Agric. Biol Chem., 47 (1), 39-46, 1983 39

The Dismutation of Aldehydes by a Bacterial Nobuo Kato, Kamon Shirakawa, Hisataka Kobayashi and Chikahiro Sakazawa Departmentof Environmental Chemistry and Technology, Tottori University, Tottori 680, Japan Received June 17, 1982

Formaldehyde-resistant Pseudomonas putida F61 isolated from a soil sample showed high activities of dehydrogenase (EC 1.2.1.1) and a specific enzyme catalyzing the dismutation of formaldehyde to form and formate. The latter enzyme, given the trivial name of formaldehyde , was purified to electrophoretic homogeneity from a cell-free extract of P. putida F61. The enzymewas a tetramer with a molecular weight of approximately 2.2 x 105, and an isoelectric point of 4.8. The enzyme catalyzed the stoichiometric dismutation of formaldehyde and acetaldehyde to form a half mol of each of the corresponding alcohol and acid without addition of an electron acceptor, but it did not catalyze the dismutation of pro- pionaldehyde, butyraldehyde and so on. The apparent Kmfor formaldehyde was found to be 350mM.Oneof the most unique properties of the enzyme was the catalytic activity of cross- dismutation between two different aldehydes, such as formaldehyde/acetaldehyde, formaldehyde/ propionaldehyde, and so on.

Formaldehyde occurs ubiquitously in nature and characterization of this enzymewhich was in the process of microbial decomposition of given the trivial name of "formaldehyde organic compounds. It is known that several dismutase. " methylotrophs can utilize formaldehyde as a sole source of carbon and energy.1~4) On the other hand, some microorganisms have been MATERIALS AND METHODS reported to oxidize formaldehyde but not to Chemicals. DEAE-Sephacel and Phenyl-Sepharose were assimilate it.5>6) In the preceding paper,6) we purchased from Pharmacia Fine Chemicals, Uppsala, described that formaldehyde-resistant yeasts Sweden; Bio-Gel A-1.5m (200~400 mesh) was from Bio- oxidized formaldehyde, which was used as an Rad Laboratories, CA, U.S.A.; Hydroxyapatite was from energy source during their growth on a me- Seikagaku Kogyo Co. Ltd., Tokyo, Japan. Porapak type Q was a of Waters Associates. Inc., Mass., U.S.A. dium containing glucose as a carbon source. Lactate dehydrogenase was a product of Sigma Chemical Subsequently, we have examined formal- Co., St. Louis, U.S.A. Formaldehyde dehydrogenase8} was dehyde oxidation by resistant bacteria which a gift from the Department of Biochemical Engineering, can grow on medium with 0.2% formaldehyde. Toyobo Co. Ltd., Tsuruga, Japan. Formaldehyde was These bacteria exhibited extremely high form- prepared as described in a previous paper.6) The other activity7) and simul- chemicals were analytical-grade reagents and were ob- taneously the cell-free extracts exhibited sig- tained from Nakarai Chemicals, Kyoto, Japan. nificantly high formaldehyde disappearance Isolation of formaldehyde-resistant bacteria. Form- activity without addition of any electron ac- aldehyde-resistant bacteria were isolated from soil ceptors. In this work, we demonstrated that samples as described previously.6) the latter activity was the result of the form- aldehyde dismutation reaction catalyzed by Organism and cultivation. A formaldehyde-resistant bac- terium, strain F61, was isolated from a soil sample, and a specific enzyme, to form methanol and for- was identified as Pseudomonas putida by the typing service mate, and here we describe the purification for identification of the National Collection of Industrial 40 N. Kato et al.

