Vol. 171, No. 9

Cloning, Nucleotide Sequence, and Expression in Escherichia coli ofthe Bacillus stearothermophilus Peroxidase Gene (perA)

SUVIT LOPRASERT, SEIJI NEGORO, AND HIROSUKE OKADA*

Department of Fermentation Technology, Osaka University, Yamada-oka, Suita-shi, Osaka 565, Japan

Received 6 March 1989/Accepted 9 June 1989

The gene encoding a thermostable peroxidase was cloned from the chromosomal DNA of Bacillusstearothermophilus IAM11001 in Escherichia coli. The nucleotide sequence of the 3.1-kilobase EcoRI fragmentcontaining the peroxidase gene (perA) and its flanking region was determined. A 2,193-base-pair open readingframe encoding a peroxidase of 731 amino acid residues (Mr, 82,963) was observed. A Shine-Dalgarno sequence

was found 9 base pairs upstream from the translational starting site. The deduced amino acid sequence

coincides with those of the amino terminus and four peptides derived from the purified peroxidase of B.stearothermophilus IAM11001. E. coli harboring a recombinant plasmid containing perA produced a largeamount of thermostable peroxidase which comigrated on polyacrylamide gel electrophoresis with the B.stearothermophilus peroxidase. The peroxidase of B. stearothermophilus showed 48% homology in the aminoacid sequence to the catalase-peroxidase of E. coli.

Peroxidases are important enzymes used in clinical andanalytical measurements. Typical peroxidases are hemopro-teins and catalyze the oxidation of a large number ofaromatic compounds in the presence of hydrogen peroxide.The best-studied peroxidase is horseradish peroxidase (29).Some peroxidases of microbial origins, such as those fromPseudomonas fluorescens (15), Streptococcus faecalis (5),Escherichia coli (3), Halobacterium halobium (8), Rhodo-pseudomonas capsulata (11), Pellicularia filamentosa (13),and Saccharomyces cerevisiae (6), have been purified andcharacterized, but little information about thermostable per-oxidases is available. We have isolated a thermostablecatalase-peroxidase of Bacillus stearothermophilus which isa dimeric enzyme having one molecule of protoheme IX permolecule (16). It has an optimum temperature at 70°C and isstable for a month at 30°C. Because of its remarkablestability, it is potentially useful for practical applications.

In this paper, we describe the cloning of the peroxidasegene of B. stearothermophilus in E. c6li, the determinationof the nucleotide sequence, and the high expression of theperoxidase gene. The homology of the amino acid sequencewith that of the catalase-peroxidase of E. coli is also dis-cussed.

MATERIALS AND METHODSBacterial strains, plasmids, media, and culture conditions.

B. stearothermophilus IAM11001 (ATCC 8005), which pro-

duces thermostable peroxidase (16), was obtained from theInstitute of Applied Microbiology, University of Tokyo,Japan. The peroxidase-deficient mutant, E. coli UM228, waskindly provided by P. C. Loewen, University of Manitoba,Winnipeg, Canada (27). E. coli JM103 was a host strain forthe M13 bacteriophage derivatives mpl8 and mpl9 (19).Plasmids pBR322 (2) and pUC19 (30) were used in thecloning experiment. Bacteria were grown on Luria-Bertanimedium containing tryptone (10 g), yeast extract (5 g), andNaCl (10 g) in 1 liter (pH 7.3) at 55°C for B. stearothermo-philus or 37°C for E. coli. When necessary, ampicillin (50,ug/ml) or tetracycline (10 ,ug/ml) was added to the medium.

Electrophoresis. Electrophoresis of DNA digested with

* Corresponding author.

restriction endonucleases was carried out as previouslydescribed (17). Polyacrylamide gel (7.5%) electrophoresis ofproteins was carried out at pH 8.0 by the method of Gabriel(9). Proteins in polyacrylamide gel were stained withCoomassie brilliant blue R250 (10).

