Extremophiles (2001) 5:423429 Digital Object Identier (DOI) 10.

1007/s007920100218

Springer-Verlag 2001

ORIGINAL PAPER
Marinka Gudelj Gilbert Otto Fruhwirth Andreas Paar Fritz Lottspeich Karl-Heinz Robra Artut Cavaco-Paulo Georg Max Gbitz

A catalase-peroxidase from a newly isolated thermoalkaliphilic Bacillus sp. with potential for the treatment of textile bleaching efuents

Received: March 14, 2001 / Accepted: June 8, 2001 / Published online: October 16, 2001
Communicated by G. Antranikian

Abstract A new thermoalkaliphilic bacterium was isolated from a textile wastewater drain and identied as a new Bacillus sp. (Bacillus SF). Because of its high pH stability and thermostability, a catalase-peroxidase (CP) from this strain has potential for the treatment of textile bleaching efuents. The CP from Bacillus SF was puried to more than 70.3-fold homogeneity using fractionated ammonium sulfate precipitation, hydrophobic interaction, and anionexchange and gel-ltration chromatography. The native CP had a molecular mass of 165 kDa and was composed of two identical subunits. The isoelectric point of the protein was at pH 6.0. Peptide mass mapping using matrix-assisted laser desorption ionizationmass spectrometry showed a homology between the CP from Bacillus SF and the CP from Bacillus stearothermophilus. The apparent Km value of the catalase activity for H2O2 was 2.6 mM and the kcat value was 11,475 s1. The enzyme showed high catalase activity and an appreciable peroxidase activity with guaiacol and odianisidine. The enzyme was stable at high pH, with a halflife of 104 h at pH 10 and 25C and 14 h at 50C. The enzyme was inhibited by azide and cyanide, in a competitive manner, but not by the catalase-specic inhibitor 3-amino-1,2,4triazole.
Key words Thermostable Alkalistable Bacillus sp. Catalase Peroxidase Textile bleaching

Introduction
The application of catalases for the elimination of hydrogen peroxide in textile bleaching efuents has been suggested recently (Tzanov et al. 2001a, b). This process would allow recycling of the water used for dyeing, for example, as many dyes would be oxidized and decolorized by hydrogen peroxide. Consequently, the enormous water consumption (100 l/kg of fabric) of the textile processing industry could be reduced (Hillenbrand 1999). The application of catalases for this purpose has the advantage over the use of chemicals such as sodium bisulte or hydrosulte in that they are only needed in catalytic quantities, avoiding unfavorable high salt concentrations in the process. Several companies have recently marketed their catalases for the treatment of bleaching baths; however, most commercial catalases would hardly withstand the conditions used during textile bleaching. Therefore, new thermoalkalistable enzymes acting at temperatures above 50C and pH values above 9 are required. We have isolated several thermoalkaliphilic bacteria from textile processing efuents, and a catalase preparation of a new Bacillus sp. (Bacillus SF) has shown promising stabilities at high pH and temperature (Paar et al. 2001). Catalase (EC 1.11.1.6) is an enzyme, present in all aerobic cells, that decomposes hydrogen peroxide to molecular oxygen and water. Its main function is to protect cells from the toxic effect of hydrogen peroxide. In eukaryotic organisms and in some prokaryotes, catalase is a molecule composed of four identical subunits. Each of the subunits binds one protoheme IX group (Murthy et al. 1981; von Ossowski et al. 1993). This enzyme can also act as a peroxidase (EC 1.11.1.7) for which several organic substances can act as a hydrogen donor. Therefore, about 20 years ago, a new class of bacterial enzymes, the catalase-peroxidases (CP), was identied. These enzymes are considered to be ancestral forms of catalase or peroxidase in evolutionary ow (Youn et al. 1995). It has been proposed that CPs may act as catalases because the substrates for peroxidatic activity are not found in cytoplasm (Claiborne and Fridovich 1979). Although they show high catalase activity, these bifunctional enzymes have little sequence homology with typical

