Production of aldehyde oxidases by microorganisms and their enzymatic properties

JOURNAL OFBIOSCIENCE ANDBIOENGINEEIUNG Vol. 94, No. 2, 124129.2002

Production of Aldehyde Oxidases by Microorganisms and Their Enzymatic Properties AKINORI YASUHARA,l MIHO AKIBA-GOTO,l IUNYA FUJISHIRO; HIROYUKI UCHIDA, TAKAYIJKI UWAJIMA,3 AND KAZUO AISAKA’* Tokyo Research Laboratories, Kyowa Hakko Kogvo Co. Ltd., 3-6-6 Asahimachi, Machida-shi, Tokyo 194-8533, Japan,’ Kyowa Medex Co. Ltd., 2-l-l Irtjune, Chuo-ku, Tokyo 104-0042, Japan2 andFact&v of Engineering, Fukui University, 3-9-l Bunkyo, Fukui 910-8507, Japan’ Received 5 March ZOOZ/Accepted17 May 2002

In order to establish an efftcient process to decompose environmentally toxic ahlehydes, dioxygen-dependent aldehyde oxidase (ALOD) from microorganisms was first sought, and some bacteria and actinomycetes were found to produce the enzyme in their cells. MethyZobucillus sp., Pseudomonas sp., and Strepomyces moderutus were selected as the representative ALOD-producing strains, and their enzymes were partially purified and characterized. The three ALODs could oxidize a wide range of aldehydes including formaldehyde, aliphatic aldehydes, and aromatic aldehydes, though their preferences differ depending on their producing strains. The other enxymatic properties were also determined with regard to their producing strains. Methylobucilhs sp. ALOD had the most acidic optimum pH for its activity and stability, and Pseudomonas sp. ALOD had the highest stability against heat treatment. Three native ALODs had molecular weights ranging from 140 to 148 kDa, and were composed of three subunits of different sixes: large (85 to 88 kDa), medium-sized (37 to 39 kDa), and small (18 to 23 kDa). [Key words: aldehyde oxidase, formaldehyde, Methylobacillus sp., Pseudomonas sp., Streptomyces moderatus]

cerevisiae (9), and Aspergillus nidulans ( 10) are involved in the catabolism of ethanol, and those in Pseudomonas sp. (11) and Vibrio harveyi (12) are involved in the metabolisms of aromatic aldehydes and long-chain aliphatic aldehydes, respectively. The oxidation of vanillin (4-hydroxy-3-methoxybenzaldehyde) to vanillic acid (4-hydroxy-3-methoxybenzoic acid) has also been reported in Streptomyces viridosporus (13), although the enzymatic properties have not been fully investigated. In recent years, some aldehydes have gained increasing attention as environmental pollutants. For example, formaldehyde and acetaldehyde are hazardous air pollutants that are known to cause nasal cancer in test animals (14, 15). Therefore, it is of great significance to develop biotechnological methods for removing contaminant aldehydes from environment. In such a case, an oxygen-dependent ALOD is better than NAD(P)-dependent enzymes from an economical standpoint, because it does not require any expensive cofactors. In the present study, we perform the screening of a novel oxygen-dependent ALOD in microorganisms. Furthermore, we purify some microbial ALODs, and compare their properties among the producing strains.

Aldehyde oxidase (aldehyde : oxygen oxidoreductase, ALOD; EC 1.2.3.1) catalyzes the oxidation of various aldehydes to their corresponding carboxylic acids with the reduction of molecular oxygen to hydrogen peroxide according to Eq. 1. Aldehyde+O,+H,O

+ acid+H,O,

Saccharomyces

(1)

