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Cloning, sequencing and expression analysis of a gene encoding alcohol oxidase in Paenibacillus sp. AIU 311
Journal of Bioscience and Bioengineering VOL. 110 No. 2, 147 – 151, 2010 www.elsevier.com/locate/jbiosc
Cloning, sequencing and expression analysis of a gene encoding alcohol oxidase in Paenibacillus sp. AIU 311 Yasutaka Sasaki,1 Michihiko Kataoka,2 Nobuyuki Urano,2 Jun Ogawa,2 Akira Iwasaki,3 Junzo Hasegawa,3 Kimiyasu Isobe,1,⁎ and Sakayu Shimizu2 The United Graduate School of Agricultural Sciences, Iwate University, 18-8, Ueda 3-chome, Morioka 020-8550, Japan 1 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan 2 and Life Science R & D Center, Kaneka Co., Miyamae-machi, Takasaga-cho, Takasago 676-8688, Japan 3 Received 17 December 2009; accepted 25 January 2010 Available online 23 February 2010
We have cloned a gene encoding an alcohol oxidase (AOD) specific to aldehyde alcohols from Paenibacillus sp. AIU 311. The AOD gene contains an open reading frame consisting of 618 nucleotides corresponding to 205 amino acid residues. The deduced amino acid sequence exhibits a high similarity to that of manganese superoxide dismutases (SODs). We expressed the cloned gene as an active product in Escherichia coli BL21 cells. The productivity (total units per culture broth volume) of the recombinant AOD expressed in E. coli BL21 is 26,000-fold higher than that of AOD in Paenibacillus sp. AIU 311. The recombinant AOD also exhibits aldehyde alcohol oxidase activity and SOD activity. The recombinant cells described in this study have utility for the production of glyoxal from glycolaldehyde. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Glycolaldehyde; Glyoxal; Aldehyde alcohol; Alcohol oxidase; Superoxide dismutase; Paenibacillus sp.]
Recently, we demonstrated that Paenibacillus sp. AIU 311 isolated by our group produced an alcohol oxidase (AOD) with high activity for aldehyde alcohols such as glycolaldehyde, glyceraldehyde, and aldotetrose, but not for methanol, ethanol, ethylene glycol or glycerol (1). The N-terminal amino acid sequence of this AOD exhibited no similarity to that of AODs from methylotrophic yeasts, such as Candida and Pichia, which catalyze the oxidation of primary alcohols to their respective aldehydes (2–5). We therefore concluded in our previous report that Paenibacillus AOD is a novel enzyme with high specificity to a hydroxy group of aldehyde alcohols (1). In further studies of the Paenibacillus AOD, we obtained new findings that the N-terminal amino acid sequence of the Paenibacillus AOD is homologous to that of superoxide dismutases (SODs) and the AOD exhibits SOD activity (6). Our previous report also demonstrated that SODs containing manganese, iron, or copper and zinc as cofactors exhibit oxidase activity with aldehyde alcohols, such as glycolaldehyde, glyceraldehyde, and aldotetrose (6). In this paper, we report the cloning and sequence analysis of the AOD gene from Paenibacillus sp. AIU 311. We also characterize the expression of the AOD gene in Escherichia coli as well as the production of glyoxal from glycolaldehyde using recombinant cells. MATERIALS AND METHODS Chemicals Glycolaldehyde dimer was purchased from Sigma Japan (Tokyo, Japan). Peroxidase was obtained from Amano Enzymes (Nagoya, Japan). All other
⁎ Corresponding author. Tel./fax: +81 19 621 6155. E-mail address: [email protected] (K. Isobe).
chemicals used were commercial products of the highest grade available. Ni-charged resin was purchased from Bio-Rad Laboratories Japan (Tokyo, Japan). Medium and culture conditions Paenibacillus sp. AIU 311, which is a source of chromosomal DNA, was incubated in an ethylene glycol medium consisting of 1% ethylene glycol, 0.2% NH4NO3, 0.1% K2HPO4, 0.1% NaH2PO4, 0.05% MgSO4·7H2O, 0.02% CaCl2·2H2O, and 0.05% yeast extract, pH 7.0, at 30 °C for 3 days (d). A transformant carrying the AOD gene was incubated in 5.0 ml of a Luria-Bertani (LB) medium (1% peptone, 0.5% yeast extract, and 0.5% NaCl, pH 7.0) containing ampicillin (50 μg/ml) at 30 °C for 5 h, and then isopropyl-β-D-thiogalactopyranoside (IPTG, 0.5 mM) was added to the culture medium and further incubated at 30 °C for 20 h. Degenerate PCR The primers for degenerate PCR were designed based on the N-terminal amino acid sequence (1), (HIDAQTMEI) and the internal amino acid sequence, (LAVTSTPNQDNPLMEGQTPVLGLDVWEHAYYLK), which was obtained by a lysyl endopeptidase digestion of the purified AOD from Paenibacillus sp. AIU 311. Thus, the following oligonucleotides were designed as the sense primer (S1) and the antisense primer (A1): 5′-CA(T,C)AT(A,T,C)GA(T,C)GCICA(A,G)AC(A,T,C,G)ATGGA(A, G)AT-3′ and 5′-TA(A,G)TA(A,T,C,G)GC(A,G)TG(T,C)TCCCA(A,T,C,G)AC(A,G)TCIA-3′, respectively (see Fig. 1). The PCR was carried out in a 50-μl volume consisting of 2.5 units of TaKaRa Ex Taq DNA polymerase, 20 nmol of dNTP, 0.5 μmol of each primer, and 0.5 μg of the extracted chromosomal DNA. The reaction mixture was first heated at 94 °C for 5 min, followed by 30 cycles of amplification (a denaturation step at 94 °C for 30 s, an annealing step at 55 °C for 30 s, and an extension step at 72 °C for 1 min). Southern hybridization The chromosomal DNA from Paenibacillus sp. AIU 311 was digested with BamHI, HindIII, NdeI, PstI, SacI, SalI, SpeI, SphI or XbaI, and the digested DNA was subjected to gel electrophoresis on a 1% agarose gel in TEA buffer at 50 V for 3 h. The DNA fragments on the gel were transferred to a nitrocellulose membrane by capillary blotting and incubated at 55 °C for 24 h with the labeled DNA fragment homologous to the AOD gene (described above). Colony hybridization A ligation mixture (10 μl) consisting of Ligation Solution I (DNA Ligation Kit Ver. 2.1, Takara Bio), 1 μg of a chromosomal DNA digested with NdeI and 100 ng of cloning vector DNA (pT 7 Blue cloning vector, Novagen) digested with NdeI was incubated at 16 °C overnight. The resulting plasmids were subsequently
1389-1723/$ - see front matter © 2010, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2010.01.019
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FIG. 1. Nucleotide sequence of a part of the NdeI fragment containing the AOD gene from Paenibacillus sp. AIU 311. The initiation and stop codons are indicated by a box. S1-S3 and A1A3 indicate the sequences used for the primers. The deduced amino acids are also indicated under the DNA sequence.
