- Email: [email protected]
A new aldehyde oxidase catalyzing the conversion of glycolaldehyde to glycolate from Burkholderia sp. AIU 129
Journal of Bioscience and Bioengineering VOL. 119 No. 4, 410e415, 2015 www.elsevier.com/locate/jbiosc
A new aldehyde oxidase catalyzing the conversion of glycolaldehyde to glycolate from Burkholderia sp. AIU 129 Miwa Yamada,1 Keika Adachi,1 Natsumi Ogawa,1 Shigenobu Kishino,2 Jun Ogawa,2 Michihiko Kataoka,3 Sakayu Shimizu,2, 4 and Kimiyasu Isobe1, * Department of Biological Chemistry and Food Science, Iwate University, Ueda-3, Morioka 020-8550, Japan,1 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan,2 Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 599-8531, Japan,3 and Faculty of Bioenvironmental Science, Kyoto Gakuen University, Sogabe-cho, Kameoka 621-8555, Japan4 Received 8 July 2014; accepted 3 September 2014 Available online 3 October 2014
We found a new aldehyde oxidase (ALOD), which catalyzes the conversion of glycolaldehyde to glycolate, from Burkholderia sp. AIU 129. The enzyme further oxidized aliphatic aldehydes, an aromatic aldehyde, and glyoxal, but not glycolate or alcohols. The molecular mass of this enzyme was 130 kDa, and it was composed of three different subunits (abg structure), in which the a, b, and g subunits were 76 kDa, 36 kDa, and 14 kDa, respectively. The N-terminal amino acid sequences of each subunit showed high similarity to those of putative subunits of xanthine dehydrogenase. Metals (copper, iron and molybdenum) and chelating reagents (a,a0 -dipyridyl and 8-hydroxyquinoline) inhibited the ALOD activity. The ALOD showed highest activity at pH 6.0 and 50 C. Twenty mM glycolaldehyde was completely converted to glycolate by incubation at 30 C for 3 h, suggesting that the ALOD found in this study would be useful for enzymatic production of glycolate. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Aldehyde oxidase; Glycolaldehyde; Glycolic acid; Burkholderia sp.; Xanthine oxidase/dehydrogenase]
Glycolate, which is the smallest a-hydroxy acid, is used as a dyeing and tanning agent in the textile industry, a flavoring agent and preservative in the food processing industry, and a skin care agent in the pharmaceutical industry. It is also useful for the production of polyglycolate and other biocompatible copolymers. Currently, ethylene glycol is utilized as an inexpensive starting material for the production of glycolate by oxidation reaction, while the chemical oxidation of ethylene glycol has certain drawbacks, such as the formation of formaldehyde as a by-product. Microbial methods for the production of glycolate from ethylene glycol by resting-cell reaction have also been studied using cells from Acetobacter, Gluconobacter, or Hansenula (1,2). Kataoka et al. (3) screened several microorganisms to produce a high concentration of glycolate in a medium containing ethylene glycol. Those results indicated that the microorganisms produced enzymes that catalyzed the conversion of ethylene glycol into glycolate via glycolaldehyde. However, the enzymes responsible for this reaction have not been purified and characterized. In terms of enzymes that are known to be involved in the oxidation of ethylene glycol, we have previously shown that the alcohol oxidases (EC 1.1.3.13) from methanolytic yeasts such as Candida sp. and Pichia pastoris (4) or glycerol oxidase from Aspergillus japonicas (5) catalyzed the oxidation of ethylene glycol to glyoxal via glycolaldehyde, and the reaction rate of ethylene glycol
* Corresponding author. Tel./fax: þ81 766 88 2280. E-mail address: [email protected] (K. Isobe).
oxidation was much faster than that of glycolaldehyde oxidation (6). Thus, those alcohol oxidases and glycerol oxidase were useful for accumulating a high concentration of glycolaldehyde from ethylene glycol (6). In our subsequent research, we have focused on a new enzymatic method for the production of glycolate from ethylene glycol utilizing two oxidases; ethylene glycol is first converted to glycolaldehyde by the alcohol oxidases from Candida sp. and P. pastoris or the glycerol oxidase from A. japonicas, and the resulting glycolaldehyde is then oxidized to glycolate by an aldehyde oxidase (ALOD) catalyzing the conversion of glycolaldehyde to glycolate. When this new enzymatic method utilizing two oxidases is fully developed, it will be applicable for the production of glyoxylate from ethylene glycol by addition of another alcohol oxidase with glycolate-oxidizing activity, which has already found in Ochrobactrum sp. AIU 033 by our group (7). Thus, it will be possible to produce glyoxylate, an attractive raw material for the chemical synthesis of vanillin, antibiotics or agrochemicals, using a combination of three microbial oxidases. ALODs exhibiting oxidase activity for glycolaldehyde have already reported from Pseudomonas sp. AIU 362 (8) and Pseudomonas sp. MX-058 (9), but the oxidation products were not identified. We therefore isolated a new bacterial strain that produces an enzyme catalyzing the oxidation of glycolaldehyde into glycolate, and revealed certain properties of both the strain and enzyme. The present paper describes our isolation of the new bacterial strain, and the remarkable characteristics of the enzyme it produces. The enzymatic production of glycolate from glycolaldehyde using the new ALOD is also described.
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.09.005
VOL. 119, 2015
NEW ALDEHYDE OXIDASE FROM BURKHOLDERIA SP. AIU 129 MATERIALS AND METHODS
TABLE 1. Purification of ALOD from Burkholderia sp. AIU 129. Step
Chemicals 2-Methoxyethanol, glycolate sodium salt, glyoxylate monohydrate, glycolaldehyde, and other aldehydes and alcohols were purchased from Wako Pure Chemical Industries (Osaka, Japan). Horseradish peroxidase (EC 1.11.1.7) was obtained from Amano Enzyme (Nagoya, Japan). All other chemicals used were of the highest grade commercially available. Isolation of the microorganism The enrichment culture was carried out three times using a 2-methoxyethanol medium containing 1% 2-methoxyethanol, 0.2% NH4NO3, 0.1% K2HPO4, 0.1% NaH2PO4, 0.02% MgSO4$7H2O, 0.01% CaCl2$2H2O, and 0.05% yeast extract, pH 6.0. The microorganisms grown in the 2-methoxyethanol medium were cultivated on an agar plate of the 2-methoxyethanol medium at 30 C for 2e3 days. The strains exhibiting glycolaldehyde-oxidizing activity were selected by evaluation of hydrogen peroxide formation on the agar plate. The formation of hydrogen peroxide was detected using a color development solution containing 0.64 mmol of 4-aminoantipyrine, 1.94 mmol of N-ethyl-N-(2-hydroxy-3sulfopropyl)-3-methylaniline sodium salt dihydrase (TOOS), 6.7 units of peroxidase, and 20 mmol of glycolaldehyde in 1.0 ml of 0.1 M of potassium phosphate, pH 7.0. The isolated strains were incubated in a test tube (16 1.5 cm diameter) containing 5 ml of the 2-methoxyethanol medium at 30 C for 2 days. Subsequently, a cell-free extract was prepared by disrupting the cells at below 5 C for 8 min using a Multibeads shocker (Yasui Kikai, Osaka, Japan). The strain exhibiting the highest activity on glycolaldehyde was selected, and used in this study. Identification of the isolated strain Identification of the newly isolated strain was performed at NCIMB Japan Co., Ltd. (Shizuoka, Japan). Cultivation of the strain The isolated strain was incubated in 5 ml of the 2methoxyethanol medium at 30 C for 2 days with shaking (120 strokes/min). The culture (1.5 ml) was inoculated into a 500-ml culture flask containing 150 ml of the medium, then incubated at 30 C for 2 days with shaking. Twenty milliliter of the second culture was transferred into a 3-l culture flask containing 2 l of the 2methoxyethanol medium, followed by cultivation at 30 C for 5 days with shaking. Assay of enzyme activity ALOD activity was assayed by measuring the initial rate of hydrogen peroxide formation at 30 C. The standard reaction mixture contained 20 mmol of glycolaldehyde or acetaldehyde, 0.6 mmol of 4-aminoantipyrine, 1.94 mmol of TOOS, 6.7 units of peroxidase, 0.1 mmol of potassium phosphate (pH 7.0), and an appropriate amount of enzyme in a final volume of 1.0 ml. The assay of enzyme activity was started by the addition of enzyme solution, and formation of hydrogen peroxidase was spectrophotometrically followed at 30 C for 5 min by measuring the absorbance at 555 nm. One unit of enzyme activity was defined as the amount of enzyme catalyzing the formation of 1 mmol of hydrogen peroxide per min. A molar absorptivity value of 16.5 103 M1 cm1 was used for calculation of the enzyme activity. Purification of the enzyme All procedures were performed at 5e10 C using potassium phosphate buffer (pH 6.0). The cells from 40 l of culture broth (34 g of wet weight) were disrupted with glass beads in 10 mM buffer solution by a Multi-beads shocker, and the supernatant was collected by centrifugation at 10,000 g for 30 min. Solid ammonium sulfate was added to the supernatant to reach 40% saturation with stirring, and it was further added to the supernatant to reach 60% saturation. The precipitate was dissolved with 10 mM buffer solution and dialyzed against 10 mM buffer solution. The dialyzed solution was applied onto a DEAEToyopearl 650 M column (20 3 cm) equilibrated with 10 mM buffer solution
411
Activity (unit)
Protein (mg)
Cell-free extract 12.2 15100 Ammonium sulfate 8.73 901 DEAE-Toyopearl 7.88 78.7 Phenyl-Toyopearl 2.25 1.86 Q-Sepharose 1.16 0.51 Hydroxyapatite 0.45 0.13 Gel filtration 0.215 0.06
Specific activity Recovery Purification (unit/mg of protein) (%) (fold) 0.00081 0.00968 0.100 1.21 2.27 3.41 3.42
100 71.6 64.8 18.5 9.53 3.70 1.77
1 12 124 1500 2820 4210 4220
ALOD activity was assayed under standard conditions using 20 mM glycolaldehyde. Specific activity is expressed as units per milligram of protein.