ALOD is present in the organs of various animal species (1). For example, bovine ALOD is expressed at high levels in the liver and lung, and has been implicated in the detoxification of certain types of environmental pollutants and xenobiotics (2). Furthermore, in animals, ALOD may also play a role in biosynthetic processes such as retinoic acid synthesis (3). In plants, ALOD seems to be involved in hormone biosyntheses, such as those of indole-3-acetic acid (4) and abscisic acid (5). In microorganisms, however, ALODs using molecular oxygen as an electron acceptor have not yet been reported. However, aldehyde oxidoreductase using 2,6-dichlorophenol-indophenol as an electron acceptor has been isolated and characterized from a sulfate-reducing bacterium of the genus Desulfovibrio (6, 7). Furthermore, aldehyde dehydrogenases (EC 1.2.1.3, 1.2.1.4, and 1.2.1.5) that use NAD and/or NADP as a coenzyme have already been reported in some microorganisms. Enzymes in Escherichia coli (8),

MATERIALS AND METHODS Screening The distribution of aldehyde oxidase (ALOD) was examined using 46 strains of bacteria, 66 strains of actinomycetes, 67 strains of yeasts, and 173 strains of molds in our culture collection. The bacteria were inoculated into yeast bouillon medium (pH7.0) consisting of 2% bouillon and 0.5% yeast ex-

* Corresponding author. e-mail: [email protected] phone: +81-(0)42-725-2555 fax: +81-(0)42-726-8330 Abbreviation: ALOD, aldehyde oxidase. 124

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tract. The actinomycetes were inoculated into Bennett’s medium (ATCC medium 174). The molds and yeasts were inoculated into YM medium (ATCC medium 200). For meth~o~-~similating bacteria and yeasts, 1% methanol was added to each medium. Cultivation was carried out at 28’C for l-5 d with shaking in test tubes (2.0 x20 cm) with 10 ml of each medium. The cells were collected by centrifugation at 10,OOOxg for 15 min or by filtration. The harvested cells were washed with 10 mM potassium phosphate buffer (PH 7.0) and suspended in the same buffer. The cell suspensions of bacteria and a~tinomycetes were dis~pted by sonication for 5 min, and those of molds and yeasts were homogenized for IO min with 0.25 mmd, glass beads in a homogenizer. After centrifugation at 15,OOOxg for 15 min, the resultant supernatants were used as enzyme preparations. ALOD activity was assayed by Assay of aldebyde oxidase measuring hydrogen peroxide generated during the oxidation of aldehydes. The standard reaction mixture contained I5 mM formaldehyde, 50 mM potassium phosphate buffer (pH 7.0), 0.36 mM 4-aminoantipyrine, 6.3 mM phenol, 7 units/ml peroxidase, and enzyme in a total volume of 2.0 ml. After incubation at 37°C for S15 min, the formation of the quinoneimine dye was estimated by measuring the absorbance at 500nm with a spectrophotometer (16). One enzyme unit was defined as the amount of enzyme that produces 1 ,umol of hydrogen peroxide per min. All operations were carPurification of aldehyde oxidases ried out at 4°C and 1OmM potassium phosphate buffer (pH7.0) was used. The culture broths (2400ml each) from Methylobacillus sp., Pseudomonas sp. and Streptomyces moderotus were centrifuged at 12,000xg for I5 min to collect each cell. The cells were disrupted by sonication, and centrifuged at 12,000 xg. (NH&SO, were added to the resulting supernatants (cell-free extracts) up to 60% saturation, and the precipitates were collected by centrifugation at 12,OOOxg for 15 min, and dialyzed against the buffer overnight. The dialyzed enzyme solutions were applied onto a column (5 x 15 cm) of DEAE-Toyopearl 650M, which was pre-equilibrated with the buffer. After the column was washed with the buffer, the enzyme was eluted with a linear gradient of NaCl from 0 to 0.5 M. The active fractions were combined, and concentrated with an Amicon YMlO membrane (Millipore, Bedford, MA, USA). Then, (NH,),SO, was added to the enzyme solution up to 30% saturation, and applied onto a column (3 x 15 cm) of phenyl-Toyopearl 650M, which was pre-equilibrated with the buffer containing 30% saturated ~H~)*SO~. After the column was washed with 30% ~H~)~SO~-saturated buffer, the enzyme was eluted with a reverse linear gradient of (NH&SO, saturation from 30% to 0%. The active fractions were combined, dialyzed against the buffer, and concentrated with a YMlO membrane. The concentrates were used as the sample enzymes for determining their enzymatic properties. ALODs from ~ethy~obaciz~~s sp., Pseudomonas sp., and S. moderator were pained 590-fold with 19.5% yield, 538-fold with 20.9% yield, and 107-fold with 12.2% yield, respectively. The purities of the final preparations obtained were suggested to be in the range of 30-50% from the results of SDS-PAGE. In order to clarify its subunit composition, further purification of the ALOD from Methylobacillus sp. was carried out. The partially purified enzyme obtained above was further purified by Resource Q (6 ml) anion exchange c~omatography and Superdex 2OOpg (26160) gel filtration, which were carried out essentially under the same conditions described above. The SDS-PAGE of the final preparation gave three major protein bands and a few contaminant-like protein bands, as shown in Fig. 1. Gel filtration was carried out Molecular weight estimation with a TSK-gel G3000SW column (0.75 x60 cm; Tosoh, Tokyo) at a flow rate of 0.3 mVmin with 200 mM pot~sium phosphate buffer (pH 7.0) containing 0.1 M NaCl as eluant. The elution profiles of

