World Journal of Microbiology & Biotechnology 16: 691±694, 2000.
Ó 2001 Kluwer Academic Publishers. Printed in the Netherlands.
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Biochemical characterization of neutral trehalase activity in Saccharomyces boulardii Antero S.R. de Andrade1, Raquel G. dos Santos1, Jacques R. Nicoli2 and Maria J. Neves1,* 1 LaboratoÂrio de Radiobiologia, Centro de Desenvolvimento da Tecnologia Nuclear/ComissaÄo Nacional de Energia Nuclear (CDTN/CNEN), Belo Horizonte, Minas Gerais, Brazil 2 Departamento de Microbiologia, Instituto de CieÃncias BioloÂgicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil *Author for correspondence: R. Prof. MaÂrio Werneck S/N, Cidade UniversitaÂria ± Pampulha, Campus UFMG, Caixa Postal 941, CEP 30123-970, Belo Horizonte ± MG, Brazil. Tel.: +55 (31) 499 31 82, Fax: +55 (31) 499 33 80, E-mail:
[email protected] Received 4 May 2000; accepted 24 October 2000
Keywords: Neutral trehalase, Saccharomyces boulardii, yeast
Summary Lyophilized cells of the non-pathogenic yeast Saccharomyces boulardii are used in many countries for the treatment of several types of diarrhoea and other gastrointestinal diseases. Although the cells must be viable, their mechanism of action is unknown. The disaccharide trehalose is a protectant against several forms of environmental stress in yeast and is involved in maintaining cell viability. There is no information on the enzymes involved in degradation of trehalose in S. boulardii. The aim of the present study was to characterize trehalase activity in this yeast. Cells of S. boulardii grown in glucose exhibited neutral trehalase activity only in the exponential phase. Acidic trehalase was not detected in glucose medium. Cells grown in trehalose exhibited acid and neutral trehalase activities at all growth stages, particularly in the exponential phase. The optimum pH and temperature values for neutral trehalase activity were determined as 6.5 and 30 °C respectively, the half-life being approximately 3 min at 45 °C. The relative molecular mass of neutral trehalase is 80 kDa and the Km 6.4 mM (0.6). Neutral trehalase activity at pH 6.5 was weakly inhibited by 5 mM EDTA and strongly inhibited by ATP, as well as the divalent ions Cu++, Fe++ and Zn++. Enzyme activity was stimulated by Mg++ and Ca++ only in the absence of cAMP. The presence of cAMP with no ion additions increased activity by 40%. Introduction Lyophilized cells of the yeast Saccharomyces boulardii have been widely used in Europe, Africa and Latin America to treat several types of diarrhoea, as well as in the prevention of antibiotic-associated diarrhoea (McFarland & Bernasconi 1993), millions of doses being sold each year. Each 100 mg capsule contains 2.8 ´ 109 cells which remain viable for at least 2 years (McFarland & Bernasconi 1993).This medication is taken orally and its ecacy is linked to the viability of the cells (Vidon et al. 1986). The mechanisms by which the cells of this yeast act are unknown. However, some hypothesis could explain the protective eect of the biotherapeutic such as the inhibition of action or production of bacterial toxins (Castagliuolo et al. 1996) and/or the immunomodulation of the host (Machado-Caetano et al. 1986). In both cases, the cells must be viable to provide an advantageous eect to the host. The disaccharide trehalose (a-D -glucopyranosyl-a-D glucopyranose) is a reserve carbohydrate that has been
found in a wide variety of species, particularly in cells under anhydrobiotic conditions (Elbein 1974; Singer & Lindquist 1998). The mobilization of trehalose accumulated under these conditions signals the end of the quiescent period and activation of the normal growth cycle. Trehalose is also crucial for cell survival when cells are exposed to heat shock, dehydration, oxidative stress and other adverse conditions (Van Laere 1989). The principal enzyme involved in degradation of trehalose is trehalase (a, a-trehalose-1D -glucohydrolase, EC 3.2.1.28). Two different trehalases, known as regulatory (or neutral) and non-regulatory (or acid) trehalase, have been described in several species of fungi (Thevelein 1984). Both are strictly speci®c for trehalose as their substrate but differ in biochemical parameters, regulation and subcellular localization. Neutral trehalase is a cytosolic enzyme encoded by the gene NHT (Nwaka 1995) and most active at pH 6.7±7.0. Its activity is tightly regulated by the RAS/adenylate cyclase signal transduction pathway, which converts the inactive enzyme to its phosphorylated active form (Thevelein
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1988). Acid trehalase occurs in cell vacuoles and is encoded by the ATH gene. This enzyme is glycosylated and its optimum pH is 4.0±5.0 (Destruelle et al. 1995). Acid trehalase is not activated by phosphorylation and is apparently specialized in the hydrolysis of extracellular trehalose that can serve as a carbon source (Jorge et al. 1997). Neutral trehalase appears to specialize in the degradation of cytosolic trehalose during the germination of spores and other vital functions in yeast, and also in the degradation of trehalose accumulated in cells submitted to various forms of stress (e.g. exposure to toxins, high temperatures, freezing and desiccation) (Nwaka et al. 1995). The gene sequence of ATH shows no homology with that of NTH (Destruelle et al. 1995). The aim of the present study was to characterize the enzyme responsible for the degradation of cytosolic trehalose.
oxidase procedure (Bergmeyer & Bernt 1974). Trehalase speci®c activity is expressed as mU (nmol of glucose released/min/mg protein).
