial growth under both culture PEG 1000 0.06 0.12 0.29 0.67 PEG 2000 0.15 0.19 0.30 1.33 conditions. This indicated that all of EGs and PEG 4000 0.06 0.22 0.21 0.90 PEGs, regardless of their molecular size, were PEG 6000 0.10 0.29 0.36 1.42 not dissimilated by this bacterium under PEG 20000 0.10 0.31 0.34 1.10 aerobic-dark and anaerobic-light conditions. Benzyl alcohol* 0.06 0.24