Bacteria, Aberdeen, Scotland. Cultivation was carried out for 30min. in a nutrient medium containing 10g peptone, 5g (ii) Protamine treatment. To the supernatant 25 g pro- beef extract, 1g K2HPO4, 5g NaCl and 1g formalde- tamine sulfate dissolved in 500ml water was added drop- hyde (per liter), pH 7.0. P. putida F61 grown at 30°C for wise with mechanical stirring. After 30 min of stirring the 24hr in a test tube containing 5ml of the mediumwas solution was centrifuged (83,000xg, 30 min) and the inoculated into a 2-liter flask containing 1 liter of the precipitate was discarded. medium and incubated with shaking at 30°C for 48hr. (iii) Ammoniumsulfate fractionation. The supernatant The harvested cells were washed twice with 10 mMpotas- solution (1500ml) was brought to 60%saturation with sium phosphate buffer (pH 7.0). ammoniumsulfate at pH 7.0, and the resulting precipitate was removedby centrifugation. Ammoniumsulfate was Enzymeassay. Formaldehyde dismutase activity was added to the supernatant solution up to 90%saturation. assayed with a standard reaction mixture containing The precipitate collected by centrifugation was dissolved 20mMformaldehyde, 100him KC1 and enzyme in a final in 200ml of 10mMpotassium phosphate buffer (pH 7.0). volume of 10 ml. The reaction was carried out at 30°C with (iv) Phenyl-Sepharose CL-4B column chromatography. stirring under N2 gas and the formation of acid (formic To the enzyme solution (220ml) was added solid NaCl to a acid) was measured by pH-stat titration with 10mM concentration of 4m, and the mixture wasapplied to a NaOHat pH 7.0. Automatic titration was carried out with Phenyl-Sepharose column (4 by 50 cm) equilibrated with a TOApH-stat model HSM-10A. One unit ofenzyme was 10 mMpotassium phosphate buffer (pH 7.0) containing 4 m defined as the amount of enzyme that catalyzed the NaCl. After washing the columnwith the equilibrating formation of 1 /zmol of formic acid per min. buffer, elution was carried out with a gradient of decreas- Formaldehyde disappearance activity was assayed with ing NaCl concentration and increasing ethylene glycol a reaction mixture containing 50 mMpotassium phosphate concentration (their final concentrations were 0 and 50%, buffer, pH 7.5, 5mMformaldehyde and enzyme in a total respectively; total volume, 6 liters) at a flow rate of 19cm/ volumeof 1 ml. The mixture was incubated at 30°C for 20 hr. The active fractions were collected and dialyzed against min. After termination of the reaction by addition of 10 mMpotassium phosphate buffer (pH 7.0). 0.2ml of 4 n HC1, the remaining formaldehyde was deter- (v) DEAE-Sephacel column chromatography. The dia- mined by Nash's method.9) lyzed solution was concentrated to 100ml by ultra fil- Formaldehyde dehydrogenase (EC -1.2. 1. 1) activity was tration with a Minimodule (Asahikasei Co. Ltd., Tokyo, measured as described previously.10) Dehydrogenation Japan), and applied to a DEAE-Sephacel column (2.5 by reactions linked with several electron acceptors were car- 45 cm) buffered with 10mMpotassium phosphate buffer ried out according to Colby and Zatman.n) (pH 7.0). The column was washed once with 2.5 liters of the equilibrating buffer, and then with 2 liters of the buffer Other methods. Protein was estimated by the Bio-Rad containing 100mMNaCl. The enzyme was eluted with a Protein Assay (Bio-Rad Laboratories) with bovine serum linear gradient of increasing NaClconcentration between albumin as the standard. The molecular weights of the 100 and 300mM(total volume, 2.5 liters) at a flow rate of native enzyme and its subunit were determined by gel lO cm/hr. The active fractions were collected and dialyzed filtration and SDS-disc gel electrophoresis, respectively, as against two changes of 100 volumes of 10mMpotassium described previously. 