transformed into E. coli JM 109. The positive clones were selected on a LB-agar plate containing X-gal/IPTG and ampicillin by blue-white selection. DNA sequence analysis The DNA sequences were determined by the dideoxy chain termination method using a DNA sequencer CEQ2000 XL (Beckman Coulter Inc., CA, U.S.A.) with a CEQ Dye Terminator Cycle Sequencing Kit (Beckman Coulter Inc.). Two sequence primers, 5′-CAGGTTGGAGATCAGCTCTTCAAGGC-3′ (S2) and 5′CATGGCTCGTCGTGGACAAGAGCGGC-3′ (A2) were designed to sequence the 5′ and 3′ regions of the AOD gene, respectively, in the NdeI fragment (see Fig. 1). DNA sequence analysis software (GENETYX) was used to identify the open reading frame and restriction enzyme sites as well as for the analysis of the deduced amino acid sequence. Expression of the Paenibacillus AOD in E. coli On the basis of the sequence obtained from the NdeI fragment, two oligonucleotides, 5′-CCCATATGGCATTTCAATTACCACC-3′ (S3) and 5′-CCCTCGAGCGGCTGTAC GATTTCCT-3′ (A3), were designed as a sense primer containing an NdeI site and an antisense primer containing a XhoI site, respectively (see Fig. 1). The AOD gene was amplified by PCR using these primers and ligated into an NdeI–XhoI site of the pET-21a(+) expression vector, containing a histidine-tag site. The pET-21a(+) plasmid containing the AOD gene (pET-GAOX) was transformed into E. coli BL21 cells (E. coli BL21/pET-GAOX), as described above.
Purification of recombinant AOD The recombinant cells (170 mg of wet weight) obtained at 25 h of cultivation from 100 ml of culture broth were suspended in 3 ml of 10 mM phosphate buffer, pH 7.0, and disrupted with glass beads by a Multi-beads shocker (Yasui Kikai, Osaka) for 10 min. The crude enzyme solution (5.0 ml) was applied to a Ni-charged resin column (1.0 × 2.0 cm), equilibrated with 50 mM sodium phosphate buffer, pH 8.0, containing 0.3 M NaCl, and eluted with 50 mM sodium phosphate buffer, pH 8.0, containing 0.3 M NaCl and 0.25 M imidazole. Oxidation reaction of glycolaldehyde with recombinant cells The recombinant cells (27 mg of wet weight) of E. coli BL21/pET-GAOX were harvested from 5 ml of the LB medium containing ampicillin and IPTG after 25 h of incubation at 30 °C. The cells were then suspended in 1.0 ml of 0.2 M phosphate buffer, pH 7.0, and 0.25 ml of the cell suspension was added to 1.5 ml of 100 mM glycolaldehyde. The reaction was carried out at 30 °C for 3 d with shaking at 120 strokes per min. Assay of enzyme activity Enzyme activities of glycolaldehyde oxidase and SOD were assayed by measuring the rate of hydrogen peroxide formation at 30 °C according to our previous report (6). Assay of glyoxal The concentration of glyoxal was assayed according to the method of Isobe and Nishise (7).
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Cloning of the gene encoding AOD from Paenibacillus sp. AIU 311 A partial DNA fragment of approximately 0.5 kb, containing 415 nucleotides of the AOD gene, was amplified from the chromosomal DNA of Paenibacillus sp. AIU 311 by PCR using S1 and A1 primers. The chromosomal DNA of Paenibacillus sp. AIU 311 was then digested with BamHI, HindIII, NdeI, PstI, SacI, SalI, SpeI, SphI or XbaI, and a DNA fragment containing the AOD gene was analyzed by Southern hybridization using the amplified DNA fragment (described above) as a probe. Because the AOD gene was contained in DNA fragments digested with NdeI, the NdeI fragments (5.0 kb) from chromosomal DNA were then ligated to a pT 7 blue cloning vector digested with NdeI and the resulting plasmids were transformed into E. coli JM 109. The positive clones were selected on an LB-agar plate containing X-gal/IPTG and ampicillin. Sequence analysis of the AOD gene The complete gene structure of AOD was not obtained by PCR using the S1 and A1 primers; therefore, newly designed primers, S2 and A2 were used for sequencing of unknown regions of the 5′- and 3′-ends of the AOD
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gene from the NdeI fragments described above. The entire length of the AOD gene was determined from the sequencing results of degenerate PCR product and the 5′- and 3′-ends of the AOD gene from the NdeI fragment. Fig. 1 shows the nucleotide sequence containing the AOD gene, in which an ORF of 618 bp encodes a 205 amino acid polypeptide. The predicted molecular mass of the ORF is in agreement with that of a subunit of the AOD from Paenibacillus sp. AIU 311. In addition, the deduced amino acid sequence of the N-terminal region of the ORF is identical to the amino acid sequence of the Paenibacillus AOD. The amino acid sequence obtained by the lysyl endopeptidase digestion of the purified AOD was also found in the deduced amino acid sequence. We therefore concluded that the ORF of 618 bp is the complete structural gene of AOD from Paenibacillus sp. AIU 311, and the NdeI fragment was used in the following studies. The sequence data of 618 nucleotides in the ORF correspond to the AOD gene in the DDBJ nucleotide sequence databases (accession number, AB509365). The amino acid sequence of the AOD polypeptide was used to search for homologous sequence using the BLAST (Basic Local Alignment Search Tool) program. More than 50% of the deduced
FIG. 2. Alignment of the amino acid sequences of the AOD protein from Paenibacillus sp. AIU 311 and SODs from bacterial strains. The deduced amino acid sequence of AOD from Paenibacillus sp. AIU 311 (GAOX) was compared to the sequences of Mn-SOD from T. thermophilus HB 27 (TTMS) (8), B. caldotenax YT 1 (BCMS) (9), B. stearothermophilus NCA 1503 (BSMS) (10), B. halodenitrificans ATCC 49067 (BHMS) (11), E. coli AB 2463/pDT1-5 (ECMS) (12), D. radiodurans DR 1279 (DRMS) (13) and S. cerevisiae FL 100 (SCMS) (14), Fe-SOD from A. aerogenes (AAFS) (15), and Cu, Zn-SOD from E. coli (ECCZS) (16). Amino acids identical to that of AOD from Paenibacillus sp. AIU 311 are depicted by boxes.