Communicated by G. Antranikian M. Gudelj G.O. Fruhwirth K.-H. Robra G.M. Gbitz (*) Institute of Environmental Biotechnology, Graz University of Technology, Petersgasse 12, A-8018 Graz, Austria Tel. +43-31687-3831; Fax +43-316873-8815 e-mail: guebitz@ima.tu-graz.ac.at M. Gudelj A. Paar G.M. Gbitz Sucher and Holzer, Graz, Austria F. Lottspeich Max-Planck-Institute of Biochemistry, Martinsried, Germany A. Cavaco-Paulo Textile Engineering Department, University of Minho, Guimaraes, Portugal
Springer-VerlagTokyoExtremophilesExtremophiles007921431-06511433-4909s00792010021800218Springer-VerlagOriginal paper

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heme-containing monofunctional catalases but have high homology with fungal cytochrome c peroxidase and plant ascorbate peroxidase. Thus, they have been recognized as a part of the class I of the superfamily of plant, fungal, and bacterial peroxidases (Welinder 1992). CP is distinct from typical catalases because it is reduced by dithionite; like peroxidase, it is not inhibited by the catalase-specic inhibitor 3-amino-1,2,4-triazole but is inactivated by hydrogen peroxide, and it possesses a narrow pH range for its maximal activity (Regelsberger et al. 1999b; Marcinkeviciene et al. 1995; Youn et al. 1995). The most interesting feature of bifunctional catalase-peroxidase is the overwhelming catalase activity with kcat/Km values comparable with monofunctional catalases (Regelsberger et al. 2000). Until now, CP have been isolated and characterized from a variety of different bacteria including Deinococcus radiophilus (Yun and Lee 2000), Escherichia coli (Claiborne and Fridovich 1979), Septoria tritici (Levy et al. 1992), Bacillus stearothermophilus (Loprasert et al. 1988), Klebsiella pneumonia (Hochman and Goldberg 1991), Rhodobacter capsulatus (Levy et al. 1992; Forkl et al. 1993), Streptomyces sp. (Youn et al. 1995), Mycobacterium smegmatis (Marcinkeviciene et al. 1995), Mycobacterium tuberculosis (Nagy et al. 1997; Johnsson et al. 1997), and Synechocystis sp. (Regelsberger et al. 1999b). However, only very little information is available in the literature on thermostability and pH stability of catalaseperoxidases. In this article, we describe the purication of a thermoalkalistable catalase-peroxidase from this Bacillus sp. and compare the properties of this enzyme to those of other known catalase-peroxidases.