MICROBIAL ALDEHYDE OXIDASE

125

Ml234 (kDa1 94

+

Large

67 43 + Medium 30

20 +

Small

14

FIG. I. SDS-PAGE pattern of purified aidehyde oxidase from

~ethyZobociI~~ssp. Lane M, Contained molecular mass marker proteins; lanes l-4, contained purified ~e~~~Q~~cj~~~~sp. aldehyde oxidase (5, 10,25, and 50 pg, respectively). the enzymes were determined by assaying the oxidase activity of the eluates. The molecular weights of the enzymes were calculated from the mobilities of standard proteins obtained &om Oriental Yeast, Osaka. SDS-PAGE was carried out by the method of Laemmli with 0.1% SDS and 12.5% polyacrylamide gel (1 mm thick). The molecular weights of the enzymes were calculated from the relative mobilities of standard proteins (Amersham Biosciences, Piscataway, NJ, USA). Chemicals Formaldehyde was prepared by heating parafo~aldehyde (Sigma Chemicals, St. Louis, MO, USA) at 100°C for 3 h, because commercial fo~aldehyde solution contains approximately 8% methanol for stabilization. The other aldehydes and all other chemicals were of the highest analytical grade available. DEAE-Toyopearl 650M and phenyl-Toyopearl 650M were obtained from Tosoh, and Resource Q (6 ml) and Superdex 200 pg (26160) were obtained from Amersham Biosciences.

RESULTS Production of aldehyde oxidase by microorganisms The distribution of ALOD was examined using formaldehyde as a substrate against approximately 3.50 strains of microo~~isms in our culture collection. The fo~aldehyde~ oxidizing enzymes were found in several kinds of microorganisms, and were classified into two classes, depending on their substrate specificity. That is, the first class included enzymes that could oxidize only formaldehyde among the aldehydes tested, but could efficiently oxidize lower primary alcohols such as methanol and ethanol. The second class included enzymes that could oxidize various kinds of aldehydes, but could not oxidized alcohols at all. The first class of enzymes seemed to be alcohol oxidases, and was produced by molds such as Cylindrocarpon didymum KY392 and U&ago crus-galli KY2708 (data not shown). On the other hand, the second class of enzymes seemed to be real ALODs, and was found in cells of some strains of the genus Pseudomonas, methanol-assimilating bacteria such as the

126

J. BIOSCI.BIOENG.,

YASUHARA ET AL. TABLE 1. Distribution of aldehyde oxidase in microorganisms

Aldehyde oxidase activity” (mu/ml)

Microorganism Methylobacillus extorquens Methylobacillus sp. Methylophaga thalassica Pseudomonas parvonacea I? stutzeri Pseudomonas sp. Streptomyces moderatus S. ochraceiscleroticus S. rimosus

Initial mediumb DSM1337 KY4400 ATCC33 146 KY3991 IF012695 KY4690 ATCC23443 ATCCl5814 ATCC 10970

