Polyethylene Glycol Dehydrogenating Activity Demonstrated By

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 polyacrylamide 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 0.35 0.88 Vanillyl alcohol* 0.15 0.77 0.33 0.92 Detection of PEGdehydrogenating activities Ethanol* 0.76 0.76 1.32 2.56 Crude extracts prepared from the aerobically 1-Propanol* 0.35 0.53 1.05 1.52 grown cells on the malate-vanillyl alcohol 1-Butanol* 0.51 0.73 1.40 1.76 mediumwere examinedto detect the dehydro- Cultures were done for 96 hr. All experiments were done genating activity on polyethylene glycols in the in duplicate. Values wereexpressed as averages of two runs. presence of phenazine methosulfate and * 5mM. 2,6-dichlorophenol indophenol as artificial electron acceptors. The results are shown in Table II. Activity on Ethylene Glycols and Table II. Surprisingly, the crude extracts clearly Polyethylene Glycols (Crude Extracts) had dehydrogenating activities on a series of Substrate Specific activity ethylene glycols and polyethylene glycols. (5 him) (units/mg of protein) x 1000 Tetraethylene glycol (TEG) was selected as the Ethylene glycol substrate for the PEG-dehydrogenating activ- Diethylene glycol 32 ity. Now, it is a great question whether a specific Triethylene glycol 217 PEGdehydrogenase would be produced as a Tetraethylene glycol 265 PEG 400 214 separate enzymeunderthese culture conditions PEG 600 216 or induced as an accompanying activity PEG 1000 191 together with the alcohol dehydrogenase. If this PEG 2000 119 so-called PEG dehydrogenase did exist and was PEG 4000 146 inducible, addition of EGs or PEGs should PEG 6000 170 188 affect the induction of the two dehydrogenase PEG 20000 activities. As summarized in the Table III, Specific activity on vanillyl alcohol was 245. addition ofEGs or PEGs to the malate medium did not inhibit the induction of alcohol dehydrogenase and TEG dehydrogenating alcohol to the basal medium greatly enhanced activities. Addition of PEGdid not demon- the production of aromatic alcohol dehydroge- strate the PEG-specific dehydrogenating activ- nase as noted previously.8) At the same time, ity. In contrast to PEG, addition of vanillyl dehydrogenating activities on TEGand PEG 840 K. Yamanaka

Table III. Induction of PMS-Linked Alcohol Dehydrogenase under Aerobic-Dark Conditions Specific activity (u/protein) x 1000 Carbon source Bacterial Protein (0.3%) growth in extracts (^650nm) (mg) AAD* Butanol TEG*

Acetate 1.04 15.3 nd* nd nd nd nd Butyrate 1.92 21.4 nd nd nd nd nd Malate 0.27 4.8 170 1720 190 200 140 Vanillyl alcohol 0.15 6.3 50 50 60 30 Malate + VAL 0.60 8.7 250 1400 300 270 170 Malate + butanol 1.23 18.1 nd nd nd nd nd

Malate + EG 0.44 8.5 130 1160 150 150 70 Malate + diEG 0.37 5.0 260 2000 250 260 140 Malate + triEG 0.29 6.3 160 950 140 120 60 Malate + TEG 0.21 1.6 590 3400 590 550 260 Malate+PEG 400 0.15 2.4 140 620 160 190 70 Malate+PEG 600 0.10 1.5 110 600 130 150 60 Malate+PEG 1000 0.20 2.7 180 770 160 190 90 Malate+PEG 2000 0.26 5.4 150 760 170 160 70 Malate+ PEG 4000 0.25 6.4 180 820 190 160 80 Malate+ PEG 6000 0.22 3.7 330 1820 290 280 190 Malate + PEG 20000 0.24 4.3 230 1530 220 230 150

Note: nd, not detected; VAL; vanillyl alcohol; AAD, m-anisaldehyde; EG, ethylene glycol; TEG, tetraethylene glycol.