Isolation of DNA, transformation, and enzymes. Chromo-somal DNA of B. stearothermophilus IAM11001 was pre-pared by the method of Saito and Miura (22). PlasmidspBR322 and pUC19 were prepared by the procedure ofBirnboim and Doly (1) followed by purification by cesiumchloride-ethidium bromide density gradient centrifugation.E. coli strains were transformed by the method of Cohen etal. (4). Digestion with restriction enzymes and ligation werecarried out by the procedures of Maniatis et al. (17). Restric-tion endonucleases, T4 DNA ligase, and alkaline phos-phatase were purchased from Toyobo Co., Ltd., Osaka,Japan.

Colony assay. Colonies on filter paper were treated with 10mM Tris and 1 mM EDTA containing 2 mg of lysozyme perml at 30°C for 20 min. The cells were lysed by 1% TritonX-100 at room temperature for 10 mmn and then flooded withthe reaction mixture containing 0.5 mM 3-methyl-2-ben-zothiazolinonehydrazone, 0.5 mM primaquine diphosphate,10 mM H202, and 20 mM sodium/potassium phosphate

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FIG. 1. Restriction map of and sequencing strategy for theperoxidase gene. Plasmid pOD10 is a hybrid plasmid consisting ofpBR322 (-) and the 3.1-kilobase EcoRI fragment containing theperoxidase gene of B. stearothermophilus ( ). The open readingframe for peroxidase which starts from the ATG codon and termi-nates at the TAA codon is also shown. DNA was deleted by B.al 31nuclease to obtain small fragments for sequencing. The DNAsequence was determined by the dideoxy method. Arrows indicatethe directions and extent of the sequencing. bp, Base pairs.

4871

JOURNAL OF BACTERIOLOGY, Sept. 1989, p. 4871-48750021-9193/89/094871-05$02.00/0Copyright C) 1989, American Society for Microbiology

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FIG. 2. Nucleotide and amino acid sequences of the peroxidase gene of B. stearothermophilus. The nucleotide sequence -is presented-fromposition 550 to 3100. The possible Shine-Dalgarno sequence (SD) and promoter region (-10 and -35 regions) are indicated by broken lines.The amino acid sequences, which are identical to the sequences determined by automated Edman sequencing of the purified enzyme andpeptides, are underlined.

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B. STEAROTHERMOPHILUS perA IN E. COLI 4873

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FIG. 3. Activity and protein staining of peroxidase on polyacryl-amide gel. Crude-enzyme solution after heat treatment was sub-jected to electrophoresis on polyacrylamide gel (pH 8.0). Lanes 1 to3, Stained with 3,3'-diaminobenzidine tetrahydrochloride for perox-idase activity; lanes 4 to 6, stained with Coomassie brilliant blue forprotein. Lanes 1 and 4, B. stearothermophilus (50 and 30 jig ofprotein, respectively); lanes 2 and 5, E. coli UM228(pBR322) (100and 20 ,ug of protein, respectively); lanes 3 and 6, E. coliUM228(pOD10) (50 and 20 p.g of protein, respectively). The perox-idase bands are indicated by arrows.

buffer (pH 7.0). The red color of the colony developed within1 min.Enzyme assay. Peroxidase activity was determined by

using a mixture of 2,4-dichlorophenol and 4-aminoantipyrineas substrate (16). One unit of peroxidase activity was definedas the amount of the enzyme consuming 1 ,imol of 4-aminoantipyrine or 2,4-dichlorophenol under the assay con-ditions. To detect the peroxidase activity on polyacrylamidegel, the gel was incubated with 1.26 mM 3,3'-diaminobenzi-dine tetrahydrochloride, 20 mM H202, and 50 mM sodium/

potassium phosphate buffer (pH 7.0) at room temperature for10 min.

Protein concentration. The protein concentration was de-termined from the A280 with A 1% 280 nm = 16.2 aspreviously described (16).

