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Purification and partial characterisation of a 1.57 kDa thermostable esterase from Bacillus stearothermophilus
FEMS Microbiology Letters 147 (1997) 151^156
Puri¢cation and partial characterisation of a 1.57 kDa thermostable esterase from Bacillus stearothermophilus Davina de C.M. Simoes *, David McNeill, Bjorn Kristiansen, Michael Mattey Department of Bioscience and Biotechnology, The Todd Centre, University of Strathclyde, 31 Taylor Street, Glasgow G4 0NR, UK
Received 11 July 1996; revised 9 December 1996; accepted 9 December 1996
Abstract
The molecular mass of esterases usually falls in the range of 20^160 kDa, although an esterase of 5.7 kDa from Candida has been described. Three other enzymes smaller than 10 kDa have been reported, all of which were more thermostable than their higher molecular mass counterparts. This paper describes the purification of an extracellular esterase hydrolysing fluorescein dibutyrate from Bacillus stearothermophilus NCIMB 13335. The esterase had a molecular mass of 1.57 kDa when analysed by SDS-PAGE, gel filtration and MALDI-TOF spectrometry. This enzyme retained more than 90% of its activity after incubation at 90³C for 2 h. lipolytica
Keywords : Bacillus stearothermophilus
; Low molecular mass enzyme; Thermostability; Esterase
1. Introduction
Esterases are widely distributed enzymes in various kinds of living organisms [1]. In aqueous solution, the esterases catalyse the hydrolytic cleavage of esters, forming the constituent acid and alcohol [1,2]. In contrast to the lipases, their action is generally restricted to short chain fatty acid esters [1,3]. Their molecular mass is usually in the range of 20^160 kDa. One esterase of just 5.7 kDa from Candida lipolytica has been described [4] and at least three other enzymes smaller than 10 kDa (microenzymes) have been mentioned in the literature [5^7]. However, only * Corresponding author. Present address: University of Sunderland, School of Health Sciences, Chester Road, Sunderland SR1 3SD, UK. Tel.: +44 (191) 515 2482; fax: +44 (191) 515 2502; e-mail: [email protected]
the esterase from C. lipolytica and the rennin from a thermophilic actinomycete [4,5] have been characterised to the extent of an amino acid composition. The amino acid composition of these enzymes was characterised by a high content of proline, glutamic acid and glycine. In addition, all of them were more thermostable than their higher molecular mass counterparts. The extracellular esterase of 42^47 kDa described by Matsunaga et al. [3] was not produced by B. stearothermophilus NCIMB 13335 [6]. However, preliminary observations of growth on solid medium with emulsi¢ed tributyrin showed large clearing zones indicative of substantial extracellular esterase activity, although direct assay suggested rather low esterase activity. One possible explanation for this observation was the presence of a very small but stable enzyme, which would have a faster di¡usion
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rate in solid medium than a conventional sized enzyme and hence give a large clearing zone. The purpose of this study was therefore to purify and determine the molecular mass of the extracellular esterase from B. stearothermophilus NCIMB 13335. 2. Materials and methods
2.1. Bacteria and culture conditions
Stock cultures of B. stearothermophilus NCIMB 13335 were grown under the conditions described previously [6], except that yeast extract in the medium was replaced by an amino acids supplement (ICN Biomedicals Inc.) at a ¢nal concentration of 150 WM of each essential and non-essential amino acid. The fermentation was interrupted when the level of enzyme activity was maximal, after 22 h of growth. 2.2. Puri¢cation of esterase