Purication of the catalase-peroxidase from Bacillus SF Cells were harvested at the end of the exponential phase of growth, centrifuged (3,000 g, 15 min), and the pellet was suspended with phosphate buffer (50 mM, pH 7.0). Cell disruption was carried out using sonication (Sonoplus HD 70; Bandelin, Berlin, Germany) monitoring progress under the microscope. Cell debris was removed by centrifugation (6,500 g, 20 min) and the remaining supernatant was used for purication. The cell lysate was subjected to fractionation by ammonium sulfate precipitation at 40% saturation to remove impurities, followed by 70% saturation in a second step to precipitate the CP. The precipitated proteins were collected by centrifugation, and the pellet was dissolved in phosphate buffer (50 mM, pH 7.0) with 1.0 M (NH4)2SO4. The solution was applied to a Phenyl Sepharose High Performance column (Pharmacia, Uppsala, Sweden) equilibrated with 1.0 M (NH4)2SO4 in phosphate buffer (as described earlier). Fractions containing protein were eluted with a linear gradient of 1.00 M (NH4)2SO4. Catalase active fractions were pooled and loaded onto a UNO Q6 column (Bio-Rad, Hercules, CA, USA) equilibrated with phosphate buffer (50 mM, pH 7.0). After adsorption, proteins were eluted with a linear gradient of 01 M NaCl in the same buffer; the catalase was eluted at about 0.25 M NaCl. Active fractions were pooled, concentrated in an ultraltration cell (Sartorius, Epsom, UK) using a membrane with a 100-kDa cutoff, and subjected to a Superdex 200 HR 10/30 column (Pharmacia) previously equilibrated with phosphate buffer (50 mM, pH 7.0) containing 0.15 M NaCl. The column was run with the same buffer, and active fractions were collected and stored at 20C until further use. The chromatographic purication of the enzyme was performed with the FPLC system AEKTA from Pharmacia. Protein characterization Electrophoresis Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) was carried out in 12% polyacrylamide gels using broad molecular weight markers from Pharmacia as standards. Proteins on the gel were stained with Coomassie brilliant blue. Native PAGE was performed in 10% polyacrylamide gels that were stained for catalase activity as described previously (Harris and Hopkinson 1976) using 0.001% H2O2. Peroxidase activity was determined according to the modied method of Graham et al. (1964) using guaiacol or o-dianisidine as a substrate. Isoelectric focusing (IEF) was carried out with the Mini-PROTEAN 3 cell from Bio-Rad using IEF Ready Gels (pH 58; Bio-Rad), and protein was located by Coomassie brilliant blue staining. Amino acid sequence analysis For internal amino acid sequence analysis, using matrixassisted laser desorption ionizationmass spectrometry (MALDI-MS), the excised gel band of catalase-peroxidase

Materials and methods

Screening and cultivation A sample (1 ml) from a wastewater drain from a textile nishing company was added to 50 ml Standard I nutrient broth (Merck, Darmstadt, Germany), buffered with 50 mM NaHCO3/Na2CO3 at pH 10.0, and incubated in 100-ml Erlenmeyer asks with chicanes in a rotary shaker at 60C and 160 rpm. Growth was monitored under the microscope, and microorganisms (1 ml) were transferred to fresh culture medium after 2 days. To isolate various strains, a dilution row was spread on Standard I nutrient agar (Merck) buffered with 50 mM NaHCO3/Na2CO3 (pH 10). Agar plates were incubated at 60C and constant humidity, and the isolated strain was deposited and identied by the German culture collection DSMZ (Braunschweig, Germany). Bacillus SF was grown in a medium containing 8 g l1 yeast extract, 8 g l1 extract from meat (Merck), and 1 g l1 KH2PO4 at 65C and pH 10.0, buffered with 50 mM NaHCO3/Na2CO3. Then, 1% (w/w) of a trace element solution (2.5 g l1 Na2EDTA, 0.1 g l1 ZnSO47H2O, 0.030 g l1 MnCl24H2O, 0.3 g l1 H3BO3, 0.2 g l1 CaCl26H2O, 0.01 g l1 CuCl22H2O, 0.02 g l1 NiCl26H2O, 0.9 g l1 Na2MoO42H2O, 0.03 g l1 Na2SO35H2O, and 1 g l1 FeSO47H2O) was added to the incubation mixture.