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FIG. 3. SDS-PAGE of recombinant AOD. Line A, cell-free extract from E.coli BL21; line B, cell-free extract from E.coli BL21/pET-GAOX; line C, purified recombinant AOD; line D, standard proteins.
amino acid sequence of the Paenibacillus AOD is identical to that of the manganese-containing SOD (Mn-SOD) from Thermus thermophilus HB 27 (8) and the putative Mn-SODs from Bacillus caldotenax YT 1(9) and Bacillus stearothermophilus NCA 1503 (10). The Paenibacillus AOD also exhibits high similarity to Mn-SODs from Bacillus halodenitrificans ATCC 49067 (11) and E. coli AB 2463/pDT1-5 (12) in the region of 140 residues from the Nterminus. In contrast, the amino acid sequence of the Paenibacillus AOD exhibits low similarity to that of Mn-SODs from Deinococcus radiodurans DR 1279 (13) and Saccharomyces cerevisiae FL 100 (14), and Fe-SOD from Aerobacter aerogenes IFO 3317 (15); however, these SODs exhibit high similarity in the region of 44 residues from the N-terminus. The Paenibacillus AOD shows no similarity to Cu, Zn-SOD from E. coli QC 871 (16) (Fig. 2). Thus, the deduced amino acid sequence of the AOD from Paenibacillus sp. AIU 311 shows high similarity to that of some Mn-SODs but not to other SODs and primary alcohol oxidases. Overexpression of the gene encoding AOD in E. coli cells The E. coli BL21/pET-GAOX transformant cells, which are E. coli BL21 cells harboring a pET-21a(+) plasmid containing the AOD gene, were incubated in the LB medium as described in Materials and Methods. The AOD protein was expressed in a soluble histidine-tagged form and
FIG. 4. Effects of pH, temperature and the cell amount on glyoxal production. (A) Effect of pH. The cell suspension and 100 mM glycolaldehyde solution were prepared using 0.2 M phosphate buffer, pH 5.0–8.5, and the cell reactions were carried out at 30 °C for 3 d with shaking. The relative amount was expressed as a percentage of the highest amount of glyoxal produced at pH 5.5. (B) Effect of temperature. The cell suspension and a 100-mM glycolaldehyde solution were prepared using 0.2 M phosphate buffer, pH 5.5, and the reactions were carried out at the indicated temperatures for 3 d with shaking. The relative amount was expressed as a percentage of the highest amount of glyoxal produced at 50 °C. (C) Effect of the cell amounts. The indicated amounts of recombinant cells were incubated with 85 mM glycolaldehyde at 50 °C and pH 5.5 for 7 d. Open triangles, 1. 7 mg of cells; closed triangles, 3. 4 mg of cells; open circles, 5. 1 mg of cells; closed circles, 6. 8 mg of cells.
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Purification of recombinant AOD The recombinant cells obtained at 25 h of cultivation from 100 ml of culture broth were disrupted with glass beads by a Multi-beads shocker and the supernatant was applied to a Ni-charged resin column as described in Materials and methods. The recombinant AOD protein was purified to a homogeneous state by column chromatography (Fig. 3). The molecular mass of the recombinant enzyme is approximately 24 kDa (deduced by SDS-PAGE), which is in agreement with that of the Paenibacillus AOD protein. The purified enzyme exhibited glycolaldehyde oxidase activity (0.83 unit per mg of protein) and SOD activity (1.28 units per mg of protein). The ratio of SOD activity to glycolaldehyde oxidase activity is also in agreement with that of the Paenibacillus AOD protein. Production of glyoxal by E. coli BL21/pET-GAOX cells We observed the formation of glyoxal when the E. coli BL21/pET-GAOX cells (6.8 mg of wet weight) were incubated with 85 mM glycolaldehyde at 30 °C and pH 7.0 for 3 d (data not shown). We then investigated the optimal conditions for glyoxal production. When the reaction pH was varied between 5.0 and 8.5, we found glyoxal production was highest at pH 5.5–6.0 (Fig. 4A). When the reaction was carried out at pH 5.5, the amount of glyoxal was highest at 50 °C after 3 d, when the reaction temperature was varied between 20 and 60 °C (Fig. 4B). On the basis of these results, cell amounts were increased at pH 5.5 and 50 °C. The amount of glyoxal produced was increased by increasing cell amounts and approximately 35 mM of glyoxal was produced from 85 mM glycolaldehyde after 7 d of incubation at 50 °C and pH 5.5 (Fig. 4C). DISCUSSION We recently reported that the N-terminal amino acid sequence of the Paenibacillus AOD protein is homologous to that of SODs, the Paenibacillus AOD exhibits SOD activity, and SODs from the other sources also exhibit oxidase activity with aldehyde alcohols, such as glycolaldehyde, glyceraldehyde, and aldotetrose (6). Therefore, we cloned and sequenced the AOD gene. The deduced amino acid sequence was compared to the other SODs. More than 50% of the deduced amino acid sequence of the Paenibacillus AOD is identical to the Mn-SOD from T. thermophilus (8) as well as putative Mn-SODs from B. caldotenax (9) and B. stearothermophilus (10). However, the amino acid sequence similarity between the Paenibacillus AOD and Fe-SOD or Cu, Zn-SOD is lower than 25%. These results support our previous results described in Ref. (1) and (6), and furthermore, this study suggests that the Paenibacillus AOD is a new member of MnSOD. The amino acid sequence analysis also demonstrates that the similarity of the N-terminal region is much higher than that of the Cterminal region in the above homologous enzymes. These results provide strong evidence for the future investigation relating the structure and enzyme activity of AOD and SOD in these enzymes. Studies of glyoxal production from glycolaldehyde using cells of or AOD from Paenibacillus sp. AIU 311 have been difficult due to the very low productivity of AOD by Paenibacillus sp. AIU 311. However, in the present study, the AOD from Paenibacillus is produced approximately 26,000-fold higher in the cells of E. coli BL21/pETGAOX. We therefore investigated the production of glyoxal from glycolaldehyde using recombinant cells. Glyoxal production favored an optimum pH of 5.5, which is a more acidic condition than that of the purified enzyme (1). This difference is most likely due to the pH stability of the enzyme. When the cell reaction was carried out using
