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Instituto de Investigaciones Bioquímicas de La Plata, CONICET, UNLP, La Plata, Argentina
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Entomopathogenic fungi adapt to growth in a culture medium containing an insect-like hydrocarbon as the sole carbon source inducing the ß-oxidation pathway during the alkane degradation. The effect of two carbon sources on the catalase activity was studied in the entomopathogenic fungus Beauveria bassiana. Catalase activity was detected both in the peroxisomal and cytosolic fraction. A significant increment in the specific activity of the peroxisomal fraction (12.6-fold) was observed when glucose was replaced by an insect-like hydrocarbon, whereas the specific activity in the cytosol diminished more than 1.2-fold in the same culture condition. After purification to homogeneity by gel filtration and strong anion exchange chromatography, an apparent molecular mass of 54.7 and 84.0 kDa per subunit were determined respectively for the peroxisomal and cytosolic catalase. The enzymes showed different biochemical and kinetic characteristics, but both were inhibited by 3-amino-1,2,4 triazole. Peroxisomal catalase was sensitive to pH, heat and high concentration of the hydrogen peroxide substrate. Inversely the cytosolic isoform exhibited a broad range of optimal pH (6.0–10.0), high thermostability (<55 C) and remained fully active at least up to 70 mM hydrogen peroxide. Measurement of catalase activity is a new approach for evaluating fungal ability to degrade hydrocarbons.
Key words: Beauveria bassiana, catalase, entomopathogenic fungi, very long chain hydrocarbons
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, b). The ability of entomopathogenic fungi to degrade insect cuticular hydrocarbons and to use them for energy production and incorporation into cellular components was shown first in this laboratory (Napolitano and Juárez 1997, Crespo et al 2000). When Beauveria bassiana (Deuteromycotina: Hyphomycetes) was adapted to grow on an insect-like hydrocarbon source, bioassays employing the bean weevil Acanthoscelides obtectus produced a twofold increment in host mortality compared to glucose-grown cultures (Crespo et al 2002). A 17% reduction in the time to kill the Chagas disease vector Triatoma infestans (Pedrini and Juárez unpublished) was detected in similar incubation conditions. In B. bassiana hydrocarbon catabolism proceeds through complete ß-oxidation (Crespo et al 2000, Juárez et al 2004). Peroxisomes are known to be the site of ß-oxidation in fungi (Kunau et al 1996). Peroxisomal proliferation, together with a marked induction of the ß-oxidation system, was reported to be related to alkane-growth adaptation in yeasts (Fukui and Tanaka 1979, Tanaka et al 1982) and in the fungus B. bassiana (Crespo et al 2000). Filamentous fungal ß-oxidation enzymes also were shown to be induced in oleate-enriched medium (Valenciano et al 1996). Catalases are typical markers of peroxisomes, however, nonperoxisomal location has been demonstrated in different eukaryotic systems (Taub et al 1999, Bussink and Oliver 2001). According to their function, there are two kinds of catalases: bifunctional catalase-peroxidases and monofunctional or true catalases. The latter group is one of the best characterized both in eukaryota and prokaryota, 74 sequences were classified into three different groups (I, II, III), corresponding to three different gene families, after phylogenetic analyses. Bacterial catalases are ubiquitous in all three groups, plant catalases belong to group I, large-subunit catalases from filamentous fungi belong to group II, whereas most peroxisome-localized catalases from animal, yeast and filamentous fungi are reported in group III (Klotz et al 1997). However many of these catalases have not been purified and physical and kinetical characterization have not been reported. Large-subunit catalases, present only in bacteria and fungi, exhibit enhanced resistance to heat and chaotropic agents, presumably due to an extended N-terminal sequence of 70 residues and a C-terminal domain of about 150 residues (Klotz et al 1997). In filamentous fungi, catalases are homotetramers and constitute a group with diverse structure, function and cell localization (Natvig et al 1996). In Aspergillus niger, an extracellular catalase protects cells from exogenous H2O2 (Witteveen et al 1992). Differential expression of a cytosolic catalase was observed under stress-growth condition in Cladosporium fulvum (Bussink and Oliver 2001) and during cell-differentiation processes in Neurospora crassa (Díaz et al 2001). In the present study the role of two different catalases in ß-oxidation processes related to alkane use was investigated in the entomopathogenic fungus B. bassiana.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Subcellular fractionation.— –