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from Bacillus SF was digested with endoproteinase Lys-C, separated by high-performance liquid chromatography (HPLC) with spectrometric mass determination, and the amino acid sequence of peptides was determined as described previously (Eckerskorn and Lottspeich 1989). For direct N-terminal amino acid sequencing of protein, the gel band was blotted onto poly(vinylidene diuoride) membranes (ProBlottTM PVDF-membrane; Applied Biosystems, Weiterstadt, Germany) by the semidry method, as described previously (Eckerskorn et al. 1988). The blotted polypeptides were stained with Coomassie blue and excised, and the N-terminal amino acid sequence was determined in a protein sequencer model 477 A (Applied Biosystems). Peptide mass mapping The proteolytic digest pattern derived from the puried catalase-peroxidase was compared with virtual digest of the CP of B. stearothermophilus using the Peptide Mapping software, ProFound, available via the PROWL World Wide Web server, at the URL address (http://129.85.19.192/ profound_bin/WebProFound.exe). Enzyme assay Catalase activity was monitored spectrophotometrically. The assay mixture contained 100 l enzyme preparation and 900 l phosphate buffer (50 mM, pH 7.0). The reaction was initiated by addition of 1,000 l 26 mM H2O2 solution monitoring the decrease in absorbance at 240 nm at room temperature. The linear range of the reaction (30 s) was used to calculate the rate of the reaction. One unit of enzyme activity was dened as 1 mol H2O2 decomposed per minute. Kinetic parameters (mean SD for three assays) of the CP were determined using the standard assay with different substrate concentrations. Km and vmax were calculated by nonlinear analysis using the program Origin, version 4.10. Inhibition of the Bacillus SF catalase-peroxidase by 3amino-1,2,4-triazol, azide, and cyanide was measured as already described, except that the enzyme was preincubated for 5 min at 20C with the inhibitor. Peroxidase activity was assayed in an incubation mixture containing 1 mM peroxoacetic acid and 1 mM of either guaiacol or o-dianisidine in phosphate buffer (50 mM, pH 7.0). The reaction was monitored spectrophotometrically as described previously (Cendrin et al. 1994; Hochman and Goldberg 1991). Other procedures The pH optimum of the CP was determined in 50 mM potassium phosphate buffer (pH 6.08.5) and 50 mM NaHCO3/ Na2CO3 for pH 9. To determine enzyme stabilities, 1 ml of enzyme preparation was diluted with 9 ml buffer in test tubes that were shaken at 50 rpm in a water bath. Samples were withdrawn at various time intervals, and remaining enzyme activity was measured as already described. The molecular mass of the puried enzyme was determined by gel ltration chromatography on a Superdex

200 HR 10/30 column (Pharmacia), equilibrated with potassium phosphate buffer (50 mM, pH 7.0) containing 150 mM NaCl, and calibrated with a high molecular weight gel ltration calibration kit from Pharmacia. Protein concentrations were determined by the method of Bradford (1976) (Bio-Rad) using bovine serum albumin as a standard. UV-visible spectra were recorded on a Hitachi U 2001 spectrophotometer (Hitachi, Tokyo, Japan).

Results
Thermoalkaliphilic Bacillus SF A thermoalkaliphilic bacterium was isolated from textile nishing efuent. The cells were gram positive, catalase positive, non-spore-forming, and rod shaped. Growth occurred at pH values ranging from 7.5 to 10.1, and optimum growth occurred around pH 9.5. The optimum temperature for growth was around 60C and the upper temperature limit for growth was 65C. Standard analysis of the cellular fatty acids indicated that the isolated bacterium belongs to a thermophilic Bacillus sp., according to the DSMZ database. Similarly, the physiological conditions determined for the organism clearly pointed to an alkalophilic Bacillus sp. A sequence analysis of the 16S rRNA gene revealed that the new organism is closely related phylogenetically to members of the genus Bacillus. Purication of catalase-peroxidase Bacillus SF was cultivated in a 10-l bioreactor, and a crude enzyme preparation was obtained showing catalase activities of 48 U mg1. Figure 1A indicates that cytosolic fractions of the thermoalkaliphilic Bacillus SF contain only one isoform of an enzyme with hydroperoxidase activity because native PAGE gave only single bands of identical migration characteristics detectable with staining procedures for either catalase (Fig. 1A) or peroxidase activity (not shown). Table 1 summarizes the four steps of the purication procedure of this enzyme, yielding a homogenous protein puried approximately 70.3-fold over the crude extract and giving a nal recovery of 11.3% of catalase activity. During the various purication steps, both catalase and peroxidase activity were always eluted as a sharp and single peak. SDSPAGE of the puried enzyme gave a single band at about 82 kDa (Fig. 1B), whereas molecular mass analysis by gel ltration on a Superdex 200 HR 10/30 column gave a molecular mass of about 165 10 kDa (data not shown), indicating that the enzyme is composed of two subunits of identical size. The isoelectric point of the enzyme protein was determined by IEF to be at pH 6.0. Physical and chemical characterization of the CP from Bacillus SF The temperature optimum for catalase activity at pH 9.5 was observed at 55C. The activity decreased by 10% at