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the E. coli BL21/pET-GAOX cells at 50 °C for 7 d, approximately 40% of glycolaldehyde was converted to glyoxal. These results indicate that glycolaldehyde was converted to glyoxal by the E. coli BL21/ pET-GAOX cells. In a future study, we intend to apply these recombinant cells or the AOD enzyme to the production of glyoxylic acid from ethylene glycol by coupling with alcohol oxidase catalyzing the conversion of ethylene glycol to glycolaldehyde (7) and aldehyde oxidase catalyzing the conversion of glyoxal to glyoxylic acid (17). References 1. Isobe, K., Kato, A., Sasaki, Y., Suzuki, S., Kataoka, M., Ogawa, J., Iwasaki, A., Hasegawa, J., and Shimizu, S.: Purification and characterization of a novel alcohol oxidase from Paenibacillus sp. AIU311, J. Biosci. Bioeng., 104, 124–128 (2007). 2. Ledeboer, A. M., Edens, L., Maat, J., Visser, C., Bos, J. W., Verrips, C. T., Janowicz, Z., Eckart, M., Roggenkamp, R., and Hollenberg, C. P.: Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha, Nucleic Acids Res., 13, 3063–3082 (1985). 3. Koutz, P., Davis, G. R., Stillman, C., Barring, K., Cregg, J., and Thill, G.: Structural comparison of the Pichia pastoris alcohol oxidase gene, Yeast, 5, 167–177 (1989). 4. Sakai, Y. and Tani, Y.: Cloning and sequencing of the alcohol oxidase-encoding gene (AOD1) from the formaldehyde-producing asporogeneous methylotrophic yeast, Candida boidinii S2, Gene, 114, 67–73 (1992). 5. Nakagawa, T., Mukaiyama, H., Yurimoto, H., Sakai, Y., and Kato, N.: Alcohol oxidase hybrid oligomers formed in vivo and in vitro, Yeast, 15, 1223–1230 (1999). 6. Isobe, K., Kato, A., Sasaki, Y., Kataoka, M., Ogawa, J., Iwasaki, A., Hasegawa, J., and Shimizu, S.: Superoxide dismutases exhibit oxidase activity on aldehyde alcohols similar to alcohol oxidase from Paenibacillus sp. AIU 311, J. Biosci. Bioeng., 105, 666–670 (2008). 7. Isobe, K. and Nishise, H.: Enzymatic production of glyoxal from ethylene glycol using alcohol oxidase from methanol yeast, Biosci. Biotechnol. Biochem., 58, 170–173 (1994). 8. Ludwig, M. L., Metzger, A. L., Pattridge, K. A., and Stallings, W. C.: Manganese superoxide dismutase from Thermus thermophilus. A structural model refined at 1.8 Å resolution, J. Mol. Biol., 219, 335–358 (1991). 9. Chambers, S. P., Brehm, J. K., Michael, N. P., Atkinson, T., and Minton, N. P.: Physical characterisation and over-expression of the Bacillus caldotenax superoxide dismutase gene, FEMS Microbiol. Lett., 70, 277–284 (1992). 10. Brehm, J. K., Chambers, S. P., Bown, K. J., Atkinson, T., and Minton, N. P.: Molecular cloning and nucleotide sequence determination of the Bacillus stearothermophilus NCA 1503 superoxide dismutase gene and its overexpression in Eschericia coli, Appl. Microbiol. Biotechnol., 36, 358–363 (1991). 11. Liao, J., Li, M., Liu, M. Y., and Chang, W. R.: Crystallization and preliminary crystallographic analysis of manganese superoxide dismutase from Bacillus halodenitrificans, Biochem. Biophys. Res. Commun., 294, 60–62 (2002). 12. Edwards, R. A., Baker, H. M., Whittaker, M. M., Whittaker, J. W., Jameson, G. B., and Baker, E. N.: Crystal structure of Escherichia coli manganese superoxide dismutase at 2.1-Å resolution, J. Biol. Inorg. Chem., 3, 161–171 (1998). 13. Dennis, R. J., Micossi, E., McCarthy, J., Moe, E., Gordon, E. J., KozielskiStuhrmann, S., Leonard, G. A., and McSweeney, S.: Structure of the manganese superoxide dismutase from Deinococcus radiodurans in two crystal forms, Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 62, 325–329 (2006). 14. Marres, C. A., Van Loon, A. P., Oudshoorn, P., Van Steeg, H., Grivell, L. A., and Slater, E. C.: Nucleotide sequence analysis of the nuclear gene coding for manganese superoxide dismutase of yeast mitochondria, a gene previously assumed to code for the Rieske iron-sulphur protein, Eur. J. Biochem., 147, 153–161 (1985). 15. Lee, S. O., Kim, S. W., Uno, I., and Lee, T. H.: Direct sequencing of superoxide dismutase genes from two bacterial strains amplified by polymerase chain reaction, Biosci. Biotechnol. Biochem., 57, 1454–1460 (1993). 16. Battistoni, A., Folcarelli, S., Gabbianelli, R., Capo, C., and Rotilio, G.: The Cu,Zn superoxide dismutase from Escherichia coli retains monomeric structure at high protein concentration. Evidence for altered subunit interaction in all the bacteriocupreins, Biochem. J., 320, 713–716 (1996). 17. Sasaki, Y., Isobe, K., Kataoka, M., Ogawa, J., Iwasaki, A., Hasegawa, J., and Shimizu, S.: Purification and characterization of a novel aldehyde oxidase from Pseudomonas sp. AIU 362, J. Biosci. Bioeng., 106, 297–302 (2008).