12) Isoelectric focusing was performed phosphate buffer (pH 7.0). with an LKB1 10ml column, which was set up according (vi) Hydroxyapatite column chromatography. The en- to the method of Vesterberg13); 46hr, at 4°C, maximum zyme solution (50ml) was placed on a hydroxyapatite load of about 1 W (2mA, 500V), using 0.77% Ampholyte column (2.5 by 21 cm) preequilibrated with the above- (LKB), pH 3~ 10. The formaldehyde solution was stan- mentioned buffer. Elution was carried out by stepwisely dardized with formaldehyde dehydrogenase.8) Aldehydes increasing the concentration of potassium phosphate buf- were determined spectrophotometrically with 3-methyl-2- fer to 10, 50, 100 and 200mM,at a flow rate of8 cm/hr. benzotiazolone hydrazone-hydrochloride.14) Formic acid The enzyme activity appeared in the fractions eluted with was assayed enzymatically as described previously.15) 200mMpotassium phosphate buffer (pH 7.0). These frac- Alcohols, aldehydes and acids were also determined by tions were collected, dialyzed against two changes of 100 gas chromatography (Shimadzu GC-8A) with a column volumes of 10mMpotassium phosphate buffer (pH 7.0), containing Porapak Q according to Baker et al.i6) and concentrated by ultra filtration with a Minimodule. The purified enzymesolution was stored at 5°C. Purification of formaldehyde dismutase. All the pro- cedures were carried out at 0~5°C. (i) Enzyme extraction. The bacterial cells (200g wet RESULTS weight) from 28 liters of culture were suspended in 10mM potassium phosphate buffer, pH 7.0 (1 liter), and then Growth and enzyme activity of isolated bacteria disrupted with an ultrasonic oscillator (19kHz, 30 min). Pseudomonasputida F61 could grow on the Cell debris was removed by centrifugation at 16,000 x g nutrient mediumcontaining up to 0.2% form- Dismutation of Aldehydes 41 aldehyde, but not on the mediumcontaining VM15A, P. putida (IFO 12653), P. aureo- formaldehyde and mineral salts as described faciens (IFO 3521), P. dimuta (IFO 12697), previously.6) Figure 1 shows the growth of P. P.flnorescens (IFO 3507), P. fragi (IFO 3458) putida F61 in the nutrient mediumwith or or P. stutzeri (IFO 12695), showed the form- without 0. 1 % of formaldehyde, the decrease of aldehyde dismutase activity, or could grow formaldehyde in the culture broth, and activ- on the medium containing 0.1% formal- ities of the formaldehyde dismutase and form- dehyde. On the other hand, 12 strains isolated aldehyde dehydrogenase in the growing cells. from different soil samples as formaldehyde- The latter enzyme was formed inducibly by the resistant bacteria exhibited high activities of addition of formaldehyde, whereas the form- formaldehyde dehydrogenase (10 to 20 U/mg aldehyde dismutase was formed constitu- of protein) and the formaldehyde dismutase (1 tively. Noneof the type strains of pseudomo- to 3 U/mg). nads examined, such as Pseudomonas putida Purification offormaldehyde dismutase Through the purification procedures de- _10 - >--* scribed in Materials and Methods, the formaldehyde dismutase was purified 75.4-fold from the cell-free extract (Table I). This en-