Improved medium

4.4 6.4 0.5 23.0 24.0 26.0 9.4 2.0 6.2

ND’ 18.0” ND ND ND 115.0* 41.3’ ND ND

a Aldehyde oxidase activity was assayed using formaldehyde as a substrate, and is indicated as mu per ml of broth. b Each initial medium is described in Materials and Methods. c Yeast bouillon medium supplemented with 1% sucrose, 0.2% glutamate, 0.2% aspartate, 0.2% alanine, and 0.01% MnSO,.7H,O was used. * Yeast bouillon medium supplemented with 1% maltose, 0.2% valine, 0.2% leucine, 0.2% isoleucine, and 0.01% MnSO,.7H,O was used. c Bennett’s medium supplemented with 0.5% glucose was used. f ND means “not determined”. genera Methylobacillus and Methylobacterium, and the genus Streptomyces (Table 1). Of these, Methylobacillus sp. KY4400, Pseudomonas sp. KY4690, and S. moderatus

ATCC23443 were selected as representative strains for tI.rrther study, because of their higher activities and stable reproducibilities. The effect of various kinds of carbon, nitrogen, and metal compounds on the production of ALOD by three strains of microorganisms was investigated. First the three strains were cultivated in each basal medium described in Materials and Methods, which was supplemented with ten metal salts of 0.01% concentration, and the ALOD activities of their cell-free extracts were assayed. The addition of manganese ion resulted in about 3.2-fold, 1S-fold, and 0.91-fold increase in the enzyme production of Methylobacillus sp., Pseudomonas sp., and S. moderatus, respectively. On the other hand, the addition of molybdenum ion, which is known to be a cofactor in animal and plant ALODs (17), did not affect the enzyme production in all the strains. However, the addition of tungsten ion, which is known to have an antagonistic effect on MO-dependent enzymes (18, 19), resulted in the reduction of their enzyme production to 6.1%, 5.7%, and 16% in Methylobacillus sp., Pseudomonas sp., and S. moderatus, respectively. Furthermore, the addition of sugar and amino acids to the basal medium resulted in the increase in the enzyme production, because of their growth-stimulating effects. On the other hand, the addition of various aldehydes did not show a positive effect on the enzyme production. Finally, the improved cultivating conditions, which are described in Table 1, brought about 2.8-fold, 4.4-fold, and 4.4-fold increase in the enzyme production, compared with those in the initial screening (Table 1). Properties of microbial aldehyde oxidases In order to determine the enzymatic properties of the mirobial ALODs, a brief purification was carried out. The cell-free extracts of the three strains were purified using ammonium sulfate fractionation, anion exchange and hydrophobic chromatographies, as described in Materials and Methods. Then, the partially purified enzymes were used for the characterization of each ALOD. The oxidizing acitivities against various aldehydes of three

ALODs were examined, and were expressed relative to that against formaldehyde (Table 2). Methylobacillus sp. ALOD showed a high activity against formaldehyde. Pseudomonas sp. ALOD showed a high activity against aliphatic aldehydes. S. moderatus ALOD showed high activities against both aliphatic and aromatic aldehydes. On the other hand, all of the three ALODs did not show an activity against xanthine, purine, and IV’-methylnicotinamide, which are substrates of xanthine oxidase (EC 1.1.3.22), and lower primary alcohols such as methanol and ethanol, which are subTABLE 2. Substrate specificity of three microbial aldehyde oxidases Relative activityb Aldehyde” Formaldehyde Acetaldehyde Propionaldehyde Butylaldehyde Valeraldehyde Hexylaldehyde Heptylaldehyde Octylaldehyde Decylaldehyde Phenylacetaldehyde Cimramaldehyde Benzaldehyde p-Hydroxybenzaldehyde Salicylaldehyde p-Anisaldehyde Veratraldehyde Acrolein Crotonaldehyde Citral DL-Glyceraldehyde Glutalaldehyde 2-Ethylbutylaldehyde Indol-3-carboxyaldehyde

Methylobacillus’ 1.0 0.35 0.23 0.31 0.56 0.74 0.76 0.64 0.41 0.26 1.7 1.3 2.0 0.22 1.8 1.2 0.08 0.61 2.1 0.28 0.18 0.51 0.20