Cloning, sequencing and expression analysis of a gene encoding alcohol oxidase in Paenibacillus sp. AIU 311 Yasutaka Sasaki,1 Michihiko Kataoka,2 Nobuyuki Urano,2 Jun Ogawa,2 Akira Iwasaki,3 Junzo Hasegawa,3 Kimiyasu Isobe,1,⁎ and Sakayu Shimizu2 The United Graduate School of Agricultural Sciences, Iwate University, 18-8, Ueda 3-chome, Morioka 020-8550, Japan 1 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan 2 and Life Science R & D Center, Kaneka Co., Miyamae-machi, Takasaga-cho, Takasago 676-8688, Japan 3 Received 17 December 2009; accepted 25 January 2010 Available online 23 February 2010
We have cloned a gene encoding an alcohol oxidase (AOD) specific to aldehyde alcohols from Paenibacillus sp. AIU 311. The AOD gene contains an open reading frame consisting of 618 nucleotides corresponding to 205 amino acid residues. The deduced amino acid sequence exhibits a high similarity to that of manganese superoxide dismutases (SODs). We expressed the cloned gene as an active product in Escherichia coli BL21 cells. The productivity (total units per culture broth volume) of the recombinant AOD expressed in E. coli BL21 is 26,000-fold higher than that of AOD in Paenibacillus sp. AIU 311. The recombinant AOD also exhibits aldehyde alcohol oxidase activity and SOD activity. The recombinant cells described in this study have utility for the production of glyoxal from glycolaldehyde. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Glycolaldehyde; Glyoxal; Aldehyde alcohol; Alcohol oxidase; Superoxide dismutase; Paenibacillus sp.]
Recently, we demonstrated that Paenibacillus sp. AIU 311 isolated by our group produced an alcohol oxidase (AOD) with high activity for aldehyde alcohols such as glycolaldehyde, glyceraldehyde, and aldotetrose, but not for methanol, ethanol, ethylene glycol or glycerol (1). The N-terminal amino acid sequence of this AOD exhibited no similarity to that of AODs from methylotrophic yeasts, such as Candida and Pichia, which catalyze the oxidation of primary alcohols to their respective aldehydes (2–5). We therefore concluded in our previous report that Paenibacillus AOD is a novel enzyme with high specificity to a hydroxy group of aldehyde alcohols (1). In further studies of the Paenibacillus AOD, we obtained new findings that the N-terminal amino acid sequence of the Paenibacillus AOD is homologous to that of superoxide dismutases (SODs) and the AOD exhibits SOD activity (6). Our previous report also demonstrated that SODs containing manganese, iron, or copper and zinc as cofactors exhibit oxidase activity with aldehyde alcohols, such as glycolaldehyde, glyceraldehyde, and aldotetrose (6). In this paper, we report the cloning and sequence analysis of the AOD gene from Paenibacillus sp. AIU 311. We also characterize the expression of the AOD gene in Escherichia coli as well as the production of glyoxal from glycolaldehyde using recombinant cells. MATERIALS AND METHODS Chemicals Glycolaldehyde dimer was purchased from Sigma Japan (Tokyo, Japan). Peroxidase was obtained from Amano Enzymes (Nagoya, Japan). All other
⁎ Corresponding author. Tel./fax: +81 19 621 6155. E-mail address: [email protected] (K. Isobe).
chemicals used were commercial products of the highest grade available. Ni-charged resin was purchased from Bio-Rad Laboratories Japan (Tokyo, Japan). Medium and culture conditions Paenibacillus sp. AIU 311, which is a source of chromosomal DNA, was incubated in an ethylene glycol medium consisting of 1% ethylene glycol, 0.2% NH4NO3, 0.1% K2HPO4, 0.1% NaH2PO4, 0.05% MgSO4·7H2O, 0.02% CaCl2·2H2O, and 0.05% yeast extract, pH 7.0, at 30 °C for 3 days (d). A transformant carrying the AOD gene was incubated in 5.0 ml of a Luria-Bertani (LB) medium (1% peptone, 0.5% yeast extract, and 0.5% NaCl, pH 7.0) containing ampicillin (50 μg/ml) at 30 °C for 5 h, and then isopropyl-β-D-thiogalactopyranoside (IPTG, 0.5 mM) was added to the culture medium and further incubated at 30 °C for 20 h. Degenerate PCR The primers for degenerate PCR were designed based on the N-terminal amino acid sequence (1), (HIDAQTMEI) and the internal amino acid sequence, (LAVTSTPNQDNPLMEGQTPVLGLDVWEHAYYLK), which was obtained by a lysyl endopeptidase digestion of the purified AOD from Paenibacillus sp. AIU 311. Thus, the following oligonucleotides were designed as the sense primer (S1) and the antisense primer (A1): 5′-CA(T,C)AT(A,T,C)GA(T,C)GCICA(A,G)AC(A,T,C,G)ATGGA(A, G)AT-3′ and 5′-TA(A,G)TA(A,T,C,G)GC(A,G)TG(T,C)TCCCA(A,T,C,G)AC(A,G)TCIA-3′, respectively (see Fig. 1). The PCR was carried out in a 50-μl volume consisting of 2.5 units of TaKaRa Ex Taq DNA polymerase, 20 nmol of dNTP, 0.5 μmol of each primer, and 0.5 μg of the extracted chromosomal DNA. The reaction mixture was first heated at 94 °C for 5 min, followed by 30 cycles of amplification (a denaturation step at 94 °C for 30 s, an annealing step at 55 °C for 30 s, and an extension step at 72 °C for 1 min). Southern hybridization The chromosomal DNA from Paenibacillus sp. AIU 311 was digested with BamHI, HindIII, NdeI, PstI, SacI, SalI, SpeI, SphI or XbaI, and the digested DNA was subjected to gel electrophoresis on a 1% agarose gel in TEA buffer at 50 V for 3 h. The DNA fragments on the gel were transferred to a nitrocellulose membrane by capillary blotting and incubated at 55 °C for 24 h with the labeled DNA fragment homologous to the AOD gene (described above). Colony hybridization A ligation mixture (10 μl) consisting of Ligation Solution I (DNA Ligation Kit Ver. 2.1, Takara Bio), 1 μg of a chromosomal DNA digested with NdeI and 100 ng of cloning vector DNA (pT 7 Blue cloning vector, Novagen) digested with NdeI was incubated at 16 °C overnight. The resulting plasmids were subsequently
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FIG. 1. Nucleotide sequence of a part of the NdeI fragment containing the AOD gene from Paenibacillus sp. AIU 311. The initiation and stop codons are indicated by a box. S1-S3 and A1A3 indicate the sequences used for the primers. The deduced amino acids are also indicated under the DNA sequence.