Table IV. Purification of Aromatic Alcohol Dehydrogenase Aromatic alcohol dehydrogenase Protein Purification step ( mg) . Specific Yield Total units activity. . Fold ....(%)

Crude extract 9,390 0.10 1.0 100 Ammoniumsulfate 9,080 454.2 0.05 0.5 58.3 precipitate (30-80%) DEAE-cellulose (pH 7.5) 4,580 644.7 0.14 1.5 82.8 DEAE-cellulose (pH 8.0) 276 466.2 1.69 17.6 59.9 Bio-Gel HTP 69 422.0 6.12 63.8 54.2

6000 were also accelerated. Ratios of dehydro- the dye-linked aromatic alcohol dehydrogenase genating activities on vanillyl alcohol, 1- itself has high PEG-dehydrogenating activity. butanol, TEG, and PEG 6000 were nearly Analysis of activity staining on polyacrylamide constant as 1:1:1:0.5 in every extract, gel electrophoresis also coincided with this regardless of the addition of PEGs to the observation. A single band having the identical culture medium. However, acetate, butyrate, Rm(0.23-0.24) was always detected by activity and butanol were excellent carbon sources to staining with vanillyl alcohol, butanol, TEG, promote the bacterial growth, but no dehydro- and PEG6000 as substrate. Therefore, genase activities were detected. There is no it can be concluded that the putative PEG- evidence for the existence of a specific PEG specific dehydrogenase was not demonstrated dehydrogenase in these bacterial extracts, but in these extracts. 778.5 PEG Dehydrogenating Activity by PMS-Alcohol Dehydrogenase 841 Induction of PEGdehydrogenating activity aromatic alcohol dehydrogenase was prepared Dye-linked aromatic alcohol dehydroge- by the procedures previously described, and nase was produced in the vanillyl alcohol- summarized in Table IV. Purity of the final malate medium as already reported, and preparation wasconfirmedby appearanceof a reached the maximumat 70 hr of culture. After single band of protein on polyacrylamide gel vanillyl alcohol was consumed, aromatic electrophoresis. Relative activities on various alcohol dehydrogenase activity rapidly de- ethylene glycols and polyethylene glycols were creased. TEGdehydrogenating activity was assayed with the purified enzyme preparation also detected at every interval of the culture, and summarizedin Table V. It was clear that and also reached the maximumafter 70hr of the purified aromatic alcohol dehydrogenase culture. The ratio of the two activities remained constant during the whole culture period as illustrated in Fig. 1. Whenculture was done on the TEG-malate medium, bacterial growth was inhibited to some extent, but the two dehydrogenating activities also appeared, delayed but reaching a maximum after 200 hr of culture as shown in Fig. 2. In this case, the ratio of the two activities was also constant during the whole culture period. In consequence, addition of TEG to the medium did not enhance the induction of TEG-dehydrogenating activity and alcohol dehydrogenase activity. Fig. 1. Production of Dye-linked Aromatic Alcohol and Dehydrogenating activity on EGs and PEGs Tetraethylene Glycol Dehydrogenating Activities on the The substrate specificity of dye-linked Vanillyl Alcohol-Malate Medium. Cultures were done under aerobic-dark conditions. £, aromatic alcohol dehydrogenase has been vanillyl alcohol dehydrogenase (units); O, tetraethylene already reported in a previous paper,8) but glycol dehydrogenating activity; A, bacterial growth activities on ethylene glycols and polyethylene (^650nm)j D» ratio of two activities (vainllyl alcohol glycols had not been measured. Purified dehydrogenase/TEG dehydrogenating activity).