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A B kDa 200,00 97,40 66,20 45,00

31.00

21,50 14,40 6,50 1

1A,B

Fig. 1A,B. Native and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of the puried catalase-peroxidase (CP) from Bacillus SF. A Native 10% polyacrylamide gel stained for catalase activity according to Harris and Hopkinson (1976). B Denaturing SDS 12% polyacrylamide gel stained for protein with Coomassie brilliant blue. Lane 1, puried CP; lane 2, Pharmacia molecular mass standard. Arrow indicates application of the catalase

Fig. 2. Effect of pH on the catalase activity of the puried CP from Bacillus SF. Catalytic activity was assayed at 25C as described in Materials and methods
2

Table 1. Purication of catalase peroxidase from the thermoalkaliphilic Bacillus SF Step Total protein (mg) 200 98 35 1.45 0.32 Tota activity (U) 9,600 8,540 5,600 1,560 1,080 Specic activity (U/mg) 48 87.1 160 1,076 3,375 Purication (-fold) Yield (%)

Crude extract 40%70% (NH4)2SO4 PhenylSepharose UNOQ-6 Superdex 200

1 1.8 3.3 22.4 70.3

100 88.8 58.3 16.3 11.3 Fig. 3. Absorption spectrum of puried CP from Bacillus SF. Thick line, 9 M native enzyme; thin line, the cyano-adduct obtained by the addition of 25 mM KCN to the native enzyme. Inset is an expanded scale of the visible portion of the spectrum. Experimental conditions were as described in Materials and methods
3

60C and by 50% at 70C. The enzyme was active at pH 6.0 10.0; however, a sharp activity maximum appeared at pH 7.0 (25C). Activity rapidly decreased until it reached 25% at pH 8.0, where it remained almost constant until pH 9.0; at pH 10, only 4% activity remained (Fig. 2). The CP from Bacillus SF was quite stable at pH 10 and 25C, showing half-lives of at least 4 days (Table 2). Interestingly, at this temperature the CP showed substantially higher stabilities at pH 8 and pH 9 than at pH 7. At 60C, the CP had the longest half-lives at pH 8.0 and pH 9.0, and its half-life decreased to only 5 h at pH 10.

Table 2. Stability of a catalase peroxidase from Bacillus SF Temperature (C) pH 7.0 25 50 60 Data are half-life in hours 190 15 22 8.0 260 35 40 9.0 240 48 38 10.0 104 14 5

Catalytic and spectral properties The visible optical density spectrum of thermoalkaliphilic CP from Bacillus SF showed a strong Soret band at 405 nm and a peak at 623 nm. The OD405/OD280 ratio of the enzyme preparations was 0.36, which is consistent with one heme per homodimer. On addition of cyanide, the Soret band

shifted from 405 to 417 nm, indicating a high to low spin transition of the iron center of the heme (Fig. 3). The catalase-peroxidase was reduced by dithionite, as indicated by two new absorption maxima at 435 and 553 nm. The catalytic activity of the puried enzyme was 3,375 U mg1 protein. The apparent Km was 2.6 0.1 mM H2O2, and the turnover number (kcat) was 11,475 s1. The observed kcat/Km was in the same order of magnitude as that from Escherichia coli HP1 (Table 3) and reects the high