Pseudomonasd StreptomyceSe 1.0 3.5 3.4 3.6 3.6 3.6 3.5 3.5 0.52 1.3 2.4 1.6 2.5 1.2 1.9 2.2 1.4 2.3 2.6 0.77 0.64 3.5 0.08

1.0 1.7 2.2 2.6 3.5 4.3 4.8 4.3 4.0 2.3 4.1 4.1 3.2 2.6 5.0 6.1 1.3 3.4 4.3 0.73 0.20 1.7 1.0

BThe standard concentration of the aldehydes used was 15 mM, and aldehydes with lower solubility were used at the saturation concentration. b The activity is expressed relative to that against formaldehyde. c Aldehyde oxidase from Methylobacillus sp. KY4400. * Aldehyde oxidase from Pseudomonas sp. KY4690. e Aldehyde oxidase from Streptomyces moderatus ATCC23443.

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VOL. 94,2002

127

TABLE 3. K,,, values of three microbial aldehyde oxidases to various aldehydes K- fmM) Aldehyde

Methylobacillus”

Pseudomonasb Streptomycesc

4.2 1.4 1.8 0.07 0.48 0.12 0.57

Formaldehyde Acetaldehyde Hexylaldehyde Sal~~yialdehyde Acrotein Citral 2-Ethylbutylaldehyde

21.2 1.4 0.15 0.70 1.4 2.1 3.5

3.6 0.13 0.26 0.34 0.74 0.06 2.2

a Aldehyde oxidase from Methylobacillus sp. KY4400. b Aldehyde oxidase from Pseudomonas sp. KY4690. ’ Aldehyde oxidase from Streptomyces moderatus ATCC23443.

of alcohol oxidase (EC 1.1.3.13). The affinities for seven representative aldehydes of the three ALODs were examined (Table 3). Methylobacillus sp. ALOD showed high affinities for formaldehyde (the smallest aldehyde), salicylaldehyde (an aromatic aldehyde), and acrolein and citral (double-bond-confining aldehydes). Pseudomonas sp. ALOD showed high aftinity for hexylaldehyde (a long aiiphatic aldehyde), but showed rather low afftnity for formaldehyde (the smallest aldehyde). S. moderatus ALOD showed high affinities for formaldehyde and acetaldehyde (small aldehydes), and acrolein and citral. Furthermore, among the aldehydes tested, acrolein showed a strong substrate inhibition at a concentration more than 2 mM only for Methylobacillus sp. ALOD. The effect of temperature on the enzyme activity was examined (Fig. 2a). The apparent optimum temperatures for a IO-min reaction were 50°C 45”C, and 30°C for ~ethylob~~i~~us sp., Pseudomonus sp., and S. moderatus, respectively. The thermal stabilities of the enzymes were examined (Fig. 2b). The enzymes from Methylobacilius sp., Pseudomonas sp., and S. moderatus showed residual activities of more than 90% at 6O”C, 80°C and 70°C, when treated at pH 7.0 for 15 min. ~ethy~obacil~us sp. ALOD was strates

020

30

40

50

TeBRerature

60

(“cl

70

OOW

Temperature

60

CC)

100

FIG. 2. Effects of temperature on the activity and stability of three microbial aldehyde oxidases. (a) Each enzyme reaction was carried out for 1Omin under the standard conditions except for the incubation temperature, which is indicated in the figure. (b) Each enzyme was treated for 15 min in 10 mM potassium phosphate buffer (pH 7.0), and then the residual enzyme activity was assayed under the standard conditions. The results are expressed as a percentage of the activity of each native enzyme. Open circles, ~ethy~obac~~~~~sp. ALOD; closed circles, Pseudomonas sp. ALOD; open triangles, S. moderatus ALOD.