Fig. 2. Production of Dehydrogenating Activities on the Tetraethylene Glycol-Malate Medium. Cultures were done under aerobic-dark conditions. #, vanillyl alcohol dehydrogenase (units); O, tetraethylene glycol dehydrogenating activity; A, bacterial growth (>465Onm); å¡> rati° of two activities (vanillyl alcohol dehydrogenase/TEG dehydrogenating activity). 842 K. Yamanaka Table V. Substrate Specificity of Aromatic PEG-dehydrogenases reported by Kawai et al. Alcohol Dehydrogenase were inducible.10~12) For instance, on the Substrate Relative activity synergistic, mixed culture of two PEG-using (5 HIM) (%) bacteria, Flavobacterium sp. and Pseudomonas sp., addition of PEG 300-20000 was necessary Vanillyl alcohol 100.0 Ethylene glycol 0.0 for the bacterial growth on TEG-mediumand Diethylene glycol 7.3 for the specific PEGdehydrogenase produc- Triethylene glycol 44.3 tion.12) Unexpectedly, PEG-induced purified Tetraethylene glycol 75.0 PEG-dehydrogenases showed wide substrate PEG 400 55.0 PEG 1000 56.6 specificity toward not only PEGmolecules, but PEG 4000 32.7 also primary aliphatic alcohols of C3-C12, the PEG 6000 32.7 corresponding aldehydes C3-C7, and aromatic PEG 20000 17.7 alcohols and aldehydes.1145'16) Rhodopseudomonasacidophila belongs to the Family of Rhodospirillacea, purple nonsulfur that was induced by vanillyl alcohol was very photosynthetic bacteria and Pseudomonasand active on these artificial synthetic polymer Flavobacterium are classified as non-photo- alcohols, ethylene glycols with different affini- trophic, and Gram-negative rod, respectively ties. The enzyme did not show any affinity for aerobic and facultatively anaerobic. Therefore, ethylene glycol, but was active on those from the microorganisms are taxonomically distinct diethylene glycol to PEG20000. The enzyme and different from each other in several was most active on tetraethylene glycol. characteristics, such as morphological and Activity gradually decreased with increasing physiological characteristics, and habitats. molecular weight of PEG. Beyond our They are capable of producing the dehydroge- expectation, it was greatly surprising that the nases having similar properties under different aromatic alcohol dehydrogenase had high culture conditions. However, resemblance of activity on PEG 20000 at 18% of the original two alcohol dehydrogenases between Rho- activity on vanillyl alcohol. dopseudomonas acidophila, strain M402and the polyethylene glycol degrading bacteria, Flavo- Discussion bacterium sp. and Pseudomonassp. was evident. Their substrate specificities will pose several In this paper, we described how the fascinating evolutionary and metabolic ques- polyethylene glycol dehydrogenating activity tions. Table VI shows the ubiquity of the originated from the aromatic alcohol dehydro- substrate specificity of both enzymes. Two genase. This activity was derived from the enzymeswere active on commonsubstrates, a surprising broadness of substrate specificity of series of straight chain and aromatic alcohols the alcohol dehydrogenase. Consequently, and aldehydes and PEGs, but profiles of the none of the polyethylene glycols tested can ratio of relative activities on these substrates serve as a carbon source for this bacterial including PEGs were not identical between growth and also as inducer for the enzyme both enzymes. Although, two enzymes are production. The enzyme can be obtained in the similar in manyaspects, they maydiffer in cells growtn on malate, succinate, or fumarate. several distinctive properties. For instance, Therefore, addition of aromatic or straight aromatic alcohol dehydrogenase is PMS- chain primary alcohols to the medium en- dependent, but PEG dehydrogenase is not. It hanced the production of this PEGdehydro- is coupled with DCPIP. Molecular properties, genating activity. PEGwas completely inert for such as molecular weight and subunit structure the production of this activity. In contrast to were distinctively different. These facts indicate the enzyme activity of R. acidophila, all that two enzymesare distinct proteins and two PEG Dehydrogenating Activity by PMS-Alcohol Dehydrogenase 843 Table