Cells were removed from the medium by centrifugation at 15 000Ug for 15 min at 4³C. The supernatant was ¢ltered through Whatman No. 1 paper to remove excess tributyrin. Subsequently the medium was ¢ltered through a tangential £ow ¢lter (Miniultrasette, Filtron Corporation) with a nominal cuto¡ of 10 kDa. The ¢ltrate was loaded on to a BioGel P-6 column (1.1U91 cm, Bio-Rad Laboratories) for size exclusion chromatography. The column was pre-equilibrated with 100 mM ammonium bicarbonate (pH 8.5), and eluted with the same bu¡er at a £ow rate of 0.07 ml min31 . All the fractions (1 ml) were analysed for esterase activity (£uorimetric assay) and protein content. Fractions which showed esterase activity were pooled, freeze dried, and their composition was analysed by HPLC using a Hamilton PRP-3 reverse-phase column (10 Wm, 300 Aî, 150U4.1 mm, Phenomenex). The detection was at 214 nm, the mobile phase was 0.1% tri£uoroacetic acid, and the £ow rate was 0.25 ml min31 . 2.3. Enzyme assays
A £uorimetric assay [8,9] was used for esterase activity. Fluorescein dibutyrate was dissolved in 2-
methoxyethanol at a concentration of 5U1034 M. 30 Wl of this substrate solution was added to 3.0 ml of 0.1 M Tris-HCl bu¡er at 40³C (pH 8.0) giving a ¢nal concentration of 5U1036 M. The substrate showed appreciable spontaneous decomposition under the assay conditions, so the mixture without added enzyme was used to measure the non-catalysed rate of breakdown. The enzymic hydrolysis of tributyrin was measured by titration of the acids liberated with sodium hydroxide. The substrate was prepared by emulsifying 10 g of tributyrin in 90 ml of 10% gum acacia in water, using a top-drive homogeniser at maximum speed for 5 min. Substrate (0.5 ml) and 0.5 ml of 0.1 M phosphate bu¡er (pH 8.0) were added to 1.0 ml of 6 10 kDa ¢ltrate (containing 37.5 mg of protein) and adjusted to pH 8.0 with NaOH. The mixture was incubated at 40³C for 2 h in a shaking water bath. The reaction was stopped by the addition of 10 ml of an acetone:ethanol (1:1) mixture. The liberated butyric acid was then determined by titration with 0.1 M NaOH. 2.4. Estimation of protein content
Protein content, unless otherwise stated, was determined by the Lowry method, using lysozyme as standard. Detection of peptides after gel ¢ltration was carried out by mixing the gel ¢ltration samples (50 Wl) with 950 Wl of 0.2% ninhydrin solution in ethanol and incubating for 15 min at 90³C. The absorbance was monitored at 540 nm using a Pye Unicam SP8-100 UV spectrophotometer. Calibration was performed using leucine. 2.5. Estimation of molecular mass by SDS-PAGE
Discontinuous SDS-PAGE was performed using a 16.5% total monomer concentration and 6% crosslinker (bisacrylamide) acrylamide slab gel [10]. Samples after puri¢cation were freeze dried and subsequently resuspended in distilled water. Protein bands were located by silver staining [11]. The molecular mass was estimated using the following standards: bradykinin (1.1 kDa), myoglobin III (2.5 kDa), myoglobin II (6.2 kDa), myoglobin I (8.2 kDa), myoglobin I and II (14.4 kDa), myoglobin (16.9 kDa). The myoglobin peptides were in a calibration kit for PAGE, from BDH.
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153
acids generated were identi¢ed on-line employing a C18 reverse phase narrow bore cartridge. The system was calibrated using Pierce Standard H with norleucine as the internal standard (250 pmol of each amino acid) for derivatisation and 24 pmol (420 Wg) myoglobin standard for hydrolysis. 2.9. Determination of thermostability
Fig. 1. Elution pro¢le of the 6 10 kDa ¢ltrate, containing 20 mg protein, on a Bio-Gel P-6 column. Calibration of the column (1.1U91 cm) was performed at 0.07 ml min31 with ammonium bicarbonate (pH 8.5). The protein standards were bradykinin (1.1 kDa), insulin chain A (2.5 kDa), insulin chain B (3.5 kDa) and insulin (5.7 kDa). The ninhydrin reaction was calibrated with 10 mM leucine. Esterase activity: solid line; ninhydrin reaction: dotted line.