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Table 3. Kinetic parameters of various catalase-peroxidases Organism Bacillus SF Bacillus stearothermophilus Alkalophilic Bacillus Rhodobacter capsulatus Mycobacterium smegmatis Mycobacterium tuberculosis Mycobacterium tuberculosis Anacystis nidulans Synechocystis 7942 Synechocystis 6301 Synechococcus 6803 Escherichia coli HP1 Km (mM) 2.6 4.4 6.8 4.2 1.4 30 5.2 4.8 4.2 4.3 4.8 3.9 kcat (s1) 11,475 1,400 n.d. n.d. 2,380 2,300 10,140 8,850 26,000 7,200 3,450 16,300 kcat/Km (s1 M1) 4.41 106 0.32 106 n.d. n.d. 1.70 106 0.076 106 1.95 106 1.8 106 6.19 106 1.66 106 0.72 106 4.17 106 Reference This study Kobayashi and Suga (1997) Yumoto et al. (1990) Hochman and Goldberg (1991) Marcinkeviciene et al. 1995) Nagy et al. (1997) Johnsson et al. (1997) Engleder et al. (2000) Mutsuda et al. (1996) Obinger et al. (1997) Regelsberger et al. (1999a) Claiborne and Fridovich (1979)

catalytic efciency of the reaction. The puried enzyme was irreversibly inhibited by high hydrogen peroxide concentrations. Starting at concentrations of 15 mM H2O2, progressive inhibition of the enzyme was seen (data not shown). In contrast to monofunctional catalases, 3-amino1,2,4-triazole did not inhibit the catalase-peroxidase from Bacillus SF. However, addition of cyanide and azide caused pronounced inhibition of the enzyme activity. For both azide and cyanide, the inhibition was competitive with a KIEI of 15 M and 1.5 M for the dissociation of the enzyme-inhibitor complex, respectively (calculated from Dixon plots and the method of EadeeHofsted). Both guaiacol and o-dianisidine functioned as electron donors for Bacillus SF catalase-peroxidase after compound I formation with peroxoacetic acid; o-dianisidine was the better substrate (data not shown).

Discussion
A thermoalkaliphilic bacterium was isolated from textile nishing efuent and identied as a new representative of the genus Bacillus. There are only a few reports on thermoalkaliphilic Bacillus species, such as Bacillus sp. TAR-1 (Takahashi et al. 2000), Bacillus thermocatenulatus (Rua et al. 1997), Bacillus thermoalcaliphilus (Sarkar and Upadhyay 1993), or an anaerobic strain, LBS3 (Prowe et al. 1996). Although catalases, which are produced by most aerobic microorganisms, are very well studied enzymes, there are only few reports on thermostable and alkalistable catalases such as those from Thermoleophilum album (Allgood and Perry 1986). Similarly, there is only little information in the literature about the thermostability and pH stability of catalase-peroxidases. At pH 6.5 and 58C, a catalase-peroxidase from Synechococcus PCC 7942 and one from Mycobacterium tuberculosis (Mutsuda et al. 1996; Nagy et al. 1997) both showed half-life times of 30 min. A CP from an alkalophilic Bacillus sp. had a half-life of 60 min at pH 11 and 60C (Yumoto et al. 1990), whereas the CP from Bacillus SF investigated in this report showed a half-life of 300 min at the same temperature but at