Fungi were processed in 50 mM sodium phosphate buffer pH 7.0 containing 1 mM phenylmethylsulfonyl fluoride with a Mini-Bead Beater homogenizer (BioSpec, Bartlesville, Oklahoma) with glass beads (0.5 mm diam.), as suggested by the manufacturer. The homogenate fraction was obtained by centrifugation at 2000 g x 15 min; a pellet containing mitochondria and peroxisomes was obtained after ultracentrifugation at 20 000 g x 30 min. The pellet (P20 000 g) was washed once by the same procedure replacing the supernatant with sodium phosphate buffer. The supernatant was ultracentrifuged at 100 000 g x 60 min in a Beckman L8-M Ultracentrifuge (Beckman, Palo Alto, California), to obtain the cytosolic fraction (S100 000 g). All procedures were performed at 4 C. On the other hand the homogenate fraction was ultracentrifuged at 130 000 g in the presence of 30% Nycodenz to isolate peroxisomes (Ghosh and Hajra 1986). However it was not possible to get rid of the large amounts of remnant hydrocarbon altering the sedimentation parameters in FS2 cultures. The P20 000 g fraction was used as the peroxisome-containing fraction after measuring the activity of the acyl-CoA oxidase (Small et al 1985) with lignoceroyl-CoA as substrate. This peroxisomal marker enzyme was detected mostly in the P20 000 g fraction (78% total activity), supporting this choice.
Catalase activity.— –
Catalase specific activity was determined in the P20 000 g and S100 000 g fractions in FS0, FS1, and FS2 cultures. Hydrogen peroxide consumption (20 mM) was measured spectrophotometrically by A240 decrease (Beers and Sizer 1952) in a Ultrospec 2100 prospectrophotometer (Biochrom Ltd., Cambridge, UK) with a 10 mm light path quartz cuvette. One unit of catalase was defined as the amount of enzyme that decompose 1 µmol of H2O2 in 1 min at pH 7.0 and 25 C. Protein concentration was determined by the method of Bradford (1976) except during the purification steps described below, in which the micro BCA method was used (Pierce, Rockford, Illinois); in both cases bovine albumin was used as standard.
Catalase purification.— – The FS0 culture was used for catalase purification. The P20 000 g fraction was disrupted by repeated sonication steps, 3 x 1 min at 0 C, at an output of 50 W (Branson, Danbury, Connecticut) and centrifuged at 10 000 g x 20 min at 4 C. The supernatant was filtered through an Amicon ultramembrane YM50 (50 000 NMWL cut-off) (Amicon, Beverly, Massachusetts) and concentrated to 300 µL. This fraction was applied to a Superdex 200 HR 10/30 gel permeation column (Pharmacia Biotech, Uppsala, Sweden) previously equilibrated with 50 mM sodium phosphate buffer pH 7.0, eluted at 0.5 mL min–1 with the same buffer and collected in 1.5 mL fractions. The fraction with catalase activity was filtered through an Amicon ultramembrane YM50, concentrated and applied to a Mono Q HR 10/10 strong anion exchange column (Pharmacia Biotech, Uppsala, Sweden) equilibrated with 20 mM sodium acetate buffer, pH 7.0. After washing out other proteins with 30 mM sodium acetate pH 6.0 and 100 mM sodium acetate buffer pH 5.0, catalase activity was step-eluted with 1 M sodium chloride plus sodium acetate 20 mM buffer, pH 7.0.
For the cytosolic isoform purification, one volume of ethanol-chloroform (1:1, v/v) was added to 5 volumes of the S100 000 g and shaked (15 s x 3). After 20 min at room temperature the mix was centrifuged at 10 000 g 20 min at 4 C; the top, aqueous layer with catalase activity was recovered. Five volumes of cold acetone were added to one volume of the aqueous layer, kept at –20 C for 20 min and centrifuged at 10 000 g (10 min at 4 C). The resulting pellet was resuspended in 50 mM sodium phosphate buffer pH 7.0, filtered through an Amicon ultramembrane YM50 (Amicon, Beverly, Massachusetts) and concentrated to 300 µL. This fraction was applied to a Superdex 200 HR 10/30 gel permeation column and to a Mono Q HR 10/10 strong anion exchange column as described above. Protein and heme-content in each fraction were determined by measuring respectively A280 and A406. After overnight dialysis against 10 mM buffer EDTA for desalinization, the fraction with catalase activity was concentrated in Ultrafree microcentrifuge filters 30 000 Da (Sigma-Aldrich, St Louis, Missouri).