containing 60 mM NaCl. The absorbed enzyme was then eluted with a linear gradient of 60e190 mM NaCl in the 10 mM buffer. Solid ammonium sulfate was added to the active fractions to reach 140 mS/cm. Then, the enzyme solution was applied to a Phenyl-Toyopearl 650 M column (10 3 cm) equilibrated with 10 mM buffer solution containing 0.4 M ammonium sulfate, and the enzyme was eluted with a linear gradient of 0.4e0.15 M ammonium sulfate in the 10 mM buffer. The collected active fractions were dialyzed against 10 mM buffer solution, and applied to a Q-Sepharose column (10 3 cm) equilibrated with 10 mM buffer solution containing 150 mM NaCl, and the enzyme was eluted with a linear gradient of 150e250 mM NaCl in the 10 mM buffer. The active fractions were deionized by ultrafiltration. The enzyme solution was applied to a hydroxyapatite column (8 1.8 cm) equilibrated with 70 mM buffer solution, and the enzyme was eluted with a linear gradient of 70e200 mM buffer solutions. The active fractions were concentrated to 0.8 ml by ultrafiltration and applied to a Toyopearl HW-55 column (53 1.3 cm) equilibrated with 10 mM buffer solution. The protein concentration was spectrophotometrically determined by measuring the absorbance value at 1% 280 nm. An E value of 10.0 was used throughout this work. 1cm SDS-PAGE and molecular mass Sample for SDS-PAGE was incubated with 1% SDS and 5% mercaptoethanol at 100 C for 3 min. SDS-PAGE was performed according to the method of Laemmli (10). Proteins were stained with Coomassie Brilliant Blue R-250 or visualized by the silver staining method. The molecular mass of the denatured enzyme was estimated by SDS-PAGE using the molecular marker standards of Bio-Rad Japan (Tokyo, Japan). The molecular mass of intact proteins was estimated by gel filtration on a TSK gel G3000SWxL column (Tosoh, Tokyo, Japan). Analysis of N-terminal amino acid sequences After blotting subunit proteins from SDS-PAGE to a polyvinylidene fluoride membrane, the blotted protein was stained with Coomassie Brilliant Blue R-250. The protein bands were cut out, and the N-terminal amino acid sequences of three subunits were analyzed using a Shimadzu gas-phase protein sequencer equipped with an on-line reverse-phase chromatography system for identification of PTH-amino acids.
A
B
C
kDa
250 150 100 75 50 37 25 20
FIG. 1. Effect of cultivation time on enzyme production. Burkholderia sp. AIU 129 was cultured in the 2-methoxyethanol medium at 30 C. The enzyme activity was assayed under standard assay conditions. Closed circles, glycolaldehyde oxidase activity; open circles, absorbance at 660 nm of culture broth.
15 10 FIG. 2. Native-and SDS-PAGE of purified ALOD from Burkholderia sp. AIU 129. (A) Native enzyme, (B) denatured enzyme, (C) molecular makers.
412
YAMADA ET AL.
J. BIOSCI. BIOENG.,
TABLE 2. Substrate specificity and kinetic values of ALOD from Burkholderia sp. AIU 129. Substrate Glycolaldehyde Formaldehyde Acetaldehyde Propionaldehyde Butyraldehyde Benzaldehyde Glyoxal
Km (mM)
Vmax (mmol/min/mg protein)
Vmax/Km
1.00 35.9 0.34 0.74 0.10 0.06 12.3
3.42 4.38 6.42 14.6 8.83 5.58 4.51
3.42 0.122 18.9 19.7 88.3 93.0 0.367
The oxidase activity for aldehydes and alcohols was assayed under standard assay conditions using 40 mM substrate solution and 1.3 munits of ALOD activity for acetaldehyde. The ALOD showed no activity for glycolate, ethanol, 1-propanol, 2methoxyethanol, ethylene glycol, and glycerol. The Km and Vmax values were calculated by HaneseWoolf plot using 0.2e2.0 mM of glycolaldehyde, 4.0e40 mM of formaldehyde, 0.1e1.2 mM of acetaldehyde, 0.1e1.0 mM of propionaldehyde, 0.025e0.25 mM of butyraldehyde, 0.025e0.25 mM of benzaldehyde, and 2.5e15 mM of glyoxal.
TABLE 3. Effects of various compounds on enzyme activity. Compound Hydroxylamine Hydrazine Phenylhydrazine Semicarbazide o-Phenanthroline EDTA a,a0 -Dipyridyl 8-Hydroxyquinoline N-Ethylmaleimide Sodium azide Iodoacetic acid Potassium cyanide
Relative activity (%)
Metal
Relative activity (%)
99 73 6 97 109 92 49 20 100 100 100 83
None MgCl2 NiCl2 CoCl2 MnCl2 CuCl2 ZnCl2 FeCl2 MoCl5
100 93 81 100 95 4 67 0 7
Effects of chemicals and metals were assayed under standard conditions using 1.1 m units of enzyme activity for acetaldehyde and 1 mM chemicals or metals. Relative activity is indicated by percentage of enzyme activity without chemicals.
Glycolate production from glycolaldehyde Twenty millimoles of glycolaldehyde was incubated with 0.2 units of the purified enzyme and 0.1 units of catalase in 0.6 ml of 0.1 M potassium phosphate buffer (pH 7.0) at 30 C for 3 h. The concentration of glycolate in the reaction mixture was analyzed by HPLC with an ULTRON PS-80H column (Shinwa Chemical Industries, Tokyo, Japan). The elution was carried out at a flow rate of 1.0 ml per min at 60 C with perchloric acid solution (pH 2.1) for 20 min. The elution peaks of glycolate, glycolaldehyde, and glyoxylate were eluted at 9.9 min, 9.7 min, and 8.2 min, respectively, under the experimental conditions described above.
RESULTS Microorganism and culture conditions The selected strain was identified by phylogenetic analysis and biochemical properties. The results of the 16S rDNA sequence of 500 bp from the 50 -terminus showed that the isolated strain should be placed in the genus Burkholderia (data not shown). This isolated strain was not fermentative, not motile, not spore-forming, rod-shaped (0.7e0.8 2.0e2.5 mm), gram-negative, catalase-positive, and oxidase-positive. These biological characteristics supported the notion that the isolated strain belonged to the genus Burkholderia, and we therefore named it Burkholderia sp. AIU 129. The strain is currently kept at the Laboratory of Applied Microbiology, Department of Biological Chemistry and Food Science, Iwate University, Japan. When the isolated strain, Burkholderia sp. AIU 129, was incubated with the medium containing 2-methoxyethano, glucose or glycerol as a carbon source, glycolaldehyde-oxidizing activity was not detected in the cells cultured with glucose or glycerol, although cell growth was well in these medium. Thus, it was revealed that the glycolaldehyde-oxidizing enzyme was inductively produced by incubation with 2-methoxyethanol. Then, Burkholderia sp. AIU 129 was incubated in a 500-ml culture flask containing 150 ml of the 2methoxyethanol medium at 30 C for 5 days, and glycolaldehydeoxidizing activity was measured each day. The growth (absorbance at 660 nm) and glycolaldehyde-oxidizing activity increased with
A
B
C
FIG. 3. Alignment of N-terminal amino acid sequences of the a, b, and g subunits of ALOD from Burkholderia sp. AIU 129 to the homologous proteins. The N-terminal sequences were compared with sequences in GenBank, using the NCBI BLAST program (18). Protein sequences exhibiting high homology to N-terminal sequence were aligned using a CLUSTAL W program (19). Identical amino acid residues are boxed. (A) Alignment of the a subunit. Bsp AIU 129, ALOD from Burkholderia sp. AIU 129; Bp BR3459a, putative molybdopterinbinding protein subunit of xanthine dehydrogenase from Burkholderia phenoliruptrix BR3459a (90% identity) (11); Bx LB400, putative molybdopterin-binding protein subunit of xanthine dehydrogenase from Burkholderia xenovorans LB400 (71% identity) (12). (B) Alignment of the b subunit. Bsp AIU 129, ALOD from Burkholderia sp. AIU 129; Bp BR3459a; putative FAD-binding subunit of xanthine dehydrogenase from B. phenoliruptrix BR3459a (80% identity) (11); Bx LB400, putative oxidoreductase from B. xenovorans LB400 (72% identity) (12); Psp KY 4649, 39 kDa-subunit of ALOD from Pseudomonas sp. KY 4649 (53% identity) (13). (C) Alignment of the g subunit. Bsp AIU 129, ALOD from Burkholderia sp. AIU 129; Bp BR3459a; putative ironesulfur-binding subunit of xanthine dehydrogenase from B. phenoliruptrix BR3459a (80% identity) (11); Bx LB400, putative [2Fee2S] ironesulfur cluster binding protein from B. xenovorans LB400 (75% identity) (12).