FIG. 3. Effects of pH on the activity and stability of three microbial aldehyde oxidases. (a) Each enzyme reaction was carried out under the standard conditions except that 20 mM universal buffers of various pHs were used. As the color intensity of the product is dependent on the pH, appropriate corrections were made using hydrogen peroxide as a standard product. (b) Each enzyme was treated using 20 mM universal buffer of various pHs for 1.5min at 55°C for Methylobacillus sp. ALOD, at 80°C for Pseudomonas sp. ALOD, and at 60°C for S. moderatus ALOD. Then, the residual enzyme activity was assayed under the standard conditions (pH 7.0). The results are expressed as a percentage of the activity of each native enzyme. Open circles, ~ethy~obacill~s sp. ALOD; closed circles, Pseudomonas sp. ALOD; open triangles, S. moderutus ALOD.

active at temperatures higher than that suggested based on its thermal stability, and Pseudomonas sp. ALOD was highly stable against heat denaturation. The effect of pH on the enzyme activity was examined (Fig. 3a). Methylobacillus sp. ALOD showed an optimum pH of approximately 3.0 to 4.0. On the other hand, the enzymes from Pseudomonas sp. and S. moderatus showed optimum pHs of approximately 6.0 to 7.0 and 5.0 to 6.0, respectively, and showed no or only little activity at pH 3.0, The pH stabilities of the enzymes were examined (Fig. 3b). ~ethy~obacillus sp. ALOD was stable between pH 3.0 and pH 5.0. In contrast, the enzymes from Pseudomonas sp. and S. moderutus were stable between pH 5.0 and pH 9.0. The effects of SH reagents and chelating agents on the ALOD activity were examined. The addition of p-chloromercuri~~oate at 1 mM caused 88%, 94%, and 90% inhibitions of the activities of enzymes from ~ethyloba~i~~us sp., Pseudomonas sp., and S. moderatus, respectively, but iodoacetate, iodoacetamide, and N-ethylmaleimide did not inhibit the enzyme activities at the same concentration (data not shown). Chelating agents such as EDTA, o-phenanthroline and ~,ff’-dip~idyl had no effect on the enzyme activity (data not shown). The molecular weights of the above ALODs were determined to be 142 kDa for the Methylobacillus enzyme, 140 kDa for the Pseudomonas enzyme, and 148kDa for the Streptomyces enzyme, as determined by gel filtration. On the other hand, SDS-PAGE of the highly purified ~ethy~obac~lius sp. ALOD revealed that the enzyme was composed of three different subunits: large (88 kDa), medium-sized (38 kDa) and small (18 kDa) (Fig. 1). By analogy with Methylobacillus sp. ALOD, 85-, 39-, and 19-kDa proteins for Pseudomonas sp. ALOD, and 86-, 37-, and 23-kDa proteins for S. moderatus ALOD were presumed to be the subunits of ALODs from these microorg~isms.

128

YASUHARA ET AL.

J. BIOSCI.BIOENG., TABLE 4. Comparison of aldehyde oxidases with their refated enzymes Electron acceptor

Native molecular mass &Da)

Subunit molecular mass(es) &Da)

h4ethylo&acillus sp.

0,

142

88,38,18

0,

ALOD ALOD ALOD ALOR ALDH

P~e~~orno~~sp. S. m~era~s Bovineliver Maize D. gigas S. cerevisiae

140 I48 300 300 200

85,39, 19 86,37,23 147 I47 97

XDHIXOD XDH XDH

Cow’s milk p ptida 86 E. barkeri

290 550

54.6 150 91.0,46.2

Enzyme ALOD ALOD

Source

02

0, 4 DCIP NADP NAD/O, NAD

Subunit structure

Ref.

.- czgr

This

al% a& a2 a, a2 CL, a&,

This This 2 20 21 9 24 25

81,30, 17.5 a@,~~ 26 NADP 530 ALOD,Aldehydeoxidase;ALOR,aIdehydeoxidoreductase;ALDH,aldehydedehydrognase;XDH,xanthinedehydrogenase;XOD,xanthine oxidase; DCIP,2,6-dichlorophenol indophenol.

