The Dismutation of Aldehydes by a Bacterial Enzyme
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Agric. Biol Chem., 47 (1), 39-46, 1983 39 The Dismutation of Aldehydes by a Bacterial Enzyme 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 formaldehyde dehydrogenase (EC 1.2.1.1) and a specific enzyme catalyzing the dismutation of formaldehyde to form methanol and formate. The latter enzyme, given the trivial name of formaldehyde dismutase, 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 product 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 aldehyde dehydrogenase 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.