VOL. 119, 2015
NEW ALDEHYDE OXIDASE FROM BURKHOLDERIA SP. AIU 129
the cultivation time, and glycolaldehyde-oxidizing activity reached the maximum at 4 or 5 days of cultivation (Fig. 1). Therefore, we harvested the cells after 5 days of incubation to obtain the enzyme exhibiting oxidase activity for glycolaldehyde.
413
A
Purification and molecular mass of the enzyme The purification procedure is summarized in Table 1. The glycolaldehydeoxidizing enzyme was purified to an electrophoretically homogeneous state by means of five column chromatographies. An approximately 4220-fold purification was achieved with an overall yield of 1.8%. The purified enzyme showed a single protein band on native-PAGE, and it exhibited three protein bands which were estimated to be 76 (a subunit), 36 (b subunit), and 14 kDa (g subunit), respectively (Fig. 2). The molecular mass of the native enzyme was estimated to be approximately 130 kDa on a TSK gel G3000SWxL column (data not shown). These results indicated that the enzyme consisted of three heterosubunits (abg structure). Substrate specificity and kinetic parameters Among the alcohols and aldehydes tested, aldehydes such as glycolaldehyde, formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, benzaldehyde, and glyoxal were oxidized, but glycolate and alcohols such as ethanol, 1-propanol, 2-methoxyethanol, ethylene glycol and glycerol were not (Table 2). The Km and Vmax values for glycolaldehyde, formaldehyde, acetaldehyde, propionaldehyde, and butyraldehyde were summarized in Table 2. It was revealed that the enzyme exhibited broad substrate specificity for aldehydes, and short-chain aliphatic aldehydes were good substrates for this enzyme. We therefore investigated the enzymatic properties of this ALOD using acetaldehyde as substrate in the following studies. Effects of compounds on the enzyme activity The effects of various compounds on the oxidase activity for acetaldehyde was analyzed by addition of 1 mM carbonyl reagents, chelating reagents, metals or other chemicals (Table 3). Among these compounds tested, Cu2þ, Fe2þ, Mo5þ and chelating reagents such as a,a0 -dipyridyl and 8-hydroxyquinoline strongly inhibited the enzyme activity. In addition, phenylhydrazine also strongly inhibited the enzyme activity. These results indicated that metals and a carbonyl group appeared to play roles in the enzyme activity. N-terminal amino acid sequence The N-terminal sequences of the a subunit, b subunit, and g subunit were found to be IETLPALRASGVPHKDVDGRLK, MNRFSYTRANEVSQAIEQARAKGAA, and LLDVAPSDVQRVPVSFDINGKKEVF, respectively. The sequence of the a subunit was similar to that of a putative molybdopterin-binding protein subunit of xanthine dehydrogenase from Burkholderia phenoliruptrix BR3459a (90% identity) (11) or Burkholderia xenovorans LB400 (71% identity) (12) (Fig. 3A). The sequence of the b subunit was similar to that of a putative FAD-binding subunit of xanthine dehydrogenase from B. phenoliruptrix BR3459a (80% identity) (11), a putative oxidoreductase from B. xenovorans LB400 (72% identity) (12), and a 39 kDa-subunit of ALOD from Pseudomonas sp. KY 4649 (53% identity) (13) (Fig. 3B). The sequence of the g subunit had high similarity to that of a putative ironesulfur-binding subunit of xanthine dehydrogenase from B. phenoliruptrix BR3459a (80% identity) (11) and a putative [2Fee2S] ironesulfur cluster-binding protein from B. xenovorans LB400 (75% identity) (12) (Fig. 3C). Effects of temperature and pH The effect of temperature on enzyme activity was assayed at pH 7.0 using acetaldehyde as substrate. The maximal oxidase activity for acetaldehyde was obtained at 50 C (Fig. 4A). When the enzyme was incubated with 0.1 M potassium phosphate buffer (pH 7.0) at 20e70 C for 30 min, the enzyme activity remained at 20e40 C and more than 50% of the original activity still remained at 70 C. The effect of pH on enzyme activity was assayed under standard assay conditions
B
FIG. 4. Effects of temperature and pH on the activity and stability of ALOD from Burkholderia sp. AIU 129. (A) Effects of temperature: Oxidase activity for acetaldehyde was assayed under standard conditions except for reaction temperature (closed circles). The thermal stability was assayed under standard assay conditions using acetaldehyde after the enzyme solution was incubated at pH 7.0 for 30 min at indicated temperatures (open circles). Percentage of remaining activity was obtained by ratio to activity without heating. (B) Effects of pH: Oxidase activity for acetaldehyde was assayed under standard conditions except for pH values (closed circles). The pH stability was assayed under standard assay conditions using acetaldehyde after heating at 30 C for 30 min at indicated pH (open circles). Percentage of remaining activity was obtained by ratio to activity without heating.
except for the pH values. In the pH range from 6.0 to 8.5, the maximum activity was seen at pH 6.0 (Fig. 4B). The pH stability was analyzed by incubation at 30 C for 30 min between pH 6.0 and 8.5. More than 70% of the enzyme activity remained in the pH region of 7.0e8.0. Production of glycolate from glycolaldehyde Since it was revealed that the enzyme was stable in the pH region of 7.0e8.0, 20 mM glycolaldehyde was incubated with 58 mg of the purified enzyme (0.2 units) at pH 7.0 and 30 C for 3 h. The reaction mixture was then applied to HPLC analysis with an ULTRON PS-80H column. A new peak was detected at 9.9 min, which was identical to the elution time for glycolate (data not shown). Thus, it was confirmed that glycolaldehyde was converted into glycolate by the enzyme. In addition, the glycolate concentration in the reaction mixture was estimated to be 19.8 mM (conversion yield, 99%), indicating that the enzyme would be useful for the production of glycolate from glycolaldehyde. DISCUSSION In this study, we isolated Burkholderia sp. AIU 129 as a producer of ALOD, which catalyzes the oxidation of glycolaldehyde to
414
YAMADA ET AL.
J. BIOSCI. BIOENG., TABLE 4. Comparison of characteristics of ALOD from Burkholderia sp. AIU 129 with other microbial ALODs.
Origin
Burkholderia sp. AIU 129
Pseudomonas sp. AIU 362
Substrate specificity (%)a Acetaldehyde 100 100 Formaldehyde 68 21 Benzaldehyde 87 75 Glyoxal 70 95 Glycolaldehyde 53 7 Opt-pH Below 6.0 6.0 Opt-temp ( C) 50 40e45 Molecular mass (kDa) Gel filtration 130 95 SDS-PAGE 14, 36, 76 27 (Heterotrimer) (Homotetramer) Reference
This study
8
Pseudomonas sp. MX-058 F10
F13
100 e 335 160 91 3.5e7.0 65
100 e 131 149 34 3.5e7.0 60
Pseudomonas sp. KY4690
100 13 90 e e e e
150 150 132 18, 39, 88 9, 18e23, 22, 9, 18e23, (Heterotrimer) 37e39, 58 37e39, 80 (Heterotetramer) (Heteropentamer) 9 9 13
Pseudomonas stutzeri Methylobacillus Streptomyces rimosus IFO 12695 sp. KY 4400 ATCC10970
100 4 80 e e 7.0 37
100 286 371 e e 3.0e4.0 50
100 17 57 30 e 7.0 30
160 18, 38, 83 (Heterotrimer)
142 18, 38, 88 (Heterotrimer)
150 23, 39, 79 (Heterotrimer)
14
15
16
e, data not shown in reference reports. a Data of Sasaki et al. (8) (formaldehyde and glycolaldehyde), Uchida et al. (14), and Yasuhara et al. (15) are derived from the relative specificity at 20 mM, 1.7 mM, and 15 mM of substrate, respectively, and those of this study and Sasaki et al. (8) (acetaldehyde, benzaldehyde, and glyoxal), Thiwthong et al. (9), Uchida et al. (13), and Uchida et al. (16) are derived from Vmax values.