pH 10. At room temperature and pH 10, this CP was stable for 104 h. For the closely related CP from B. stearothermophilus (see following), an interesting phenomenon was observed. The CP from this organism showed an increase in kcat with a concomitant decrease of Km after heating. In contrast to these ndings, no second active form could be found on thermal activation for the CP of Bacillus SF. The catalase-peroxidase from Bacillus SF was found to share a number of structural and spectroscopic properties with other bacterial catalase-peroxidases. This enzyme is a dimer of 82-kDa subunits, containing one ferric protoporphyrin IX prosthetic group, and exhibits a high-spin ferric heme optical spectrum. High-spin to low-spin conversion was observed in the presence of CN. Homodimeric or homotetrameric structures are typical for this type of hydroperoxidase. In general, the identical subunits of catalase-peroxidases are 7885 kDa in size, as has been demonstrated for the subunits in E. coli (Claiborne and Fridovich 1979), Streptomyces sp. IMSNU-1 (Youn et al. 1995), Mycobacterium smegmatis (Marcinkeviciene et al. 1995), Mycobacterium tuberculosis (Johnsson et al. 1997), Rhodobacter capsulatus (Hochman and Goldberg 1991), and B. stearothermophilus (Loprasert et al. 1988). Other heme-containing peroxidases of fungi and plants are in most cases monomeric proteins. The double length of the bacterial peroxidases has been ascribed to gene duplication (Welinder 1992). The OD405/OD280 ratio of the enzyme preparations from Bacillus SF was 0.36, which is consistent with one heme per homodimer. It has been reported that catalase-peroxidases found in other bacterial systems also have low heme content. The OD405/OD280 ratios per subunit of typical catalaseperoxidases were 0.54 in Streptomyces sp. (Youn et al. 1995), 0.56 in M. smegmatis (Marcinkeviciene et al. 1995), 0.52 in E. coli HPI (Claiborne and Fridovich 1979), 0.3 in R. capsulatus (Hochman and Goldberg 1991), and between 0.35 and 0.46 in Synechocystis sp. (Regelsberger et al. 1999b). Additional characteristics shared with other bacterial catalase-peroxidases include the competitive inhibition by cyanide and azide and the lack of inhibition by the catalase inhibitor, 3-amino-1,2,4-triazole (Marcinkeviciene et al. 1995). The linear, competitive nature of the inhibition by

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CN suggests that the heme iron atom of the catalaseperoxidase is the site of both peroxide and cyanide binding, as is the case for other peroxidases (Dunford 1991). The KIEI of 1.5 M measured for the inhibition of the CP from Bacillus SF by cyanide was signicantly lower than the KIEI of 18.7 M reported previously for a CP from Anacystis nidulans (Engleder et al. 2000). The absorption spectrum of the Bacillus SF catalase-peroxidase obtained after addition of dithionite was comparable with data reported in the literature for other catalase-peroxidases. Dithionite reduction distinguishes the CP from typical catalases, which are not reduced by this agent, whereas typical peroxidases are rapidly reduced by dithionite (Johnsson et al. 1997; Marcinkeviciene et al. 1995; Loprasert et al. 1988). With a kcat of 11,475 s1, the catalytic activity of the puried CP from Bacillus SF is about in the same order of magnitude as that of other typical catalase-peroxidases (see Table 3). In addition to its catalase activity, the enzyme functions as a peroxidase, oxidizing guaiacol and odianisidine, for example. In contrast, no activity on guaiacol was previously found for CP from Synechocystis sp. and Klebsiella pneumoniae (Cendrin et al. 1994; Hochman and Goldberg 1991) whereas o-dianisidine seemed to be a substrate for most CP (Johnsson et al. 1997; Marcinkeviciene et al. 1995; Youn et al. 1995; Mutsuda et al. 1996). The homology of the puried protein with the catalaseperoxidase of Bacillus stearothermophilus was determined by peptide mass mapping using MALDI-MS. Thus, in conclusion, physical characterization of the catalase-peroxidase from the alkalithermophilic Bacillus SF clearly indicates that this enzyme belongs to the developing class of bacterial enzymes that catalyze both catalase- and peroxidase-like reactions. The low heme content of catalase-peroxidases is in contradiction to sequence alignment models proposing one heme binding per monomer (Welinder 1991). Nevertheless, this OD405/OD280 ratio seems to be one of the characteristics of this type of hydroperoxidase. To obtain more information about the structurefunction relationship of this enzyme, the determination of the three-dimensional structure of this new thermoalkalistable catalaseperoxidase has been initiated.
Acknowledgments The authors thank the European Commission for nancial support of project EW 98 0009.

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