Preparation of crude-enzyme solution. Cells grown on 250ml of Luria-Bertani medium to a density of 109 cells per mlwere harvested by centrifugation, suspended in 3 ml of 100mM sodium/potassium phosphate buffer (pH 7.0), and dis-rupted by sonication at 20 KHz for 4 min. The supernatantobtained by centrifugation at 20,000 x g for 10 min was usedas a crude-enzyme solution. Heat treatment was done byincubating the crude-enzyme solution at 70°C for 10 min.The supernatant was obtained after centrifugation at 20,000x g for 10 min.DNA sequencing. DNA fragments for sequencing were

obtained by deletion with Bal 31 nuclease (Toyobo) aspreviously described (14). DNA sequencing was carried outby the dideoxy-chain termination method (23) with a se-quencing kit from United States Biochemical Corp., Cleve-land, Ohio, and [a-32P]dCTP from ICN Pharmaceuticals,Inc., Irvine, Calif.Amino acid sequence analysis. The peroxidase was purified

from B. stearothermophilus IAM11001 as previously de-scribed (16). The amino-terminal sequence of the purifiedenzyme was determined by automated Edman sequencingwith a 470A sequenator (Applied Biosystems) (12). Thephenylthiohydantoin derivatives were purified by high-pres-sure liquid chromatography as previously described (28).

Lysylendopeptidase digestion. Lyophilized purified perox-idase (500 [g) was incubated with lysylendopeptidase (2.5,ug) in 50 mM Tris (pH 9.0)-2 M urea at 30°C for 16 h. Thedigested peptides were separated by high-pressure liquidchromatography on a 5C4-300 column (Nakarai Chemical,Kyoto, Japan) with an acetonitrile gradient. Four well-separated peaks (peaks 1 through 4) were collected atacetonitrile concentrations of 24, 28, 40, and 50%, respec-tively, and then evaporated and rechromatographed with a

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FIG. 4. Comparison of the deduced amino acid sequences of B. stearothermophilus peroxidase (upper) and E. coli catalase-peroxidase(lower) (26).

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4874 LOPRASERT ET AL.

5PhT-300 column (Nakarai Chemical) with an acetonitrilegradient. The purified peptides were analyzed for amino acidsequences by the method described above. Lysylendopepti-dase was from Wako Chemical, Osaka, Japan.

RESULTS

Cloning of the peroxidase gene. Chromosomal DNA of B.stearothermophilus was digested with EcoRI and ligatedwith pBR322, which had been cleaved with EcoRI andtreated with alkaline phosphatase. After transformation of E.coli UM228 with the hybrid plasmids, a peroxidase-pro-ducing clone was detected by colony assay. Of 1,500 tetra-cyline-resistant clones examined, 2 clones showed peroxi-dase activity. After isolation and characterization of therecombinant plasmids by various restriction enzymes, bothclones were found to harbor the same 3.1-kilobase insertedfragment at the EcoRI site. The recombinant plasmidpBR322 with the 3.1-kilobase insertion was designatedpOD10. The retransformation of peroxidase-deficient E. coliwith pOD10 resulted in all peroxidase-producing transfor-mants, thus confirming that pOD10 carries the structuralgene of peroxidase.

Nucleotide sequence of the peroxidase gene. The restrictionmap of and sequencing strategy for the peroxidase gene areshown in Fig. 1. The nucleotide sequence and deducedamino acid sequence are shown in Fig. 2. A 2,193-base-pairopen reading frame starting at ATG (position 761) andterminating at TAA (position 2956) which was capable ofcoding a peptide of 731 amino acid residues of 82,963 daltonswas found. This molecular weight is close to the 86,000 ofthe peroxidase subunit as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (16).The pieces of evidence which indicated that this open

reading frame encodes peroxidase from B. stearothermophi-lus were the amino acid sequences of the amino termini- ofthe enzyme- and lysylendopeptidase-digested peptides. Allsequences were identical to the deduced amino acid se-quence (underlined) in Fig. 2. The amino acids Met-Glu-Asn-Gln-Asn-Arg, which are not found in mature proteins,are presumably removed posttranslationally.A Shine-Dalgarno sequence, the ribosome-binding site

GAAAGGAG, which is complementary to the 3' ends of 16SrRNAs of Bacillus subtilis (20) and B. stearothermophilus(24), was observed 9 base pairs upstream from the initiationcodon (Fig. 2). The free-energy change of the most stableShine-Dalgarno pairing calculated for this sequence was-16.2 kcallmol (1 cal = 4.184 J) (25), which is in the rangereported (-11.6 to -21 kcal/mol) for B. subtilis 16S rRNA(18). The possible promoter sequences, TAGAAT for the- 35 region and TATTT for the -10 region, were alsoobserved upstream from the Shine-Dalgarno sequence.The G+C content of the peroxidase gene is 55%, while at

the third base of the codon, the G+C content is 61%. Thecodon usage of this gene is biased for G and C. For the aminoacids which have four or six codons (Lue, Val, Ser, Pro,Thr, Ala, Arg, and Gly), the codon with G or C as the thirdletter is preferred.