transformed into E. coli JM 109. The positive clones were selected on a LB-agar plate containing X-gal/IPTG and ampicillin by blue-white selection. DNA sequence analysis The DNA sequences were determined by the dideoxy chain termination method using a DNA sequencer CEQ2000 XL (Beckman Coulter Inc., CA, U.S.A.) with a CEQ Dye Terminator Cycle Sequencing Kit (Beckman Coulter Inc.). Two sequence primers, 5′-CAGGTTGGAGATCAGCTCTTCAAGGC-3′ (S2) and 5′CATGGCTCGTCGTGGACAAGAGCGGC-3′ (A2) were designed to sequence the 5′ and 3′ regions of the AOD gene, respectively, in the NdeI fragment (see Fig. 1). DNA sequence analysis software (GENETYX) was used to identify the open reading frame and restriction enzyme sites as well as for the analysis of the deduced amino acid sequence. Expression of the Paenibacillus AOD in E. coli On the basis of the sequence obtained from the NdeI fragment, two oligonucleotides, 5′-CCCATATGGCATTTCAATTACCACC-3′ (S3) and 5′-CCCTCGAGCGGCTGTAC GATTTCCT-3′ (A3), were designed as a sense primer containing an NdeI site and an antisense primer containing a XhoI site, respectively (see Fig. 1). The AOD gene was amplified by PCR using these primers and ligated into an NdeI–XhoI site of the pET-21a(+) expression vector, containing a histidine-tag site. The pET-21a(+) plasmid containing the AOD gene (pET-GAOX) was transformed into E. coli BL21 cells (E. coli BL21/pET-GAOX), as described above.
Purification of recombinant AOD The recombinant cells (170 mg of wet weight) obtained at 25 h of cultivation from 100 ml of culture broth were suspended in 3 ml of 10 mM phosphate buffer, pH 7.0, and disrupted with glass beads by a Multi-beads shocker (Yasui Kikai, Osaka) for 10 min. The crude enzyme solution (5.0 ml) was applied to a Ni-charged resin column (1.0 × 2.0 cm), equilibrated with 50 mM sodium phosphate buffer, pH 8.0, containing 0.3 M NaCl, and eluted with 50 mM sodium phosphate buffer, pH 8.0, containing 0.3 M NaCl and 0.25 M imidazole. Oxidation reaction of glycolaldehyde with recombinant cells The recombinant cells (27 mg of wet weight) of E. coli BL21/pET-GAOX were harvested from 5 ml of the LB medium containing ampicillin and IPTG after 25 h of incubation at 30 °C. The cells were then suspended in 1.0 ml of 0.2 M phosphate buffer, pH 7.0, and 0.25 ml of the cell suspension was added to 1.5 ml of 100 mM glycolaldehyde. The reaction was carried out at 30 °C for 3 d with shaking at 120 strokes per min. Assay of enzyme activity Enzyme activities of glycolaldehyde oxidase and SOD were assayed by measuring the rate of hydrogen peroxide formation at 30 °C according to our previous report (6). Assay of glyoxal The concentration of glyoxal was assayed according to the method of Isobe and Nishise (7).
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Cloning of the gene encoding AOD from Paenibacillus sp. AIU 311 A partial DNA fragment of approximately 0.5 kb, containing 415 nucleotides of the AOD gene, was amplified from the chromosomal DNA of Paenibacillus sp. AIU 311 by PCR using S1 and A1 primers. The chromosomal DNA of Paenibacillus sp. AIU 311 was then digested with BamHI, HindIII, NdeI, PstI, SacI, SalI, SpeI, SphI or XbaI, and a DNA fragment containing the AOD gene was analyzed by Southern hybridization using the amplified DNA fragment (described above) as a probe. Because the AOD gene was contained in DNA fragments digested with NdeI, the NdeI fragments (5.0 kb) from chromosomal DNA were then ligated to a pT 7 blue cloning vector digested with NdeI and the resulting plasmids were transformed into E. coli JM 109. The positive clones were selected on an LB-agar plate containing X-gal/IPTG and ampicillin. Sequence analysis of the AOD gene The complete gene structure of AOD was not obtained by PCR using the S1 and A1 primers; therefore, newly designed primers, S2 and A2 were used for sequencing of unknown regions of the 5′- and 3′-ends of the AOD
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gene from the NdeI fragments described above. The entire length of the AOD gene was determined from the sequencing results of degenerate PCR product and the 5′- and 3′-ends of the AOD gene from the NdeI fragment. Fig. 1 shows the nucleotide sequence containing the AOD gene, in which an ORF of 618 bp encodes a 205 amino acid polypeptide. The predicted molecular mass of the ORF is in agreement with that of a subunit of the AOD from Paenibacillus sp. AIU 311. In addition, the deduced amino acid sequence of the N-terminal region of the ORF is identical to the amino acid sequence of the Paenibacillus AOD. The amino acid sequence obtained by the lysyl endopeptidase digestion of the purified AOD was also found in the deduced amino acid sequence. We therefore concluded that the ORF of 618 bp is the complete structural gene of AOD from Paenibacillus sp. AIU 311, and the NdeI fragment was used in the following studies. The sequence data of 618 nucleotides in the ORF correspond to the AOD gene in the DDBJ nucleotide sequence databases (accession number, AB509365). The amino acid sequence of the AOD polypeptide was used to search for homologous sequence using the BLAST (Basic Local Alignment Search Tool) program. More than 50% of the deduced
FIG. 2. Alignment of the amino acid sequences of the AOD protein from Paenibacillus sp. AIU 311 and SODs from bacterial strains. The deduced amino acid sequence of AOD from Paenibacillus sp. AIU 311 (GAOX) was compared to the sequences of Mn-SOD from T. thermophilus HB 27 (TTMS) (8), B. caldotenax YT 1 (BCMS) (9), B. stearothermophilus NCA 1503 (BSMS) (10), B. halodenitrificans ATCC 49067 (BHMS) (11), E. coli AB 2463/pDT1-5 (ECMS) (12), D. radiodurans DR 1279 (DRMS) (13) and S. cerevisiae FL 100 (SCMS) (14), Fe-SOD from A. aerogenes (AAFS) (15), and Cu, Zn-SOD from E. coli (ECCZS) (16). Amino acids identical to that of AOD from Paenibacillus sp. AIU 311 are depicted by boxes.