?~3 6 å \ /^y^^^D^r*^^* zymewas completely separated from formal- å o5"5 '/T dehyde dehydrogenase by DEAE-Sephacel chromatography. The purified enzyme gave 2^-<^ / / one single band on SDS-polyacrylamide gel electrophoresis. U_CD UJ / rf*\

0 tc_-å ^-a-'^^Q

Table I. Purification of the Formaldehyde Dismutase from Pseudomonas putida ¥61

Total Total Specific y... Step protein activity activity Purification (0. (mg) (U) (U/mg). (/o)

Cell-free extract 25, 100 64,300 2.56 1.0 100 Protamine sulfate 24,000 62,400 2.60 1.0 97.0 Al^°n^ Sulfate. , 1,650 16,500 10.0 3.9 25.7 Phenyl-Sepharose(60CL-4B~95%200 1 1saturation),400 57.0 22.3 17.7 DEAE-Sephacel 99 8, 150 82.3 32. 1 12.7 Hydroxyapatite 1 8 3,470 193 75.4 5.4 42 N. Kato et al.

0 I-i 1 ' « 1 1 1 1 1 1- 250 300 350 Wavelength (nm) Fig. 2. Absorption Spectrum of the Purified Formaldehyde Dismutase.

A, 0.72mg enzyme/ml; B,400 7.2mg enzyme/ml.

Table II. Stoichiometry of Formaldehyde Dismutation The reaction mixture contained 1 mmol of KC1, 200 /miol of formaldehyde (standardized as described in Materials and Methods) and 20 units of the formaldehyde dismutase in a final volume of 10ml, and was incubated at 30°C for 30 min with pH-stat (pH 7.0) titration with 100mMNaOH. After termination of the reaction by the addition of 1 ml of 4n HC1, formaldehyde and products were determined as indicated in the table.

Assay method /miol Formaldehyde disappearance Nash's 205 Methanol produced Gas chromatography 1 10 Formic acid produced Formate dehydrogenase 98 NaOHtitration 103

Isoelectric point 2HCH0 + H2O -> CH3OH + HCOOH. The purified enzyme (2 mg) was subjected to electrofocusing. Only one sharp peak of pro- 02 requirement and dehydrogenation activity of tein showing the activity was found with a the enzyme maximumat pH 4.8. The enzyme activity was followed by the formaldehyde disappearance under several sets Stoichiometry of the reaction of reaction conditions. No formaldehyde dis- The reaction mixture, containing formal- appearance was observed in the reaction mix- dehyde as a , was incubated at 30°C, ture when the heat-denatured enzyme >yas and the reaction was followed by pH-stat used. The reaction velocity under N2-gas was titration with lOOniM NaOH. As shown in identical with that in air, and the enzyme was Table II, one mol each of methanol and for- not influenced by the addition of 1 mMKCN. mic acid was formed from 2mol of formalde- These results indicated that the reaction was hyde, indicating that the enzyme catalyzed independent of the presence of . the coupled oxidation and reduction reactions Formaldehyde dehydrogenase activity of of formaldehyde stoichiometrically as fol- the enzymewas investigated using several elec- lows; tron acceptors, such as NAD+, NADP+, acetylpyridine-NAD +, phenazine methosul- Dismutation of Aldehydes 43 fate/2,6-dichlorophenolindophenol and potas- sium ferricyanide. Noreduction of any elec- 15 - tron acceptors occurred during the formalde- hyde disappearance, and the dismutase ac- tivity was not influenced by the addition of these electron acceptors. Dehydrogenation of E10- / \ methanol and ethanol linked with these elec- tron acceptors wasnot observed. E f \ Z2. / T General properties of the enzyme Whenthe enzyme was incubated in lOmM u / I potassium phosphate buffer (pH 7.0) at vari- < / ous temperatures for lOmin, the remaining activity was 100%of the original activity at 25°C, 79% at 30°C, 49% at 35°C, 18% at45°C 0I Ià"' ' I 1 I V and none at 50°C. The maximumactivity was 4 5 6 7 8 9 10 found at 40°C. The enzyme was found to be pH relatively stable at pH 6.0 to 7.0. The pH Fig. 3. Effect of pH on the Formaldehyde Dismutase dependence of the enzymeactivity was deter- Activity. mined by pH-stat titration at various pHs. The Enzymeactivity was determined by pH-stat titration of enzyme was most active at pH 8.0 (Fig. 3). 10him NaOHin the standard reaction system at 30°C. The enzymewas very susceptible to sulfhy- 100 - dryl reagents; 0.01 niM HgCl2 (94% inhibition), A 1 mM/?-chloromercuribenzoate (29%) and 1 mMiodoacetate (51%), and was inhibited by M^ 0.1 I 1 metal ions; 0.1mM ZnSO4 (100%), 0.1mM 2 / B CuSO4 (95%) and 0.1mM FeCl3 (100%). The enzyme activity was not influenced by metal E / T 0.05" yr chelating agents, such as ethylenediaminetetra- acetate, o-phenanthroline and a,a'-dipyridyl, J 0 10 20 I s-1 at the concentration of LOmM. Ol . . 1