glycolate, by incubation with 2-methoxyethanol. We also isolated Pseudomonas sp. AIU362 as another producer of ALOD with high activity for glyoxal but a low activity for glycolaldehyde (8). These results indicate that 2-methoxyethanol is an effective inducer to isolate the ALOD-producing microorganisms. In isolation of the ALOD-producing strains, checking of the ALOD activity on the agar plate using a color development solution and substrate was effective. In this study, the strain, which produces the ALOD showing high activity for glycolaldehyde, was isolated by using a color development solution and glycolaldehyde. The ALOD from Burkholderia sp. AIU 129 consisted of three different subunits, and oxidized aliphatic and aromatic aldehydes as well as glycolaldehyde, but did not oxidize aldehyde acids, ahydroxy acid, or alcohols (Table 2). ALODs consisting of three different subunits have also been reported from Pseudomonas sp. KY4690 (13), Pseudomonas stutzeri IFO12695 (14), Methylobacillus sp. KY440 (15), and Streptomyces rimosus ATCC10970 (16). These enzymes exhibited broad substrate specificity for aliphatic and aromatic aldehydes such as formaldehyde, acetaldehyde and benzaldehyde, but oxidase activity for glycolaldehyde was not demonstrated (Table 4). To date, only the ALODs from Pseudomonas sp. AIU 362 and Pseudomonas sp. MX-058 have been reported to show oxidase activity for glycolaldehyde (Table 4). However, the former ALOD consists of four identical subunits and has a molecular mass of 27 kDa (8), and the latter ALODs have heterotetramer and heteropentamer constructions, respectively (9). Thus, we first found the ALOD which oxidized glycolaldehyde and consisted of three heterosubunits. The N-terminal amino acid sequences of the a, b and g subunits of ALOD from Burkholderia sp. AIU 129 were similar to those of a putative molybdenum-binding protein subunit, a putative FADbinding subunit, and a putative ironesulfur-binding subunit of xanthine dehydrogenase from B. phenoliruptrix BR3459a, respectively (Fig. 3). The N-terminal amino acid sequence of the b subunit of the ALOD from Burkholderia sp. AIU 129 was also similar to that of the 39 kDa-subunit of ALOD from Pseudomonas sp. KY4690 (Fig. 3B), which belongs to a xanthine oxidase family that contains the [2Fee2S] cluster and molybdenum-molybdopterin-cytosine dinucleotide complex (13). These results indicated that the ALOD from Burkholderia sp. AIU 129 might belong to the ALOD group in the xanthine oxidase family. However, the ALOD from Burkholderia sp. AIU 129 did not show the oxidase or dehydrogenase activity for xanthine. The analysis of the effects of various compounds on the enzyme activity revealed that the ALOD from Burkholderia sp. AIU 129 was
inhibited by metals such as copper, iron, and molybdenum and chelating reagents such as a,a0 -dipyridyl and 8-hydroxyquinoline, indicating that metal might play an important role in the oxidation reaction. Furthermore, the metal-induced inhibition of ALOD activity also suggested that the ALOD from Burkholderia sp. AIU 129 would contain molybdenum as a cofactor, because it has been reported that heavy metal ions inhibit the activity of molybdenumcofactor-containing enzymes by binding to the dithiolene moiety of molybdopterin (17). Thus, these results supported the above conclusion that the ALOD from Burkholderia sp. AIU 129 might belong to the xanthine oxidase family. We are currently conducting the cloning and whole sequence analysis of our ALOD to identify the cofactors, because the enzyme productivity of the wild type strain was very low. In this report, we also demonstrated that the ALOD from Burkholderia sp. AIU 129 had no activity for glycolate (Table 2), and it was useful for the production of glycolate from glycolaldehyde without further oxidation of glycolate into glyoxylate. Further studies on the optimal reaction conditions for efficient production of glycolate without by-products are also now in progress. ACKNOWLEDGMENT The work described here was supported by a Grant-in-Aid for Scientific Research of Japan (no. 25820395) (to M. Yamada), from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. References 1. Deley, J. and Kersters, K.: Oxidation of aliphatic glycols by acetic acid bacteria, Bacteriol. Rev., 28, 164e180 (1964). 2. Harada, T. and Hirabayashi, T.: Utilization of alcohols by Hansenula miso, Agr. Biol. Chem., 32, 1175e1180 (1968). 3. Kataoka, M., Sasaki, M., Hidalgo, AR. G. D., Nakano, M., and Shimizu, S.: Glycolic acid production using ethylene glycol-oxidizing microorganisms, Biosci. Biotechnol. Biochem., 65, 2265e2270 (2001). 4. Couderc, R. and Baratti, J.: Oxidation of methanol by the Yeast, Pichia pastoris. Purification and properties of the alcohol oxidase, Agric. Biol. Chem., 44, 2279e2289 (1980). 5. Uwajima, T., Akita, H., Ito, K., Mihara, A., Aisaka, K., and Terada, O.: Formation and purification of a new Enzyme, glycerol oxidase and stoichiometry of the enzyme reaction, Agric. Biol. Chem., 44, 399e406 (1980). 6. Isobe, K. and Nishise, H.: A new enzymatic method for glycolaldehyde production from ethylene glycol, J. Mol. Catal. B: Enzym., 1, 37e43 (1995). 7. Yamada, M., Higashiyama, T., Kishino, S., Kataoka, M., Ogawa, J., Shimizu, S., and Isobe, K.: Novel alcohol oxidase with glycolate oxidase activity from Ochrobactrum sp. AIU 033, J. Mol. Catal. B: Enzym., 105, 41e48 (2014).
VOL. 119, 2015 8. Sasaki, Y., Isobe, K., Kataoka, M., Ogawa, J., Iwasaki, A., Hasegawa, J., and Shimizu, S.: Purification and characterization of a new aldehyde oxidase from Pseudomonas sp. AIU 362, J. Biosci. Bioeng., 106, 297e302 (2008). 9. Thiwthong, R., Kataoka, M., Iwasaki, A., Watanabe, H., Hasegawa, J., Isobe, K., and Shimizu, S.: Aldehyde oxidase carrying an unusual subunit structure from Pseudomonas sp. MX-058, Microb. Biotechnol., 1, 395e402 (2008). 10. Laemmli, U. K.: Cleavage of structure proteins during the assembly of the head of bacteriophage T4, Nature, 227, 680e685 (1970). 11. De Oliveira Cunha, C., Goda Zuleta, L. F., Paula de Almeida, L. G., Prioli Ciapina, L., Lustrino Borges, W., Pitard, R. M., Baldani, J. I., Straliotto, R., de Faria, S. M., Hungria, M., and other 3 authors: Complete genome sequence of Burkholderia phenoliruptrix BR3459a (CLA1), a heat-tolerant, nitrogen-fixing symbiont of Mimosa flocculosa, J. Bacteriol., 194, 6675e6676 (2012). 12. Chain, P. S., Denef, V. J., Konstantinidis, K. T., Vergez, L. M., Agullo, L., Reyes, V. L., Hauser, L., Cordova, M., Gomez, L., Gonzalez, M., and other 18 authors: Burkholderia xenovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility, Proc. Natl. Acad. Sci. USA, 103, 15280e15287 (2006). 13. Uchida, H., Kondo, D., Yamashita, A., Nagaosa, Y., Sakurai, T., Fujii, Y., Fujishiro, K., Aisaka, K., and Uwajima, T.: Purification and characterization of an aldehyde oxidase from Pseudomonas sp. KY 4690, FEMS Microbiol. Lett., 229, 31e36 (2003).
NEW ALDEHYDE OXIDASE FROM BURKHOLDERIA SP. AIU 129
415
14. Uchida, H., Fukuda, T., Satoh, Y., Okamura, Y., Toriyama, A., Yamashita, A., Aisaka, K., Sakurai, T., Nagaosa, Y., and Uwajima, T.: Characterization and potential application of purified aldehyde oxidase from Pseudomonas stutzeri IFO12695, Appl. Microbiol. Biotechnol., 68, 53e56 (2005). 15. Yasuhara, A., Akiba-Goto, M., Fujishiro, K., Uchida, H., Uwajima, T., and Aisaka, K.: Production of aldehyde oxidases by microorganisms and their enzymatic properties, J. Biosci. Bioeng., 94, 124e129 (2002). 16. Uchida, H., Okamura, Y., Yamanaka, H., Fukuda, T., Haneda, S., Aisaka, K., and Fujii, Y.: Purification and some properties of an aldehyde oxidase from Streptomyces rimosus ATCC10970, World J. Microbiol. Biotechnol., 22, 469e474 (2006). 17. Neumann, M. and Leimkuhler, S.: Heavy metal ions inhibit molybdoenzyme activity by binding to the dithiolene moiety of molybdopterin in Escherichia coli, FEBS J., 275, 5678e5689 (2008). 18. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res., 25, 3389e3402 (1997). 19. Thompson, J. D., Higgins, D. G., and Gibson, T. J.: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Res., 22, 4673e4680 (1994).