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FIG. 3. SDS-PAGE of recombinant AOD. Line A, cell-free extract from E.coli BL21; line B, cell-free extract from E.coli BL21/pET-GAOX; line C, purified recombinant AOD; line D, standard proteins.
amino acid sequence of the Paenibacillus AOD is identical to that of the manganese-containing SOD (Mn-SOD) from Thermus thermophilus HB 27 (8) and the putative Mn-SODs from Bacillus caldotenax YT 1(9) and Bacillus stearothermophilus NCA 1503 (10). The Paenibacillus AOD also exhibits high similarity to Mn-SODs from Bacillus halodenitrificans ATCC 49067 (11) and E. coli AB 2463/pDT1-5 (12) in the region of 140 residues from the Nterminus. In contrast, the amino acid sequence of the Paenibacillus AOD exhibits low similarity to that of Mn-SODs from Deinococcus radiodurans DR 1279 (13) and Saccharomyces cerevisiae FL 100 (14), and Fe-SOD from Aerobacter aerogenes IFO 3317 (15); however, these SODs exhibit high similarity in the region of 44 residues from the N-terminus. The Paenibacillus AOD shows no similarity to Cu, Zn-SOD from E. coli QC 871 (16) (Fig. 2). Thus, the deduced amino acid sequence of the AOD from Paenibacillus sp. AIU 311 shows high similarity to that of some Mn-SODs but not to other SODs and primary alcohol oxidases. Overexpression of the gene encoding AOD in E. coli cells The E. coli BL21/pET-GAOX transformant cells, which are E. coli BL21 cells harboring a pET-21a(+) plasmid containing the AOD gene, were incubated in the LB medium as described in Materials and Methods. The AOD protein was expressed in a soluble histidine-tagged form and
FIG. 4. Effects of pH, temperature and the cell amount on glyoxal production. (A) Effect of pH. The cell suspension and 100 mM glycolaldehyde solution were prepared using 0.2 M phosphate buffer, pH 5.0–8.5, and the cell reactions were carried out at 30 °C for 3 d with shaking. The relative amount was expressed as a percentage of the highest amount of glyoxal produced at pH 5.5. (B) Effect of temperature. The cell suspension and a 100-mM glycolaldehyde solution were prepared using 0.2 M phosphate buffer, pH 5.5, and the reactions were carried out at the indicated temperatures for 3 d with shaking. The relative amount was expressed as a percentage of the highest amount of glyoxal produced at 50 °C. (C) Effect of the cell amounts. The indicated amounts of recombinant cells were incubated with 85 mM glycolaldehyde at 50 °C and pH 5.5 for 7 d. Open triangles, 1. 7 mg of cells; closed triangles, 3. 4 mg of cells; open circles, 5. 1 mg of cells; closed circles, 6. 8 mg of cells.
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Purification of recombinant AOD The recombinant cells obtained at 25 h of cultivation from 100 ml of culture broth were disrupted with glass beads by a Multi-beads shocker and the supernatant was applied to a Ni-charged resin column as described in Materials and methods. The recombinant AOD protein was purified to a homogeneous state by column chromatography (Fig. 3). The molecular mass of the recombinant enzyme is approximately 24 kDa (deduced by SDS-PAGE), which is in agreement with that of the Paenibacillus AOD protein. The purified enzyme exhibited glycolaldehyde oxidase activity (0.83 unit per mg of protein) and SOD activity (1.28 units per mg of protein). The ratio of SOD activity to glycolaldehyde oxidase activity is also in agreement with that of the Paenibacillus AOD protein. Production of glyoxal by E. coli BL21/pET-GAOX cells We observed the formation of glyoxal when the E. coli BL21/pET-GAOX cells (6.8 mg of wet weight) were incubated with 85 mM glycolaldehyde at 30 °C and pH 7.0 for 3 d (data not shown). We then investigated the optimal conditions for glyoxal production. When the reaction pH was varied between 5.0 and 8.5, we found glyoxal production was highest at pH 5.5–6.0 (Fig. 4A). When the reaction was carried out at pH 5.5, the amount of glyoxal was highest at 50 °C after 3 d, when the reaction temperature was varied between 20 and 60 °C (Fig. 4B). On the basis of these results, cell amounts were increased at pH 5.5 and 50 °C. The amount of glyoxal produced was increased by increasing cell amounts and approximately 35 mM of glyoxal was produced from 85 mM glycolaldehyde after 7 d of incubation at 50 °C and pH 5.5 (Fig. 4C). DISCUSSION We recently reported that the N-terminal amino acid sequence of the Paenibacillus AOD protein is homologous to that of SODs, the Paenibacillus AOD exhibits SOD activity, and SODs from the other sources also exhibit oxidase activity with aldehyde alcohols, such as glycolaldehyde, glyceraldehyde, and aldotetrose (6). Therefore, we cloned and sequenced the AOD gene. The deduced amino acid sequence was compared to the other SODs. More than 50% of the deduced amino acid sequence of the Paenibacillus AOD is identical to the Mn-SOD from T. thermophilus (8) as well as putative Mn-SODs from B. caldotenax (9) and B. stearothermophilus (10). However, the amino acid sequence similarity between the Paenibacillus AOD and Fe-SOD or Cu, Zn-SOD is lower than 25%. These results support our previous results described in Ref. (1) and (6), and furthermore, this study suggests that the Paenibacillus AOD is a new member of MnSOD. The amino acid sequence analysis also demonstrates that the similarity of the N-terminal region is much higher than that of the Cterminal region in the above homologous enzymes. These results provide strong evidence for the future investigation relating the structure and enzyme activity of AOD and SOD in these enzymes. Studies of glyoxal production from glycolaldehyde using cells of or AOD from Paenibacillus sp. AIU 311 have been difficult due to the very low productivity of AOD by Paenibacillus sp. AIU 311. However, in the present study, the AOD from Paenibacillus is produced approximately 26,000-fold higher in the cells of E. coli BL21/pETGAOX. We therefore investigated the production of glyoxal from glycolaldehyde using recombinant cells. Glyoxal production favored an optimum pH of 5.5, which is a more acidic condition than that of the purified enzyme (1). This difference is most likely due to the pH stability of the enzyme. When the cell reaction was carried out using