0 0.5 1.0 Substrate specificity S IHCHO1 (M) The ability of the enzyme to catalyze dismu- Fig. 4. Effect of Formaldehyde Concentration on the tation of various aldehydes was examined Formaldehyde Dismutase Activity (A) and the Double using the standard reaction mixture containing Reciprocal Plots (B). 20mMof each aldehyde. The enzyme was also Enzyme activity was assayed under the standard con- active toward acetaldehyde (5%) and methyl- ditions with the substrate concentration being varied. glyoxal (22%), the relative activity toward formaldehyde being 100%, but not toward substrate consumed. propionaldehyde, butyraldehyde, heptalde- hyde, glyceraldehyde, glycolaldehyde or ben- Effect of formaldehyde concentration on the zaldehyde. Half equimolar ethanol was form- activity ed from equimolar acetaldehyde consumed. As shown in Fig. 4-A, the enzyme was very Pyruvic acid could be detected as a product stable toward formaldehyde, which, in general, from methylglyoxal by the enzymatic assay binds nonspecifically with protein, and the with lactate dehydrogenase,17) but the amount Michaelis-Menten equation was almost com- ofpyruvic acid was not stoichiometric with the pletely applicable to concentrations over 1.0 m. 44 N. Kato et al.

Table III. Cross-dismutation between Two Different Aldehydes by the Formaldehyde Dismutase The reaction mixture contained 300/miol of KC1, the aldehydes indicated and 5 units of the enzyme in a final volume of 3ml, and was incubated at 30°C with pH-stat (pH 8.0) titration with 20mMNaOH. After 20 min incubation, the reaction mixture was directly injected into the gas chromatograph. Acid was determined from the titration volume of 10mMNaOH,and alcohol was determined by gas chromatography. The concentration of a product was corrected for the initial reaction volume.

Product (him) Aldehyde (him) Acid Alcohol

HCHO: 10 4.6 CH3OH: 4.5 HCHO: 10, CH3CHO: 10 8.0 C2H5OH: 7,9 HCHO: 10, C2H5CHO: 10 8.3 C3H7OH: 8.1 HCHO: 10, C2H5CHO: 5 7.3 C3H7OH: 4.7, CH3OH: 2.6 HCHO: 10, C3H7CHO: 10 5.4 C4H9OH: 2.1, CH3OH: 3.2 CH3COCHO: 10, C2H5CHO: 10 0.82 C3H7OH: 0.80 C2H5CHO: 10, C3H7CHO: 10 ND* ND

* Not detected.

[C2H5CHO] substrate were used at equimolar concen- o.i - y° trations, each second aldehyde consumed was almost completely converted to the corre- 2 y* j^°025M ? /à" %^^*- 0.050M sponding alcohol accompanied by the equi- molar oxidation of formaldehyde to formate. a. 0.05 - y/ J*^\^^^ In these cases, only a negligible amount of methanol was detected. When formaldehyde Jr ^ , and propionaldehyde were 2: 1 on a molar