A new aldehyde oxidase catalyzing the conversion of glycolaldehyde to glycolate from Burkholderia sp. AIU 129 Miwa Yamada,1 Keika Adachi,1 Natsumi Ogawa,1 Shigenobu Kishino,2 Jun Ogawa,2 Michihiko Kataoka,3 Sakayu Shimizu,2, 4 and Kimiyasu Isobe1, * Department of Biological Chemistry and Food Science, Iwate University, Ueda-3, Morioka 020-8550, Japan,1 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan,2 Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 599-8531, Japan,3 and Faculty of Bioenvironmental Science, Kyoto Gakuen University, Sogabe-cho, Kameoka 621-8555, Japan4 Received 8 July 2014; accepted 3 September 2014 Available online 3 October 2014
We found a new aldehyde oxidase (ALOD), which catalyzes the conversion of glycolaldehyde to glycolate, from Burkholderia sp. AIU 129. The enzyme further oxidized aliphatic aldehydes, an aromatic aldehyde, and glyoxal, but not glycolate or alcohols. The molecular mass of this enzyme was 130 kDa, and it was composed of three different subunits (abg structure), in which the a, b, and g subunits were 76 kDa, 36 kDa, and 14 kDa, respectively. The N-terminal amino acid sequences of each subunit showed high similarity to those of putative subunits of xanthine dehydrogenase. Metals (copper, iron and molybdenum) and chelating reagents (a,a0 -dipyridyl and 8-hydroxyquinoline) inhibited the ALOD activity. The ALOD showed highest activity at pH 6.0 and 50 C. Twenty mM glycolaldehyde was completely converted to glycolate by incubation at 30 C for 3 h, suggesting that the ALOD found in this study would be useful for enzymatic production of glycolate. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Aldehyde oxidase; Glycolaldehyde; Glycolic acid; Burkholderia sp.; Xanthine oxidase/dehydrogenase]
Glycolate, which is the smallest a-hydroxy acid, is used as a dyeing and tanning agent in the textile industry, a flavoring agent and preservative in the food processing industry, and a skin care agent in the pharmaceutical industry. It is also useful for the production of polyglycolate and other biocompatible copolymers. Currently, ethylene glycol is utilized as an inexpensive starting material for the production of glycolate by oxidation reaction, while the chemical oxidation of ethylene glycol has certain drawbacks, such as the formation of formaldehyde as a by-product. Microbial methods for the production of glycolate from ethylene glycol by resting-cell reaction have also been studied using cells from Acetobacter, Gluconobacter, or Hansenula (1,2). Kataoka et al. (3) screened several microorganisms to produce a high concentration of glycolate in a medium containing ethylene glycol. Those results indicated that the microorganisms produced enzymes that catalyzed the conversion of ethylene glycol into glycolate via glycolaldehyde. However, the enzymes responsible for this reaction have not been purified and characterized. In terms of enzymes that are known to be involved in the oxidation of ethylene glycol, we have previously shown that the alcohol oxidases (EC 1.1.3.13) from methanolytic yeasts such as Candida sp. and Pichia pastoris (4) or glycerol oxidase from Aspergillus japonicas (5) catalyzed the oxidation of ethylene glycol to glyoxal via glycolaldehyde, and the reaction rate of ethylene glycol
* Corresponding author. Tel./fax: þ81 766 88 2280. E-mail address: [email protected] (K. Isobe).
oxidation was much faster than that of glycolaldehyde oxidation (6). Thus, those alcohol oxidases and glycerol oxidase were useful for accumulating a high concentration of glycolaldehyde from ethylene glycol (6). In our subsequent research, we have focused on a new enzymatic method for the production of glycolate from ethylene glycol utilizing two oxidases; ethylene glycol is first converted to glycolaldehyde by the alcohol oxidases from Candida sp. and P. pastoris or the glycerol oxidase from A. japonicas, and the resulting glycolaldehyde is then oxidized to glycolate by an aldehyde oxidase (ALOD) catalyzing the conversion of glycolaldehyde to glycolate. When this new enzymatic method utilizing two oxidases is fully developed, it will be applicable for the production of glyoxylate from ethylene glycol by addition of another alcohol oxidase with glycolate-oxidizing activity, which has already found in Ochrobactrum sp. AIU 033 by our group (7). Thus, it will be possible to produce glyoxylate, an attractive raw material for the chemical synthesis of vanillin, antibiotics or agrochemicals, using a combination of three microbial oxidases. ALODs exhibiting oxidase activity for glycolaldehyde have already reported from Pseudomonas sp. AIU 362 (8) and Pseudomonas sp. MX-058 (9), but the oxidation products were not identified. We therefore isolated a new bacterial strain that produces an enzyme catalyzing the oxidation of glycolaldehyde into glycolate, and revealed certain properties of both the strain and enzyme. The present paper describes our isolation of the new bacterial strain, and the remarkable characteristics of the enzyme it produces. The enzymatic production of glycolate from glycolaldehyde using the new ALOD is also described.
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.09.005
VOL. 119, 2015
NEW ALDEHYDE OXIDASE FROM BURKHOLDERIA SP. AIU 129 MATERIALS AND METHODS
TABLE 1. Purification of ALOD from Burkholderia sp. AIU 129. Step
Chemicals 2-Methoxyethanol, glycolate sodium salt, glyoxylate monohydrate, glycolaldehyde, and other aldehydes and alcohols were purchased from Wako Pure Chemical Industries (Osaka, Japan). Horseradish peroxidase (EC 1.11.1.7) was obtained from Amano Enzyme (Nagoya, Japan). All other chemicals used were of the highest grade commercially available. Isolation of the microorganism The enrichment culture was carried out three times using a 2-methoxyethanol medium containing 1% 2-methoxyethanol, 0.2% NH4NO3, 0.1% K2HPO4, 0.1% NaH2PO4, 0.02% MgSO4$7H2O, 0.01% CaCl2$2H2O, and 0.05% yeast extract, pH 6.0. The microorganisms grown in the 2-methoxyethanol medium were cultivated on an agar plate of the 2-methoxyethanol medium at 30 C for 2e3 days. The strains exhibiting glycolaldehyde-oxidizing activity were selected by evaluation of hydrogen peroxide formation on the agar plate. The formation of hydrogen peroxide was detected using a color development solution containing 0.64 mmol of 4-aminoantipyrine, 1.94 mmol of N-ethyl-N-(2-hydroxy-3sulfopropyl)-3-methylaniline sodium salt dihydrase (TOOS), 6.7 units of peroxidase, and 20 mmol of glycolaldehyde in 1.0 ml of 0.1 M of potassium phosphate, pH 7.0. The isolated strains were incubated in a test tube (16 1.5 cm diameter) containing 5 ml of the 2-methoxyethanol medium at 30 C for 2 days. Subsequently, a cell-free extract was prepared by disrupting the cells at below 5 C for 8 min using a Multibeads shocker (Yasui Kikai, Osaka, Japan). The strain exhibiting the highest activity on glycolaldehyde was selected, and used in this study. Identification of the isolated strain Identification of the newly isolated strain was performed at NCIMB Japan Co., Ltd. (Shizuoka, Japan). Cultivation of the strain The isolated strain was incubated in 5 ml of the 2methoxyethanol medium at 30 C for 2 days with shaking (120 strokes/min). The culture (1.5 ml) was inoculated into a 500-ml culture flask containing 150 ml of the medium, then incubated at 30 C for 2 days with shaking. Twenty milliliter of the second culture was transferred into a 3-l culture flask containing 2 l of the 2methoxyethanol medium, followed by cultivation at 30 C for 5 days with shaking. Assay of enzyme activity ALOD activity was assayed by measuring the initial rate of hydrogen peroxide formation at 30 C. The standard reaction mixture contained 20 mmol of glycolaldehyde or acetaldehyde, 0.6 mmol of 4-aminoantipyrine, 1.94 mmol of TOOS, 6.7 units of peroxidase, 0.1 mmol of potassium phosphate (pH 7.0), and an appropriate amount of enzyme in a final volume of 1.0 ml. The assay of enzyme activity was started by the addition of enzyme solution, and formation of hydrogen peroxidase was spectrophotometrically followed at 30 C for 5 min by measuring the absorbance at 555 nm. One unit of enzyme activity was defined as the amount of enzyme catalyzing the formation of 1 mmol of hydrogen peroxide per min. A molar absorptivity value of 16.5 103 M1 cm1 was used for calculation of the enzyme activity. Purification of the enzyme All procedures were performed at 5e10 C using potassium phosphate buffer (pH 6.0). The cells from 40 l of culture broth (34 g of wet weight) were disrupted with glass beads in 10 mM buffer solution by a Multi-beads shocker, and the supernatant was collected by centrifugation at 10,000 g for 30 min. Solid ammonium sulfate was added to the supernatant to reach 40% saturation with stirring, and it was further added to the supernatant to reach 60% saturation. The precipitate was dissolved with 10 mM buffer solution and dialyzed against 10 mM buffer solution. The dialyzed solution was applied onto a DEAEToyopearl 650 M column (20 3 cm) equilibrated with 10 mM buffer solution
411
Activity (unit)
Protein (mg)
Cell-free extract 12.2 15100 Ammonium sulfate 8.73 901 DEAE-Toyopearl 7.88 78.7 Phenyl-Toyopearl 2.25 1.86 Q-Sepharose 1.16 0.51 Hydroxyapatite 0.45 0.13 Gel filtration 0.215 0.06
Specific activity Recovery Purification (unit/mg of protein) (%) (fold) 0.00081 0.00968 0.100 1.21 2.27 3.41 3.42
100 71.6 64.8 18.5 9.53 3.70 1.77
1 12 124 1500 2820 4210 4220
ALOD activity was assayed under standard conditions using 20 mM glycolaldehyde. Specific activity is expressed as units per milligram of protein.