Expression of the peroxidase gene in E. coli. The peroxidasegene from B. stearothermophilus is expressed in E. coli.Figure 3 is the electrophoretogram of crude extracts afterheat treatment at 70°C for 10 min on polyacrylamide gel.Activity and protein staining are shown. E. coli(pOD10)produced peroxidase which was thermostable and migratedat the same distance as the B. stearothermophilus peroxi-dase does. The heat-treated cell extracts were subjected to

sodium dodecyl sulfate-polyacrylamide gel electrophoresisand stained with Coomassie brilliant blue; a dense extraband was clearly observed for the cell extract of E.coli(pOD10) but was absent for that of E. coli without therecombinant plasmid. The protein of the extra band comi-grated with purified peroxidase from B. stearothermophilus.These results proved that the peroxidase gene of B. stearo-thermophilus is expressed in E. coli and that the expressedproduct is enzymatically active.The peroxidase productivities of E. coli harboring pOD10

and B. stearothermophilus were measured after heat treat-ment (70°C, 10 min). The substrate was a mixture of 2,4-dichlorophenol and 4-aminoantipyrine. E. coli(pOD10)produced the enzyme at the same level as B. stearo-thermophilus, the DNA donor (0.0112 and 0.0133 U/mg,respectively). To increase the productivity, the 3.1-kilobaseEcoRI fragment of pOD10 containing the peroxidase genewas subcloned at the unique EcoRI site of pUC19, a high-copy-number plasmid, and pOD40 was obtained. E.coli(pOD40) produced 10 times as much peroxidase (0.1280U/mg) as that produced by E. coli(pOD10) and B. stearo-thermophilus. E. coli UM228 produced <0.0003 U/mg.

DISCUSSION

The peroxidase of B. stearothermophilus is a bifunctionalenzyme having catalase and peroxidase activities (16). Thesame type of enzymes have been isolated from E. coli (3),Rhodopseudomonas capsulata (11), and Comamonas com-pransoris (21); some of them (E. coli and R. capsulataenzymes) are tetrameric enzymes, and others (B. stearother-mophilus and C. compransoris enzymes) are dimeric en-zymes. In spite of the differences in their subunit numbers,all have been found to contain one molecule of protoheme IXper two subunits. Recently, the gene of catalase-peroxidase,HP I, of E. coli has been sequenced. No homology betweenHP I and other typical catalases which have no peroxidaseactivity was found (26). The amino acid alignment betweenthe catalase-peroxidase of E. coli and that of B. stearother-mophilus is shown in Fig. 4. HP I consists of 726 aminoacids, which is very close to the 731 amino acids of B.stearothermophilus enzyme, and has 48% homology overall.High homology was observed in three regions starting fromThr-78 to Arg-139 (homology is 79%), from Met-247 toPro-434 (69%), and from Gly-627 to Leu-721 (73%). Noobvious homology is observed between B. stearothermophi-lus peroxidase and horseradish enzyme (7). These observa-tions might suggest that the group of enzymes having cata-lase and peroxidase activities is different from typicalcatalases and typical peroxidases.The peroxidase gene of B. stearothermophilus was ex-

pressed in E. coli, and the expression product had enzymeactivity, suggesting that protoheme was incorporated cor-rectly in the expression product. The enzyme also exhibitedthermostability, which is a remarkable property of B. stearo-thermophilus peroxidase.

ACKNOWLEDGMENT

We thank P. Loewen for providing E. coli UM228. We are gratefulto M. Kobayashi, 0. Nishimura, and I. Kubota for amino acidsequence analysis and I. Urabe for valuable discussion.

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