10 20 basis, methanol was not formed until pro- IHCHO]"1 (M)"1 pionaldehyde had been almost completely con- Fig. 5. Double Reciprocal Plots for the Cross- sumed, and methanol, propanol and formate in a molar ratio of nearly 0.5:1:1.5 were dismutation Reactions. Initial velocity versus formaldehyde at various constant detected as the products. In the case ofcoupled levels of propionaldehyde. Enzyme activity was assayed by substrates of formaldehyde and butyral- determination of formate formed by pH-stat (pH 8.0) dehyde, methanol and butanol were simul- titration of 10mMNaOH. taneously produced in the early stage of the reaction. Neither alcohol nor acid was formed From the double reciprocal plots (Fig. 4-B), from propionaldehyde and butyraldehyde. the apparent Kmfor formaldehyde was found Methylglyoxal could act as a partner substrate to be 350mM.for propionaldehyde in the dismutation reaction. Cross-dismutation reaction Initial velocity studies on the cross- The ability of the enzyme to catalyze a dismutation reaction were performed with re- coupled oxidation and reduction reaction be- spect to formaldehyde at a fixed concentration tween two different aldehydes was examined of propionaldehyde. As shown in Fig. 5, the (Table III), the equation for the overall re- reaction velocity increased with increasing action is represented as follows; RCHO + concentration of propionaldehyde, but the ap- R'CHO + H2O -> RCOOH + RCH2OH. parent Kmfor formaldehyde was not altered Whenformaldehyde as one substrate and acet- by the addition of propionaldehyde. aldehyde or propionaldehyde as the second Dismutation of Aldehydes 45