containing 60 mM NaCl. The absorbed enzyme was then eluted with a linear gradient of 60e190 mM NaCl in the 10 mM buffer. Solid ammonium sulfate was added to the active fractions to reach 140 mS/cm. Then, the enzyme solution was applied to a Phenyl-Toyopearl 650 M column (10 3 cm) equilibrated with 10 mM buffer solution containing 0.4 M ammonium sulfate, and the enzyme was eluted with a linear gradient of 0.4e0.15 M ammonium sulfate in the 10 mM buffer. The collected active fractions were dialyzed against 10 mM buffer solution, and applied to a Q-Sepharose column (10 3 cm) equilibrated with 10 mM buffer solution containing 150 mM NaCl, and the enzyme was eluted with a linear gradient of 150e250 mM NaCl in the 10 mM buffer. The active fractions were deionized by ultrafiltration. The enzyme solution was applied to a hydroxyapatite column (8 1.8 cm) equilibrated with 70 mM buffer solution, and the enzyme was eluted with a linear gradient of 70e200 mM buffer solutions. The active fractions were concentrated to 0.8 ml by ultrafiltration and applied to a Toyopearl HW-55 column (53 1.3 cm) equilibrated with 10 mM buffer solution. The protein concentration was spectrophotometrically determined by measuring the absorbance value at 1% 280 nm. An E value of 10.0 was used throughout this work. 1cm SDS-PAGE and molecular mass Sample for SDS-PAGE was incubated with 1% SDS and 5% mercaptoethanol at 100 C for 3 min. SDS-PAGE was performed according to the method of Laemmli (10). Proteins were stained with Coomassie Brilliant Blue R-250 or visualized by the silver staining method. The molecular mass of the denatured enzyme was estimated by SDS-PAGE using the molecular marker standards of Bio-Rad Japan (Tokyo, Japan). The molecular mass of intact proteins was estimated by gel filtration on a TSK gel G3000SWxL column (Tosoh, Tokyo, Japan). Analysis of N-terminal amino acid sequences After blotting subunit proteins from SDS-PAGE to a polyvinylidene fluoride membrane, the blotted protein was stained with Coomassie Brilliant Blue R-250. The protein bands were cut out, and the N-terminal amino acid sequences of three subunits were analyzed using a Shimadzu gas-phase protein sequencer equipped with an on-line reverse-phase chromatography system for identification of PTH-amino acids.
A
B
C
kDa
250 150 100 75 50 37 25 20
FIG. 1. Effect of cultivation time on enzyme production. Burkholderia sp. AIU 129 was cultured in the 2-methoxyethanol medium at 30 C. The enzyme activity was assayed under standard assay conditions. Closed circles, glycolaldehyde oxidase activity; open circles, absorbance at 660 nm of culture broth.
15 10 FIG. 2. Native-and SDS-PAGE of purified ALOD from Burkholderia sp. AIU 129. (A) Native enzyme, (B) denatured enzyme, (C) molecular makers.
412
YAMADA ET AL.
J. BIOSCI. BIOENG.,
TABLE 2. Substrate specificity and kinetic values of ALOD from Burkholderia sp. AIU 129. Substrate Glycolaldehyde Formaldehyde Acetaldehyde Propionaldehyde Butyraldehyde Benzaldehyde Glyoxal
Km (mM)
Vmax (mmol/min/mg protein)
Vmax/Km
1.00 35.9 0.34 0.74 0.10 0.06 12.3
3.42 4.38 6.42 14.6 8.83 5.58 4.51
3.42 0.122 18.9 19.7 88.3 93.0 0.367
The oxidase activity for aldehydes and alcohols was assayed under standard assay conditions using 40 mM substrate solution and 1.3 munits of ALOD activity for acetaldehyde. The ALOD showed no activity for glycolate, ethanol, 1-propanol, 2methoxyethanol, ethylene glycol, and glycerol. The Km and Vmax values were calculated by HaneseWoolf plot using 0.2e2.0 mM of glycolaldehyde, 4.0e40 mM of formaldehyde, 0.1e1.2 mM of acetaldehyde, 0.1e1.0 mM of propionaldehyde, 0.025e0.25 mM of butyraldehyde, 0.025e0.25 mM of benzaldehyde, and 2.5e15 mM of glyoxal.
TABLE 3. Effects of various compounds on enzyme activity. Compound Hydroxylamine Hydrazine Phenylhydrazine Semicarbazide o-Phenanthroline EDTA a,a0 -Dipyridyl 8-Hydroxyquinoline N-Ethylmaleimide Sodium azide Iodoacetic acid Potassium cyanide
Relative activity (%)
Metal
Relative activity (%)
99 73 6 97 109 92 49 20 100 100 100 83
None MgCl2 NiCl2 CoCl2 MnCl2 CuCl2 ZnCl2 FeCl2 MoCl5
100 93 81 100 95 4 67 0 7
Effects of chemicals and metals were assayed under standard conditions using 1.1 m units of enzyme activity for acetaldehyde and 1 mM chemicals or metals. Relative activity is indicated by percentage of enzyme activity without chemicals.
Glycolate production from glycolaldehyde Twenty millimoles of glycolaldehyde was incubated with 0.2 units of the purified enzyme and 0.1 units of catalase in 0.6 ml of 0.1 M potassium phosphate buffer (pH 7.0) at 30 C for 3 h. The concentration of glycolate in the reaction mixture was analyzed by HPLC with an ULTRON PS-80H column (Shinwa Chemical Industries, Tokyo, Japan). The elution was carried out at a flow rate of 1.0 ml per min at 60 C with perchloric acid solution (pH 2.1) for 20 min. The elution peaks of glycolate, glycolaldehyde, and glyoxylate were eluted at 9.9 min, 9.7 min, and 8.2 min, respectively, under the experimental conditions described above.
RESULTS Microorganism and culture conditions The selected strain was identified by phylogenetic analysis and biochemical properties. The results of the 16S rDNA sequence of 500 bp from the 50 -terminus showed that the isolated strain should be placed in the genus Burkholderia (data not shown). This isolated strain was not fermentative, not motile, not spore-forming, rod-shaped (0.7e0.8 2.0e2.5 mm), gram-negative, catalase-positive, and oxidase-positive. These biological characteristics supported the notion that the isolated strain belonged to the genus Burkholderia, and we therefore named it Burkholderia sp. AIU 129. The strain is currently kept at the Laboratory of Applied Microbiology, Department of Biological Chemistry and Food Science, Iwate University, Japan. When the isolated strain, Burkholderia sp. AIU 129, was incubated with the medium containing 2-methoxyethano, glucose or glycerol as a carbon source, glycolaldehyde-oxidizing activity was not detected in the cells cultured with glucose or glycerol, although cell growth was well in these medium. Thus, it was revealed that the glycolaldehyde-oxidizing enzyme was inductively produced by incubation with 2-methoxyethanol. Then, Burkholderia sp. AIU 129 was incubated in a 500-ml culture flask containing 150 ml of the 2methoxyethanol medium at 30 C for 5 days, and glycolaldehydeoxidizing activity was measured each day. The growth (absorbance at 660 nm) and glycolaldehyde-oxidizing activity increased with
A
B
C
FIG. 3. Alignment of N-terminal amino acid sequences of the a, b, and g subunits of ALOD from Burkholderia sp. AIU 129 to the homologous proteins. The N-terminal sequences were compared with sequences in GenBank, using the NCBI BLAST program (18). Protein sequences exhibiting high homology to N-terminal sequence were aligned using a CLUSTAL W program (19). Identical amino acid residues are boxed. (A) Alignment of the a subunit. Bsp AIU 129, ALOD from Burkholderia sp. AIU 129; Bp BR3459a, putative molybdopterinbinding protein subunit of xanthine dehydrogenase from Burkholderia phenoliruptrix BR3459a (90% identity) (11); Bx LB400, putative molybdopterin-binding protein subunit of xanthine dehydrogenase from Burkholderia xenovorans LB400 (71% identity) (12). (B) Alignment of the b subunit. Bsp AIU 129, ALOD from Burkholderia sp. AIU 129; Bp BR3459a; putative FAD-binding subunit of xanthine dehydrogenase from B. phenoliruptrix BR3459a (80% identity) (11); Bx LB400, putative oxidoreductase from B. xenovorans LB400 (72% identity) (12); Psp KY 4649, 39 kDa-subunit of ALOD from Pseudomonas sp. KY 4649 (53% identity) (13). (C) Alignment of the g subunit. Bsp AIU 129, ALOD from Burkholderia sp. AIU 129; Bp BR3459a; putative ironesulfur-binding subunit of xanthine dehydrogenase from B. phenoliruptrix BR3459a (80% identity) (11); Bx LB400, putative [2Fee2S] ironesulfur cluster binding protein from B. xenovorans LB400 (75% identity) (12).