Materials and Methods
Determination of Km
Organism and growth conditions
The Km was determined using a trehalose concentration range of 0.6±80 mM. Samples were processed for the determination of trehalase activity as described. The Km was estimated by Lineweaver±Burk double reciprocal plot.
Lyophilized S. boulardii cells were donated by Merck S.A., Rio de Janeiro, Brazil (S. boulardii 17-FloratilÒ). The lyophilized cells were inoculated into liquid medium containing 2% (w/v) peptone, 1% (w/v) yeast extract and 2% (w/v) glucose (YPG medium). Incubation and extraction conditions To measure trehalase activity, lyophilized cells were incubated in YPG medium in a shaking water bath at 30 °C and harvested during the exponential phase or stationary phase as con®rmed by presence or absence of glucose, respectively. Saccharomyces boulardii is a facultative aerobic yeast that produces ethanol by glucose fermentation. Cell extracts were prepared by homogenization with glass beads in 10 mM sodium phosphate buer, pH 7.4 containing 1 mM DTT, 1 mM PMSF, 10 mM NaF. After centrifugation at 3000 rev/min for 10 min the supernatant (crude extract) was collected and used as the source of enzyme. Activation of trehalase The extract was incubated for 30 min at 30 °C in 50 mM sodium phosphate buer pH 7.4, 10 mM NaF, 10 lM cAMP, 2.8 mM Mg-acetate, 1 mM theophylline and 0.8 mM ATP. Determination of trehalase The crude extract, activated or not by cAMP, was incubated with 0.1 M trehalose, 0.15 M McIlvaine buer pH 6.5 and 25 mM CaCl2 or other ions at the indicated concentrations. The samples were incubated for 30 min at 30 °C then boiled for 4 min. The quantity of glucose liberated was estimated by the glucose
Superose 12 chromatography The molecular weight of trehalase was estimated by Superose 12 gel ®ltration chromatography. The column was calibrated with the following molecular weight markers: b-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa). The sample volume loaded was of 250 ll and the column was equilibrated with the 0.1 M McIlvaine buer (pH 6.5) containing 0.15 M NaCl. The column was eluted at room temperature with the same buer, using a ¯ow rate of 0.5 ml/min. Fractions of 0.25 ml were collected and analysed for trehalase activity.
Protein determination Protein content was assayed by the method of Lowry using BSA as a standard. Reproducibility of the results All experiments were repeated at least three times with consistent results. Representative data are shown. Results and Discussion Trehalase activity was determined at pH 6.5 and 4.5 in crude extracts obtained from cells grown in glucosesupplemented medium. Trehalase activity at pH 6.5 was observed only for cells harvested in the logarithmic phase (Figure 1), while at pH 4.5 it was seen only in cells grown in trehalose-supplemented medium harvested during logarithmic phase (Figure 2). In the glucosesupplemented medium a very low trehalase activity was detected at pH 4.5 (results not shown). In glucose medium, a faster use of this carbohydrate by S. boulardii cells was observed when compared with wild type cells of S. cerevisiae (data not shown). The activity measured at pH 6.5 was designated as being due to neutral trehalase, as suggested by Thevelein (1984). Neutral trehalases exhibit a neutral optimum pH and low thermal stability. They are also known as regulatory enzymes, since they can be activated by cAMP-dependent phosphorylation. These results demonstrated the in¯uence of a carbon source on trehalase activity and
Neutral trehalase of Saccharomyces boulardii
Figure 1. Growth curve and trehalase activity in glucose medium. Cells of S. boulardii were inoculated in a YP medium supplemented with 2% glucose. Samples were taken at the indicated times intervals to measure growth at 660 nm (s) and to determine the activity of trehalase at pH 4.5 and 6.5. Trehalase activity appeared only at pH 6.5 (h). Speci®c activity: nmol of glucose liberated/min/mg protein.