DISCUSSION H R OH R-C^ +H2O > p (1) It is knownthat mammalianalcohol dehy- drogenase18~20) and lactate dehydrogen- \) H OH ase2i ,22j cataiyZe dismutation (disproportiona- tion) of formaldehyde and glyoxylate, respec- R OH OH tively, to form the corresponding alcohols and E-X + C åºB-XH2 + R-C. acids. In the dismutation reactions, each alde- HOH O hyde provides both an alcohol (hydrated (2) form) and aldehydic function. These dismuta- H R' H tions occur in the presence of a catalytic amount of NAD+, whose oxidoreduction E-XH2 + R'-C åºE-X+ C mediates the coupled reactions (oxidation or O H OH reduction of an aldehyde) in the enzyme- (3) bound form, E-NAD+ ;=± E-NADH + H+, but not in the free form, E + NAD+ Since formaldehyde is approximately 99.9% ^=^E + NADH + H+.19'22) The formalde- hydrated (1), it is more likely to be oxidized (2) hyde dismutation by the enzyme from P. than reduced (3). On the other hand, pro- putida is assumed to proceed by the same pionaldehyde which exists abundantly in the reaction mechanismas above, on the basis free aldehyde form is inadequate for oxidation of the equimolar formation of methanol and by the formaldehyde dismutase. In the cross- formate from formaldehyde. Acetaldehyde dismutation reaction, therefore, propional- and methylglyoxal which were hydrated to dehyde becomes a good substrate to be re- some extent were also active substrates for duced by the reduced mediator (E-XH2) re- the dismutation. However, dismutation by sulting from formaldehyde oxidation. It is thus the P. putida enzyme did not require any co- reasonable that the reaction rate of the enzyme factors. The purified enzyme showed a char- was significantly enhanced by the addition of acteristic absorbance at 320nm. It is not yet propionaldehyde. The affinity of the enzyme clear what kind of chromophore causes the ab- for formaldehyde was extremely low. In an sorption. However it may be that an un- aqueous solution, the free form of formal- known coenzyme was bound tightly to the dehyde is present in an amountof about one protein and was not released from the enzyme thousandth that of the hydrated form. If the by the purification procedure. free aldehyde form was the rate-limiting sub- Oneof the most unique catalytic properties strate in the dismutation, the apparent Kmfor of the P. putida enzyme is that cross- formaldehyde would become lower upon the dismutation occurred between two different addition of a non-hydrated aldehyde. How- aldehydes. In this case, one aldehyde able to be ever, the Km for formaldehyde was not af- hydrated was exclusively oxidized and the fected by the addition of propionaldehyde, resulting reduced enzyme complex acted to indicating that the affinity for formaldehyde is reduce another aldehyde to produce the cor- dependent on its hydrated form. responding alcohol. These coupled reactions As far as examined, the formaldehyde are represented by the following equations. dismutase was found exclusively in the Reactions (2) and (3) are mediated by the formaldehyde-resistant bacteria, indicating reduction and oxidation of 'coenzyme X' that the enzymeplays a certain physiological where E is the formaldehyde dismutase. role in the resistance. In our preceding paper,6) resistance of yeasts to formaldehyde was as- sumed to be mainly due to its oxidation cata- lyzed by formaldehyde dehydrogenase which 46 N. Kato et ah wasconstitutively formed in the yeasts. The 5) R. Numazawa,K. Watanabe and F. Fukimbara, bacterial resistance to formaldehyde is also /. Ferment. Technol, 52, 799, 805 (1974). 6) N. Kato, N. Miyawaki and C. Sakazawa, Agric. Biol. mainly explicable in terms of the oxidation by Chem., 46, 655 (1982). this dehydrogenase which was found to exhibit 7) N. Kato, N. Miyawaki and C. Sakazawa, Agric. extremely high activity in the medium contain- Biol. Chem., in press. ing formaldehyde. The extent of formalde- 8) M. Ando, T. Yoshimoto, S. Ogushi, K. Rikitake, S. Shibata and D. Tsuru, J. Biochem., 85, 1165 (1979). hyde-resistance of yeasts and bacteria is not 9) T. Nash, Biochem. J., 55, 416 (1953). always directly proportional to the specific 10) N. Kato, T. Tamaoki, Y. Tani and K. Ogata, Agric. activity of the enzyme in the cells. The form- Biol. Chem., 36, 2411 (1972). aldehyde dismutase is formed independent- ll) J. Colby and L. J. Zatman, Biochem. J., 143, 555 ly of the addition of formaldehyde to the me- (1974). dium, and its Kmfor formaldehyde exceeded 12) N. Kato, T. Higuchi, C. Sakazawa, T. Nishizawa, Y. Tani and H. Yamada, Biochim. Biophys. Ada, 715, the physiological concentration. Although 143 (1982). the enzyme might play some role in the resist- 13) O. Vesterberg, "Methods in Enzymology," Vol. 22, ance, this role is not explained by the enzyma- ed. by W. B. Jakoby, Academic Press, New York, tic properties elucidated in this work. 1971, pp. 389-412. 14) E. Sawicki, T. R. Hauser, T. W. Stanley and W. Elbert, Anal. Chem., 33, 93 (1961). Acknowledgments. We wish to thank Professor K. 15) N. Kato, M. Kano, Y. Tani and K. Ogata, Agric. Soda, Institute for Chemical Research, Kyoto University, Biol. Chem., 38, 111 (1974). and Mr. M. Shimao, Tottori University, for their valuable 16) R. N. Baker, A. L. Alenty andJ. F. Zack, Jr., J. suggestions during this work. This workwas supported in Chromatogr. ScL, 7, 312 (1969). part by a Grant-in-Aid for Scientific Research from the 17) R. Czok and W. Lamprecht, "Methods in Enzymatic Ministry of Education, Science and Culture of Japan. Analysis, 2nd Ed.," Vol. 3, ed. by H. U. Bergmeyer, Academic Press, New York, 1974, pp. 1446~ 1451. REFERENCES 18) R. H. Abeles and H. A. Lee, Jr., J. Biol. Chem., 235, 1499 (1960). 1) I. Goldberg, J. S. Rock, A. Ben-Bassat and R. I. 19) N. K. Gupta, Arch. Biochem. Biophys., 141, 632 Mateles, Biotechnol Bioeng., 18, 1657 (1976). (1970). 2) K. Sakaguchi, B. Kurane and M. Murata, Agric. 20) J. A. Hinson and R. A. Neal, J. Biol. Chem., 247, Biol. Chem., 39, 1695 (1975). 7106 (1972). 3) Y. Ohta and R. Sugimoto, Hakkokogaku Kaishi, 56, 21) C. Lluis and J. Bozal, Biochim. Biophys. Ada, 480, 258 (1978). 333 (1977). 4) W. Hirt, E. Papoutsakis, E. Krug, H. C. Lim and G. 22) R. J. Duncan, Arch. Biochem. Biophys., 201, 128 T. Tsao, Appl Env. MicrobioL, 36, 56 (1978). (1980).