VOL. 119, 2015
NEW ALDEHYDE OXIDASE FROM BURKHOLDERIA SP. AIU 129
the cultivation time, and glycolaldehyde-oxidizing activity reached the maximum at 4 or 5 days of cultivation (Fig. 1). Therefore, we harvested the cells after 5 days of incubation to obtain the enzyme exhibiting oxidase activity for glycolaldehyde.
413
A
Purification and molecular mass of the enzyme The purification procedure is summarized in Table 1. The glycolaldehydeoxidizing enzyme was purified to an electrophoretically homogeneous state by means of five column chromatographies. An approximately 4220-fold purification was achieved with an overall yield of 1.8%. The purified enzyme showed a single protein band on native-PAGE, and it exhibited three protein bands which were estimated to be 76 (a subunit), 36 (b subunit), and 14 kDa (g subunit), respectively (Fig. 2). The molecular mass of the native enzyme was estimated to be approximately 130 kDa on a TSK gel G3000SWxL column (data not shown). These results indicated that the enzyme consisted of three heterosubunits (abg structure). Substrate specificity and kinetic parameters Among the alcohols and aldehydes tested, aldehydes such as glycolaldehyde, formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, benzaldehyde, and glyoxal were oxidized, but glycolate and alcohols such as ethanol, 1-propanol, 2-methoxyethanol, ethylene glycol and glycerol were not (Table 2). The Km and Vmax values for glycolaldehyde, formaldehyde, acetaldehyde, propionaldehyde, and butyraldehyde were summarized in Table 2. It was revealed that the enzyme exhibited broad substrate specificity for aldehydes, and short-chain aliphatic aldehydes were good substrates for this enzyme. We therefore investigated the enzymatic properties of this ALOD using acetaldehyde as substrate in the following studies. Effects of compounds on the enzyme activity The effects of various compounds on the oxidase activity for acetaldehyde was analyzed by addition of 1 mM carbonyl reagents, chelating reagents, metals or other chemicals (Table 3). Among these compounds tested, Cu2þ, Fe2þ, Mo5þ and chelating reagents such as a,a0 -dipyridyl and 8-hydroxyquinoline strongly inhibited the enzyme activity. In addition, phenylhydrazine also strongly inhibited the enzyme activity. These results indicated that metals and a carbonyl group appeared to play roles in the enzyme activity. N-terminal amino acid sequence The N-terminal sequences of the a subunit, b subunit, and g subunit were found to be IETLPALRASGVPHKDVDGRLK, MNRFSYTRANEVSQAIEQARAKGAA, and LLDVAPSDVQRVPVSFDINGKKEVF, respectively. The sequence of the a subunit was similar to that of a putative molybdopterin-binding protein subunit of xanthine dehydrogenase from Burkholderia phenoliruptrix BR3459a (90% identity) (11) or Burkholderia xenovorans LB400 (71% identity) (12) (Fig. 3A). The sequence of the b subunit was similar to that of a putative FAD-binding subunit of xanthine dehydrogenase from B. phenoliruptrix BR3459a (80% identity) (11), a putative oxidoreductase from B. xenovorans LB400 (72% identity) (12), and a 39 kDa-subunit of ALOD from Pseudomonas sp. KY 4649 (53% identity) (13) (Fig. 3B). The sequence of the g subunit had high similarity to that of a putative ironesulfur-binding subunit of xanthine dehydrogenase from B. phenoliruptrix BR3459a (80% identity) (11) and a putative [2Fee2S] ironesulfur cluster-binding protein from B. xenovorans LB400 (75% identity) (12) (Fig. 3C). Effects of temperature and pH The effect of temperature on enzyme activity was assayed at pH 7.0 using acetaldehyde as substrate. The maximal oxidase activity for acetaldehyde was obtained at 50 C (Fig. 4A). When the enzyme was incubated with 0.1 M potassium phosphate buffer (pH 7.0) at 20e70 C for 30 min, the enzyme activity remained at 20e40 C and more than 50% of the original activity still remained at 70 C. The effect of pH on enzyme activity was assayed under standard assay conditions
B
FIG. 4. Effects of temperature and pH on the activity and stability of ALOD from Burkholderia sp. AIU 129. (A) Effects of temperature: Oxidase activity for acetaldehyde was assayed under standard conditions except for reaction temperature (closed circles). The thermal stability was assayed under standard assay conditions using acetaldehyde after the enzyme solution was incubated at pH 7.0 for 30 min at indicated temperatures (open circles). Percentage of remaining activity was obtained by ratio to activity without heating. (B) Effects of pH: Oxidase activity for acetaldehyde was assayed under standard conditions except for pH values (closed circles). The pH stability was assayed under standard assay conditions using acetaldehyde after heating at 30 C for 30 min at indicated pH (open circles). Percentage of remaining activity was obtained by ratio to activity without heating.
except for the pH values. In the pH range from 6.0 to 8.5, the maximum activity was seen at pH 6.0 (Fig. 4B). The pH stability was analyzed by incubation at 30 C for 30 min between pH 6.0 and 8.5. More than 70% of the enzyme activity remained in the pH region of 7.0e8.0. Production of glycolate from glycolaldehyde Since it was revealed that the enzyme was stable in the pH region of 7.0e8.0, 20 mM glycolaldehyde was incubated with 58 mg of the purified enzyme (0.2 units) at pH 7.0 and 30 C for 3 h. The reaction mixture was then applied to HPLC analysis with an ULTRON PS-80H column. A new peak was detected at 9.9 min, which was identical to the elution time for glycolate (data not shown). Thus, it was confirmed that glycolaldehyde was converted into glycolate by the enzyme. In addition, the glycolate concentration in the reaction mixture was estimated to be 19.8 mM (conversion yield, 99%), indicating that the enzyme would be useful for the production of glycolate from glycolaldehyde. DISCUSSION In this study, we isolated Burkholderia sp. AIU 129 as a producer of ALOD, which catalyzes the oxidation of glycolaldehyde to
414
YAMADA ET AL.
J. BIOSCI. BIOENG., TABLE 4. Comparison of characteristics of ALOD from Burkholderia sp. AIU 129 with other microbial ALODs.
Origin
Burkholderia sp. AIU 129
Pseudomonas sp. AIU 362
Substrate specificity (%)a Acetaldehyde 100 100 Formaldehyde 68 21 Benzaldehyde 87 75 Glyoxal 70 95 Glycolaldehyde 53 7 Opt-pH Below 6.0 6.0 Opt-temp ( C) 50 40e45 Molecular mass (kDa) Gel filtration 130 95 SDS-PAGE 14, 36, 76 27 (Heterotrimer) (Homotetramer) Reference
This study
8
Pseudomonas sp. MX-058 F10
F13
100 e 335 160 91 3.5e7.0 65
100 e 131 149 34 3.5e7.0 60
Pseudomonas sp. KY4690
100 13 90 e e e e
150 150 132 18, 39, 88 9, 18e23, 22, 9, 18e23, (Heterotrimer) 37e39, 58 37e39, 80 (Heterotetramer) (Heteropentamer) 9 9 13
Pseudomonas stutzeri Methylobacillus Streptomyces rimosus IFO 12695 sp. KY 4400 ATCC10970
100 4 80 e e 7.0 37
100 286 371 e e 3.0e4.0 50
100 17 57 30 e 7.0 30
160 18, 38, 83 (Heterotrimer)
142 18, 38, 88 (Heterotrimer)
150 23, 39, 79 (Heterotrimer)
14
15
16
e, data not shown in reference reports. a Data of Sasaki et al. (8) (formaldehyde and glycolaldehyde), Uchida et al. (14), and Yasuhara et al. (15) are derived from the relative specificity at 20 mM, 1.7 mM, and 15 mM of substrate, respectively, and those of this study and Sasaki et al. (8) (acetaldehyde, benzaldehyde, and glyoxal), Thiwthong et al. (9), Uchida et al. (13), and Uchida et al. (16) are derived from Vmax values.