Catalase characterization.— –
Gel electrophoresis..
SDS-PAGE was carried out on a polyacrylamide gel (10 and 12%) in the presence of ß-mercaptoethanol with a Mini-Protean II apparatus (BioRad, Hercules, California) by using the method of Laemmli (1970). Proteins were stained by Coomassie Brillant Blue R-250. The molecular mass was estimated with 1D Image Analysis Software (Kodak, Rochester, New York) employing appropriate molecular weight standards (Amersham Biosciences, Buckingham-shire, UK). Native gradient gels (4–20%) of both purified fractions were soaked 20 min in deionized water, then a H2O2 solution (10%) was added on to the gel (Kinoshita et al 1998). Bands of oxygen bubbles indicate catalase activity.
Effect of pH and temperature on enzymes activity. –
The optimum pH of the purified enzymes was determined at 25 C; the buffers were 50 mM sodium acetate (pH 3.8–5.5), 50 mM sodium phosphate (pH 6.0–8.2) and 50 mM sodium carbonate (pH 8.5–9.6). The optimum temperature was determined at pH 7.0 in 50 mM sodium phosphate buffer. Enzyme thermostability was evaluated after pre-incubating the purified enzymes in 50 mM sodium phosphate buffer (pH 7.0) 5 min at different temperatures (Brown-Peterson and Salin 1995); residual activity was assayed using a standard technique as described above.
Catalytic properties. –
The effect of substrate concentration on the catalase activity was examined after incubation of the purified proteins at 25 C in 50 mM of sodium phosphate buffer (pH 7.0) at varying H2O2 concentrations (5–70 mM), six replicates each. Catalase activity was measured in the presence of 20 mM 3-amino-1,2,4 triazole (3-AT) by incubating the enzymes at 37 C in 50 mM sodium phosphate buffer (pH 7.0) containing 4 mM H2O2 (Margoliash et al 1960). After 10 min incubation the remaining activity was evaluated as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). A significant reduction in the catalase specific activity was observed in the S100 000 g fraction of the FS2 culture. The specific activity was diminished from 118 ± 24 (FS0) to 52 ± 10 U mg–1 (FS2) (P < 0.0001) (TABLE I). The activity of FS1 cultures was not significantly different from that of the controls (data not shown). After several months at –70 C, the catalase activity remained unaltered in the S100 000 g fraction, whereas no activity was detected in the P20 000 g fraction (data not shown).
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). Purification of the cytosolic catalase resulted in a 150-fold purification with 6% yield; catalase content was close to 0.04% of the total cytosolic proteins (TABLE II). A single oxygen-bubble band in the presence of H2O2 was observed in native gels of each enzyme (data not shown). Apparent molecular masses of 54.7 kDa (FIG. 1a) and 84.0 kDa (FIG. 1b) were determined by SDS-PAGE for each subunit of the tetrameric peroxisomal and cytosolic enzymes, respectively.
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). Inversely, a broad optimum pH range was observed for the cytosolic catalase of pH 6.0–10.0 (FIG. 2a). Activity was determined at different temperatures; optimum values were obtained at 25–37 C for both enzymes, although residual activity was still detected at 7 C (31%) and 60 C (40%) for the cytosolic catalase (data not shown). Thermostability was assayed by a 5 min pre-incubation period at fixed temperatures, the cytosolic enzyme was shown to be fully stable up to 55 C, compared to pre-incubation at 25 C, and retained 35% of activity at 75 C (FIG. 2b). Conversely the peroxisomal catalase was more sensitive to heat; only 27% of activity was detected after pre-incubation at 45 C, whereas no activity was detected at 75 C (FIG. 2b).