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the E. coli BL21/pET-GAOX cells at 50 °C for 7 d, approximately 40% of glycolaldehyde was converted to glyoxal. These results indicate that glycolaldehyde was converted to glyoxal by the E. coli BL21/ pET-GAOX cells. In a future study, we intend to apply these recombinant cells or the AOD enzyme to the production of glyoxylic acid from ethylene glycol by coupling with alcohol oxidase catalyzing the conversion of ethylene glycol to glycolaldehyde (7) and aldehyde oxidase catalyzing the conversion of glyoxal to glyoxylic acid (17). References 1. Isobe, K., Kato, A., Sasaki, Y., Suzuki, S., Kataoka, M., Ogawa, J., Iwasaki, A., Hasegawa, J., and Shimizu, S.: Purification and characterization of a novel alcohol oxidase from Paenibacillus sp. AIU311, J. Biosci. Bioeng., 104, 124–128 (2007). 2. Ledeboer, A. M., Edens, L., Maat, J., Visser, C., Bos, J. W., Verrips, C. T., Janowicz, Z., Eckart, M., Roggenkamp, R., and Hollenberg, C. P.: Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha, Nucleic Acids Res., 13, 3063–3082 (1985). 3. Koutz, P., Davis, G. R., Stillman, C., Barring, K., Cregg, J., and Thill, G.: Structural comparison of the Pichia pastoris alcohol oxidase gene, Yeast, 5, 167–177 (1989). 4. Sakai, Y. and Tani, Y.: Cloning and sequencing of the alcohol oxidase-encoding gene (AOD1) from the formaldehyde-producing asporogeneous methylotrophic yeast, Candida boidinii S2, Gene, 114, 67–73 (1992). 5. Nakagawa, T., Mukaiyama, H., Yurimoto, H., Sakai, Y., and Kato, N.: Alcohol oxidase hybrid oligomers formed in vivo and in vitro, Yeast, 15, 1223–1230 (1999). 6. Isobe, K., Kato, A., Sasaki, Y., Kataoka, M., Ogawa, J., Iwasaki, A., Hasegawa, J., and Shimizu, S.: Superoxide dismutases exhibit oxidase activity on aldehyde alcohols similar to alcohol oxidase from Paenibacillus sp. AIU 311, J. Biosci. Bioeng., 105, 666–670 (2008). 7. Isobe, K. and Nishise, H.: Enzymatic production of glyoxal from ethylene glycol using alcohol oxidase from methanol yeast, Biosci. Biotechnol. Biochem., 58, 170–173 (1994). 8. Ludwig, M. L., Metzger, A. L., Pattridge, K. A., and Stallings, W. C.: Manganese superoxide dismutase from Thermus thermophilus. A structural model refined at 1.8 Å resolution, J. Mol. Biol., 219, 335–358 (1991). 9. Chambers, S. P., Brehm, J. K., Michael, N. P., Atkinson, T., and Minton, N. P.: Physical characterisation and over-expression of the Bacillus caldotenax superoxide dismutase gene, FEMS Microbiol. Lett., 70, 277–284 (1992). 10. Brehm, J. K., Chambers, S. P., Bown, K. J., Atkinson, T., and Minton, N. P.: Molecular cloning and nucleotide sequence determination of the Bacillus stearothermophilus NCA 1503 superoxide dismutase gene and its overexpression in Eschericia coli, Appl. Microbiol. Biotechnol., 36, 358–363 (1991). 11. Liao, J., Li, M., Liu, M. Y., and Chang, W. R.: Crystallization and preliminary crystallographic analysis of manganese superoxide dismutase from Bacillus halodenitrificans, Biochem. Biophys. Res. Commun., 294, 60–62 (2002). 12. Edwards, R. A., Baker, H. M., Whittaker, M. M., Whittaker, J. W., Jameson, G. B., and Baker, E. N.: Crystal structure of Escherichia coli manganese superoxide dismutase at 2.1-Å resolution, J. Biol. Inorg. Chem., 3, 161–171 (1998). 13. Dennis, R. J., Micossi, E., McCarthy, J., Moe, E., Gordon, E. J., KozielskiStuhrmann, S., Leonard, G. A., and McSweeney, S.: Structure of the manganese superoxide dismutase from Deinococcus radiodurans in two crystal forms, Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 62, 325–329 (2006). 14. Marres, C. A., Van Loon, A. P., Oudshoorn, P., Van Steeg, H., Grivell, L. A., and Slater, E. C.: Nucleotide sequence analysis of the nuclear gene coding for manganese superoxide dismutase of yeast mitochondria, a gene previously assumed to code for the Rieske iron-sulphur protein, Eur. J. Biochem., 147, 153–161 (1985). 15. Lee, S. O., Kim, S. W., Uno, I., and Lee, T. H.: Direct sequencing of superoxide dismutase genes from two bacterial strains amplified by polymerase chain reaction, Biosci. Biotechnol. Biochem., 57, 1454–1460 (1993). 16. Battistoni, A., Folcarelli, S., Gabbianelli, R., Capo, C., and Rotilio, G.: The Cu,Zn superoxide dismutase from Escherichia coli retains monomeric structure at high protein concentration. Evidence for altered subunit interaction in all the bacteriocupreins, Biochem. J., 320, 713–716 (1996). 17. Sasaki, Y., Isobe, K., Kataoka, M., Ogawa, J., Iwasaki, A., Hasegawa, J., and Shimizu, S.: Purification and characterization of a novel aldehyde oxidase from Pseudomonas sp. AIU 362, J. Biosci. Bioeng., 106, 297–302 (2008).