glycolate, by incubation with 2-methoxyethanol. We also isolated Pseudomonas sp. AIU362 as another producer of ALOD with high activity for glyoxal but a low activity for glycolaldehyde (8). These results indicate that 2-methoxyethanol is an effective inducer to isolate the ALOD-producing microorganisms. In isolation of the ALOD-producing strains, checking of the ALOD activity on the agar plate using a color development solution and substrate was effective. In this study, the strain, which produces the ALOD showing high activity for glycolaldehyde, was isolated by using a color development solution and glycolaldehyde. The ALOD from Burkholderia sp. AIU 129 consisted of three different subunits, and oxidized aliphatic and aromatic aldehydes as well as glycolaldehyde, but did not oxidize aldehyde acids, ahydroxy acid, or alcohols (Table 2). ALODs consisting of three different subunits have also been reported from Pseudomonas sp. KY4690 (13), Pseudomonas stutzeri IFO12695 (14), Methylobacillus sp. KY440 (15), and Streptomyces rimosus ATCC10970 (16). These enzymes exhibited broad substrate specificity for aliphatic and aromatic aldehydes such as formaldehyde, acetaldehyde and benzaldehyde, but oxidase activity for glycolaldehyde was not demonstrated (Table 4). To date, only the ALODs from Pseudomonas sp. AIU 362 and Pseudomonas sp. MX-058 have been reported to show oxidase activity for glycolaldehyde (Table 4). However, the former ALOD consists of four identical subunits and has a molecular mass of 27 kDa (8), and the latter ALODs have heterotetramer and heteropentamer constructions, respectively (9). Thus, we first found the ALOD which oxidized glycolaldehyde and consisted of three heterosubunits. The N-terminal amino acid sequences of the a, b and g subunits of ALOD from Burkholderia sp. AIU 129 were similar to those of a putative molybdenum-binding protein subunit, a putative FADbinding subunit, and a putative ironesulfur-binding subunit of xanthine dehydrogenase from B. phenoliruptrix BR3459a, respectively (Fig. 3). The N-terminal amino acid sequence of the b subunit of the ALOD from Burkholderia sp. AIU 129 was also similar to that of the 39 kDa-subunit of ALOD from Pseudomonas sp. KY4690 (Fig. 3B), which belongs to a xanthine oxidase family that contains the [2Fee2S] cluster and molybdenum-molybdopterin-cytosine dinucleotide complex (13). These results indicated that the ALOD from Burkholderia sp. AIU 129 might belong to the ALOD group in the xanthine oxidase family. However, the ALOD from Burkholderia sp. AIU 129 did not show the oxidase or dehydrogenase activity for xanthine. The analysis of the effects of various compounds on the enzyme activity revealed that the ALOD from Burkholderia sp. AIU 129 was
inhibited by metals such as copper, iron, and molybdenum and chelating reagents such as a,a0 -dipyridyl and 8-hydroxyquinoline, indicating that metal might play an important role in the oxidation reaction. Furthermore, the metal-induced inhibition of ALOD activity also suggested that the ALOD from Burkholderia sp. AIU 129 would contain molybdenum as a cofactor, because it has been reported that heavy metal ions inhibit the activity of molybdenumcofactor-containing enzymes by binding to the dithiolene moiety of molybdopterin (17). Thus, these results supported the above conclusion that the ALOD from Burkholderia sp. AIU 129 might belong to the xanthine oxidase family. We are currently conducting the cloning and whole sequence analysis of our ALOD to identify the cofactors, because the enzyme productivity of the wild type strain was very low. In this report, we also demonstrated that the ALOD from Burkholderia sp. AIU 129 had no activity for glycolate (Table 2), and it was useful for the production of glycolate from glycolaldehyde without further oxidation of glycolate into glyoxylate. Further studies on the optimal reaction conditions for efficient production of glycolate without by-products are also now in progress. ACKNOWLEDGMENT The work described here was supported by a Grant-in-Aid for Scientific Research of Japan (no. 25820395) (to M. Yamada), from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. References 1. Deley, J. and Kersters, K.: Oxidation of aliphatic glycols by acetic acid bacteria, Bacteriol. Rev., 28, 164e180 (1964). 2. Harada, T. and Hirabayashi, T.: Utilization of alcohols by Hansenula miso, Agr. Biol. Chem., 32, 1175e1180 (1968). 3. Kataoka, M., Sasaki, M., Hidalgo, AR. G. D., Nakano, M., and Shimizu, S.: Glycolic acid production using ethylene glycol-oxidizing microorganisms, Biosci. Biotechnol. Biochem., 65, 2265e2270 (2001). 4. Couderc, R. and Baratti, J.: Oxidation of methanol by the Yeast, Pichia pastoris. Purification and properties of the alcohol oxidase, Agric. Biol. Chem., 44, 2279e2289 (1980). 5. Uwajima, T., Akita, H., Ito, K., Mihara, A., Aisaka, K., and Terada, O.: Formation and purification of a new Enzyme, glycerol oxidase and stoichiometry of the enzyme reaction, Agric. Biol. Chem., 44, 399e406 (1980). 6. Isobe, K. and Nishise, H.: A new enzymatic method for glycolaldehyde production from ethylene glycol, J. Mol. Catal. B: Enzym., 1, 37e43 (1995). 7. Yamada, M., Higashiyama, T., Kishino, S., Kataoka, M., Ogawa, J., Shimizu, S., and Isobe, K.: Novel alcohol oxidase with glycolate oxidase activity from Ochrobactrum sp. AIU 033, J. Mol. Catal. B: Enzym., 105, 41e48 (2014).
VOL. 119, 2015 8. Sasaki, Y., Isobe, K., Kataoka, M., Ogawa, J., Iwasaki, A., Hasegawa, J., and Shimizu, S.: Purification and characterization of a new aldehyde oxidase from Pseudomonas sp. AIU 362, J. Biosci. Bioeng., 106, 297e302 (2008). 9. Thiwthong, R., Kataoka, M., Iwasaki, A., Watanabe, H., Hasegawa, J., Isobe, K., and Shimizu, S.: Aldehyde oxidase carrying an unusual subunit structure from Pseudomonas sp. MX-058, Microb. Biotechnol., 1, 395e402 (2008). 10. Laemmli, U. K.: Cleavage of structure proteins during the assembly of the head of bacteriophage T4, Nature, 227, 680e685 (1970). 11. De Oliveira Cunha, C., Goda Zuleta, L. F., Paula de Almeida, L. G., Prioli Ciapina, L., Lustrino Borges, W., Pitard, R. M., Baldani, J. I., Straliotto, R., de Faria, S. M., Hungria, M., and other 3 authors: Complete genome sequence of Burkholderia phenoliruptrix BR3459a (CLA1), a heat-tolerant, nitrogen-fixing symbiont of Mimosa flocculosa, J. Bacteriol., 194, 6675e6676 (2012). 12. Chain, P. S., Denef, V. J., Konstantinidis, K. T., Vergez, L. M., Agullo, L., Reyes, V. L., Hauser, L., Cordova, M., Gomez, L., Gonzalez, M., and other 18 authors: Burkholderia xenovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility, Proc. Natl. Acad. Sci. USA, 103, 15280e15287 (2006). 13. Uchida, H., Kondo, D., Yamashita, A., Nagaosa, Y., Sakurai, T., Fujii, Y., Fujishiro, K., Aisaka, K., and Uwajima, T.: Purification and characterization of an aldehyde oxidase from Pseudomonas sp. KY 4690, FEMS Microbiol. Lett., 229, 31e36 (2003).
NEW ALDEHYDE OXIDASE FROM BURKHOLDERIA SP. AIU 129
415
14. Uchida, H., Fukuda, T., Satoh, Y., Okamura, Y., Toriyama, A., Yamashita, A., Aisaka, K., Sakurai, T., Nagaosa, Y., and Uwajima, T.: Characterization and potential application of purified aldehyde oxidase from Pseudomonas stutzeri IFO12695, Appl. Microbiol. Biotechnol., 68, 53e56 (2005). 15. Yasuhara, A., Akiba-Goto, M., Fujishiro, K., Uchida, H., Uwajima, T., and Aisaka, K.: Production of aldehyde oxidases by microorganisms and their enzymatic properties, J. Biosci. Bioeng., 94, 124e129 (2002). 16. Uchida, H., Okamura, Y., Yamanaka, H., Fukuda, T., Haneda, S., Aisaka, K., and Fujii, Y.: Purification and some properties of an aldehyde oxidase from Streptomyces rimosus ATCC10970, World J. Microbiol. Biotechnol., 22, 469e474 (2006). 17. Neumann, M. and Leimkuhler, S.: Heavy metal ions inhibit molybdoenzyme activity by binding to the dithiolene moiety of molybdopterin in Escherichia coli, FEBS J., 275, 5678e5689 (2008). 18. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res., 25, 3389e3402 (1997). 19. Thompson, J. D., Higgins, D. G., and Gibson, T. J.: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Res., 22, 4673e4680 (1994).