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). At increasing H2O2 concentrations the cytosolic catalase achieved its maximal activity at 40 mM; no loss of activity was evident up to 70 mM. The peroxisomal isoform exhibited the highest activity at 20–40 mM showing a sharp decline at higher substrate concentration, with a 73% inhibition at 60 mM H2O2. After 10 min incubation with 20 mM 3-AT, a 100% and 85% inhibition was detected respectively for the peroxisomal and cytosolic catalases. The cytosolic isoform was fully inhibited at 60 mM 3-AT (data not shown). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). It is well documented that cultivation of yeasts in alkanes trigger the induction of the peroxisomal ß-oxidation system (Fukui and Tanaka 1979). It also was suggested that catalase activity might be related to hydrocarbon metabolism in yeasts (Teranishi et al 1974). The ability of entomopathogenic fungi to degrade very long chain insect-like hydrocarbons was shown first in this laboratory (Napolitano and Juárez 1997). Peroxisome proliferation was detected in alkane-grown B. bassiana cultures after staining with diaminobenzidine (Crespo et al 2000, Juárez et al 2004). In this work peroxisomal catalase induction is reported in alkane-grown B. bassiana. A high increment in the catalase activity (
14-fold) was measured in the FS2 cultures (TABLE I). Similar results were obtained for a native B. bassiana strain (Bb 10) (IMYZA, INTA, Argentina) with a significant 6.6-fold increment of 139 ± 72 to 1063 ± 190 U mg–1 for FS0 and FS2, respectively (P < 0.0001) (data not shown). FS1 cultures did not differ significantly from FS0 cultures, suggesting that a second adaptation period, FS2, was required for significant changes to be expressed in the ß-oxidation system. A second adaptation period also was needed to detect substantial peroxisome proliferation (Crespo et al 2000). Metabolic studies using radiolabeled substrates also showed that two adaptation periods were necessary to obtain significant differences in metabolite production from alkane degradation, compared to the controls (Napolitano and Juárez 1997, Crespo et al 2000, Juárez et al 2000). The cytosolic catalase exhibited a different behavior, more than twofold reduction was observed in the catalase activity of the alkane-grown GHA (TABLE I) and Bb 10 strains (data not shown). The generation of singlet oxygen was proposed to account for the oxidation of the heme group and enzyme degradation under stress in a cytosolic catalase (Cat-1) of N. crassa (Lledías et al 1998). We may speculate that the activity cut observed could be explained by enhanced enzyme degradation. Catalase and glucose oxidase (GOD) are produced simultaneously at high levels by filamentous fungi, associated with the cell wall (Petruccioli et al 1995, Fiedurek and Gromada 2000). In glucose-grown fungi, GOD catalyzes the reaction of ß-D-glucose with molecular oxygen to D-gluconic acid and H2O2. Lack of glucose in the culture medium might affect GOD production, and hence H2O2 generation, with a concomitant reduction of the cytosolic catalase expression in alkane-grown B. bassiana. Monofunctional (true) catalases are inhibited by 3-AT (Margoliash et al 1960) and they commonly are isolated from animals, plants, fungi and bacteria (Klotz et al 1997). The catalase-peroxidases are isolated only from bacteria and fungi and they are insensitive to 3-AT (Nadler et al 1986, Fraaije et al 1996). Monofunctional catalases are classified in the small subunit and large subunit groups. In A. nidulans the small subunit catalase containing the peroxisome-targeting signal was reported in the particulate fraction, along with other peroxisomal marker enzymes (Kawasaki and Aguirre 2001). Peroxisomal catalases from different systems showed a relatively small range of molecular weight, from 48 kDa for Saccharomyces cereviciae to 55 kDa both for Candida tropicalis and the filamentous fungi Aspergillus nidulans (Okada et al 1987, Kawasaki and Aguirre 2001). Present results show that the B. bassiana peroxisomal catalase fits into the small subunit group. Large subunit enzymes have been identified in bacteria and filamentous fungi but not in higher eukaryotes. They are located both in the cytosol and the cell wall, exhibiting a broad optimum pH range and enhanced resistance to heat and organic solvents (Kawasaki and Aguirre 2001). A monofunctional and large subunit enzyme is the predominant catalase in the conidia of N. crassa (Chary and Natvig 1989) and A. nidulans (Kawasaki and Aguirre 2001). B. bassiana cytosolic catalase had an apparent molecular mass (84 kDa) and kinetic and physical properties similar to these enzymes. Measurement of peroxisomal catalase activity now is shown as a new and simple approach for evaluating fungal ability to degrade hydrocarbons. Using the appropriate insect host-like hydrocarbon, this assay might be used as an alternative method to assess mycoinsecticide improvement by alkane-growth adaptation. Molecular studies will be addressed to detect the different gene expression pattern of both enzymes and to help understand the role of different carbon sources on their regulation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: mjuarez{at}isis.unlp.edu.ar
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