Figure 2. Growth curve and trehalases activities in trehalose medium. Cells of S. boulardii were inoculated in a YP medium supplemented with 1% trehalose (w/v). Samples were taken at the indicated time intervals to measure the growth at 660 nm (s) and to determinate the activity of trehalase at pH 4.5 (n) and 6.5 (h). Speci®c activity: nmol of glucose liberated/min/mg protein.
showed that S. boulardii possesses two forms of this enzyme, as do S. cerevisiae, Schizosaccharomyces pombe, Candida utilis, Torulaspora delbrueckii, Kluyveromyces lactis, Fusarium oxysporum and Mucor rouxii (Jorge et al. 1997). The eect of pH on trehalase activity was investigated using 0.1 mM McIlvaine buer. The enzyme showed maximal activity at pH 6.5 (Figure 3). The optimum temperature for trehalose hydrolysis was 30 °C (Figure 4). At pH 6.5 and 45 °C the enzyme had a half-life of approximately 3 min (Figure 4). Optimum activity within the range of the physiological growth temperatures and low thermostability are characteristics of neutral trehalases (Thevelein 1984). The Km value obtained in presence of 10 mM calcium was 6.4 (0.6) mM. The eect of several cations on neutral trehalase activity was also investigated (Table 1). A 1.4-fold activation was observed when the crude extract obtained from stationary phase cells (basal level, no additions)
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Figure 3. pH pro®le of neutral trehalase activity. Cells were inoculated in a YP glucose medium and harvested at logarithmic phase. Trehalase activity was determined in crude extracts using McIIvaine buer 0.1 M at the indicated pH values. Speci®c activity: nmol of glucose liberated/ min/mg protein.
was compared with that previously activated in vitro by cAMP. By comparing the cAMP-activated extract with the basal level without additions, trehalase activity was observed to be stimulated by calcium chloride (1.7-fold), manganese chloride (1.5-fold) and magnesium chloride (1.5-fold). Conversely, copper sulphate, zinc chloride and ferrous sulphate inhibited enzyme activity by about 13.6, 18.5 and 64%, respectively. Trehalase activity was inhibited markedly by ATP (70%) but only slightly by EDTA. Although the same parameters of activation or inhibition were obtained when ions were added to extracts previously activated by cAMP, the values obtained were not the same. The relative molecular weight of neutral trehalase was 80 kDa, estimated in the absence of Ca++ ions by Superose 12 gel ®ltration chromatography. This study revealed some important dierences between the neutral trehalases of S. boulardii and S. cerevisiae (Londesborough & Varimo 1984): (a) in S. cerevisiae the enzyme is absolutely dependent on
Figure 4. Rate of thermal inactivation and temperature optimum. Cells of S. boulardii grown in YP glucose medium were harvested at logarithmic phase. Samples (crude extract) were incubated, removed at the indicated times and cooled on ice. Residual neutral trehalase activity was tested. The insert shows the temperature curve of neutral trehalase. Cell conditions were the same as for thermal inactivation.
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Table 1. Eects of cAMP, divalents ions, ATP and EDTA on the activity of neutral trehalase. Conditions
Basal level (without cAMP)
Previously activated cAMP
No additions 10 mM Ca++ 10 mM Mn++ 10 mM Mg++ 10 mM Zn++ 10 mM Fe++ 10 mM Cu++ 2 mM ATP 5 mM EDTA
32.5 35.5 35.7 32.0 21.3 15.2 20.1 9.0 24.8
45.4 54.2 47.5 49.4 26.5 11.7 28.1 9.7 34.3
Crude extracts were obtained from cells grown in YP glucose, harvested at stationary phase and processed as described in Methods. (Results are expressed in nmol of glucose liberated/min/ mg protein).
Ca+2 or Mn+2 and Mg+2 being ineective, whereas in S. boulardii only the cAMP-activated enzyme was stimulated by Ca+2 (to about 16%), additions of Mn+2 or Mg+2 having relatively minor eects; (b) the neutral trehalase of S. cerevisiae was completely inhibited by 1 mM EDTA, while in S. boulardii 5 mM EDTA produced only about 25% of inhibition; (c) 0.1 mM of ZnCl2 totally inhibited trehalase activity in S. cerevisiae, while the presence of 10 mM ZnCl2 inhibited about 40% of the activity in S. boulardii; (d) in S. cerevisiae the basal activity of neutral trehalase (without activation by cAMP) was inhibited about 50% in the presence of 1 mM MgCl2, while the addition of 10 mM of MgCl2 was ineective in S. boulardii extracts; (e) in S. cerevisiae the activation of regulatory trehalase by cAMP was diminished in the presence of Ca+2, whereas the addition of 10 mM Ca+2 to an extract previously activated by cAMP increased trehalase activity of S. boulardii. Although these yeasts belong to the same genus and their status as separate species is disputed (McFarland 1996; McCullough et al. 1998), signi®cant catalytic dierences were observed between S. cerevisiae and S. boulardii neutral trehalases. The physiological signi®cance of these dierences needs to be better understood. Acknowledgements This research was supported by FundacËaÄo de Amparo aÁ Pesquisa do Estado de Minas Gerais (FAPEMIG) and ComissaÄo Nacional de Energia Nuclear/Centro
de Desenvolvimento da Tecnologia Nuclear (CNEN/ CDTN). Maria J. Neves is a research fellow of the Conselho Nacional de Desenvolvimento CientõÂ ®co e TecnoloÂgico (CNPq, Brazil).
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