VI. Comparison on Relative Activities the nomenclatural standpoint. of Aromatic Alcohol Dehydrogenase and Biodegradation of PEG in vitro has been PEGDehydrogenase toward Various Hydroxyl Compounds established by Kawai et al.,10~12) but another possibility for the biodegradation of PEG in Relative activity (%) vitro and in vivo may be postulated with the Dehydrogenase Aromatic facts in this paper. As seen with R. acidophilla, alcohol PEG dehydrogenase some strains of purple, non-sulfur photo- Microorganism dehydro- Purified11} Purified16) trophic bacteria would become possible de- Substrate final genase8)R. mixed Flavobac- composers of xenobiotic synthetic polymer concentration acidophila culture terium alcohols, polyethylene glycols in the natural (mM) 5 5 10 environmentwithout the expense on PEGfor Methanol 0 0 0 enzyme induction. It is difficult to draw a firm Ethanol 23 ( 25)* 1 ( 2) 0 conclusion from this Comparison. The true 1 -Propanol 115(126) 18(21) 72(32) explanation of the wide substrate specificity 91(100) 87(100) 227(100) 1 -Butanol must await the elucidation of the common 1 -Pentanol 53(58) 111(128) 267(118) 1-Hexanol 34(37) 115(132) 280(123) structure of both alcohol dehydrogenases. 1 -Heptanol 17(18) 114(131) - Genetic analysis of these enzymes would be 15( 16) 187( 82) 1 -Octanol 137( 60) most desirable for this purpose. 1 -Dodecanol 298 (130) 49( 54) Benzyl alcohol 40( 18) Vanillyl alcohol 100 (110) 119( 52) Acknowledgments. This work was supported by a Cinnamyl alcohol 40( 44) 0 Grant-in-Aid for the Combined Research Project Aon Formaldehyde 61( 67) 0 Biochemical Functions of PQQ from the Ministry of Acetaldehyde 8( 9) 6( Eduction, Science and Culture of Japan. I am greatly 60( 66) 32( indebted to Professor F. Kawai, Kobe University of Propionaldehyde 34( 15) 64( 70) 1 -Butyraldehyde 64( 28) Commercefor her cooperative comments and valuable 1 -Hexaldehyde 7( 3) discussions, and also for kind donation of polyethylene 1 -Heptaldehyde 4( 2) glycols. I amalso indebted to Messrs. T. Uchida and S. Benzaldehyde 14( 6) 0 3) Hashimoto for their cooperation during this study. Vanillin 41(45) /-Cinnamaldehyde 0 14) 7( 8) Ethylene glycol 44( 48) References 75( 82) Diethylene glycol 55( 60) 27( 12) 57( 63) G. T Triethylene glycol 0 27( 12) Sperl, H. S. Forrest and D. T. Gibson, /. 4( 5) 118( 52) Tetraethylene glycol 5( 6) BacterioL, 118, 541 (1974). 25( 29) 169( 75) PEG 400 49( 56) R. N. Patel andA. Felix, /. BacterioL, 128, 413 (1976). PEG 1000 26( 30) 131( 58) I. Goldberg, Eur. J. Biochem., 63, 233 (1976). PEG 4000 -20( 23) 100 ( 44) K. Yamanaka and K. Matsumoto, Agric. BioI. PEG 6000 33(36) 100(115) 95( 42) Chem., 41, 467 (1977). PEG 20000 18(20) 10( ll) 27( 12) R. N. Patel, C. T. Hou and A. Felix, /. BacterioL, 136, 352 (1978). Values in parenthesis are recalculated which are 6) C. W. Bamforth and J. R. Quayle, Biochem. J., 169, based the activity on 1-butanol as 100. 677 (1978). 7) C. T. Hou, R. Patel, A. I. Laskin, N. Barnabe and bacteria might have evolved them quite I. Marczak, Appl. Environ. MicrobioL, 41, 829 (1981). 8) K. Yamanaka and Y. Tsuyuki, Agric. Biol. Chem., independently. In consequence, the wide 47, 2173 (1983). substrate specificity of both enzymesprompts 9) K. Yamanaka, H. lino and T. Oikawa, Agric. Biol. us to refer to these enzymes as dye-linked Chem., in press. 0) F. Kawai,T. Kimura, M. Fukaya,Y. Tani, K. Ogata, alcohol dehydrogenase, because they are more T. Ueno and H. Fukami, Appl. Environ. MicrobioL, active on alcohols and aldehydes than ethylene 35, 679 (1978). glycols and PEGs. These facts substantially 1) F. Kawai, T. Kimura, Y. Tani, H. Yamada and M. support the denial of the existence of a poly- Kurachi, Appl. Environ. MicrobioL, 40, 701 (1980). ethylene glycol-specific dehydrogenase from 2) F. Kawai and H. Yamanaka, Arch. MicrobioL, 146, 844 K. Yamanaka

125(1986). P. (1963).J. Large and J. R. Quayle, Biochem. J., 87, 386 15) F. Kawai, H. Yamanaka, M. Ameyama, E. 16) Shinagawa, K. Matsushita and O. Adachi, Agric. Biol. Chem., 49, 1071 (1985). F.300(1989).Kawai and H. Yamanaka, /. Ferm. Bioeng., 67, H. 324(1989).Yamanaka and F. Kawai, /. Ferm. Bioeng., 67,