DISCUSSION When the cell-free extracts of various kinds of microorganisms were screened for oxidative activity against formaldehyde, some bacteria and actinomycetes were found to be able to catalyze the dioxygen-dependent oxidation of formaldehyde and many other kinds of aldehydes. Currently, the oxygen-de~ndent ALOD is known in animals (2) and plants (20), but not in microorganisms, although alternative aldehyde-metabolizing enzymes such as NAD and/or NADP-dependent aldehyde dehydrogenases (9-12) and 2,6dichrorophenol indophenol-dependent aldehyde oxidoreductases (20-23), are known in some microorganisms. The production of ALODs by micr~~~isms was limited in dis~bution, and involved low and constitutive formation. Therefore, the roles of ALODs are of interest in connection with the aldehyde metabolism in microorganisms. Mammalian and plant ALODs are well known to be molybdo-iron-sulfur flavoproteins (17). We do not know whether the microbial ALODs obtained here are molybdoiron-sulfur enzymes or not. However, although the addition of molybdate to the growth media did not essentially affect the production of microbial ALODs, the addition of tungstate strongly inhibited the production of the enzymes. These results suggest that these microbial ALODs are molybdoproteins, because tungstate has been proved to show an ~~gonistic effect on the inco~oration of molybdate into molybdate-dependent enzymes ( 18, 19). ALODs from the microorganisms were composed of three subunits that differ in sizes, and were in the ranges of 8588 kDa for the large ones, 37-39 kDa for the medium-sized ones, and 18-23 kDa for the small ones. The molecular masses of the native enzymes were in the range of 140148 kDa, which indicated that the microbial ALODs were heterotrimers. On the other hand, animal and plant ALODs are known to be homodimers, consisting of two 147-150 kDa subunits (2, 20), as summarized with other related enzymes in Table 4. These results indicate that the total molecular mass of the three subunits from the microorg~sms is about the same as that of the subunits of the animal and plant ALODs. Therefore, it is suggested that in animal and plant enzymes all the functional units are loaded into one subunit, but in microbial enzymes they are separated into

three subunits. Similar observations were made between the one subunit (150 kDa) of x~thine dehy~ogen~e (XDH) from cow’s milk (24), the two subunits (91 .O and 46.2 kDa) of XDH from Pseudomonas putidu (25) and the three subunits (81, 30, and 17.5 kDa) of XDH from Eubacterium barkeri (26) (Table 4). It remains to be solved why these microbial enzymes tend to be divided into smaller and more than one subunit. The properties of microbial AL,ODs were dependent on their producing strains. Methylobacillus sp. ALOD showed high activity and stability in the acidic-pa region, and gave high activity against formaldehyde.~Pseudomonus sp. ALOD was characterized by its extremely high heat stability, but showed only limited activity and a%nity against formaldehyde. S. modera~s ALOD showed low optimum temperature for the oxidation reaction, although its heat stability was rather high. Therefore, the enzyme from S. moderutus seemed to be a kind of intermediate between those from Mthylobacillus sp. and Pseudomonas sp. These results give us the following two important oppo~ities. First, different ALODs give us a chance to determine the structural characteristics of this type of enzyme to clarify the differences of these enzymes. Second, distinctive ALODs give us a chance to create more superior enzymes by combining each good point into one unit with the methods such as the family DNA shuffling (27). Therefore, the cloning of the ALOD genes is now under way. REFERENCES Felsted, R L., Chu, A. E., and Chaykin, S.: Purification and properties of the aldehyde oxidases from hog and rabbit livers. J. Biol. Chem., 248,2X%-2587 (1973). Caizi, M. L., Raviolo, C., Gbibaudi, E., De Gioia, L., Salmona, M., Cazzaniga, G., Kurosaki, M., Terao, M., and Garattini, E.: Purification, cDNA cloning, and tissue distribution of bovine liver aldehyde oxidase. J. Biol. Chem., 270, 31037-3104s (1995). Huang, D. Y. and Ichikawa, Y.: Two different enzymes are primarily responsible for retinoic acid synthesis in rabbit liver cytosol. Biochem. Biophys. Res. Commtm., 205, 1278-1283 (1994). Sekimoto, H., Seo, M., Kawakami, N., Komano, T., Desloire, S., Liotenberg, S., Marion-Poll, A., Caboche, M., Kamiya, Y., and Koshiba, T.: Molecular cloning and charac-

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