150 Wl of 0.1 mg pure enzyme per ml of 20 mM Tris-HCl bu¡er (pH 8.0) were placed in sealed glass tubes and incubated at 70, 80 or 90³C. Every 12 h, three tubes were removed from each incubator, left to cool at room temperature and subsequently stored at 320³C. At the end of the incubation period (4 days), samples were assayed for enzyme activity using the £uorimetric assay. Activities were expressed as a percentage of the activity at time 0 h. 2.10. Statistical analysis
2.6. Estimation of molecular mass by gel ¢ltration
Gel ¢ltration was carried out using the same BioGel P-6 column described in Section 2.2. For molecular mass determination, the column was calibrated with reference proteins. The proteins were bradykinin (1.1 kDa), insulin chain A (2.5 kDa), insulin chain B (3.5 kDa) and insulin (5.7 kDa). The void volume (Vo ) was measured with blue dextran (2000 kDa).
The probability plot correlation coe¤cient test for normality was used to determine whether data were normally distributed. Pearson's product moment correlation coe¤cient [12] was applied to express correlations between the following variables: relative mobility (Rf) and log Mr ; elution volume (Ve ) over Vo and log Mr. Statistical signi¢cance for all statistical procedures was established at P 6 0.05. The data are presented as means þ S.D. Analysis was carried out with the MINITAB statistical package (release 8.0).
2.7. Estimation of molecular mass by MALDI-TOF analysis
The molecular mass was determined using a Vestec Laser-desorption Mass Spectrometer at the University of Aberdeen Protein Sequence Facility. The time of £ight data acquired were converted to mass charge ratio by calibrating with insulin using the modi¢ed Galatica Grams/386 software. 2.8. Amino acid analysis
The analysis was carried out on an Applied Biosystems 420H amino acid analyser with automatic hydrolysis and derivatisation at the University of Aberdeen Protein Sequence Facility. The PTC amino
Fig. 2. Thermostability of esterase activity measured using the £uorimetric assay. Activity at 0 h was taken to be 100%.
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3. Results
3.1. Puri¢cation of esterase activity
Esterase activity was detected only in the 6 10 kDa ¢ltrate after ¢ltration using the tangential £ow ¢lter Mini-ultrasette. Bio-Gel P-6 resolved peptides in the range 1.1^5.7 kDa. Size exclusion chromatography of this ¢ltrate presented a single peak of esterase activity from fractions 78^82 (Fig. 1). Fractions 79^81 were pooled and HPLC analysis con¢rmed that the enzyme was homogeneous. 3.2. Enzyme activity
After puri¢cation, the esterase activity, using £uorescein dibutyrate as a substrate, was 0.11 nmol £uorescein released min31 (mg protein)31 . Increasing concentrations of £uorescein dibutyrate revealed that the esterase was saturated at a substrate concentration of 5 WM. The Km and Vmax determined from a Lineweaver-Burk plot were respectively 0.91 WM and 35 ng £uorescein released min31 (mg protein)31 . The esterase also hydrolysed tributyrin. The speci¢c activity of the enzyme using the titration assay was 15.6 Wmol acid released min31 (mg protein)31 . These results con¢rmed the presence of a catalyst for the hydrolysis of esters. 3.3. Determination of thermostability
The puri¢ed esterase was stable at elevated temperatures for periods up to 96 h (Fig. 2). More than 90% of the original activity was retained after 96 h at 70³C. At 80³C the activity decreased linearly with Table 1 Amino acid composition of esterase Amino acid Residue weight Aspartic acid 115 Glutamic acid 129 Serine 87 Glycine 57 Histidine 137 Alanine 71 Threonine 101 Total
Fig. 3. Standard curve for SDS-PAGE using 16.5% total monomer concentration and 6% cross-linker (bisacrylamide) acrylamide slab gel [10] (r = 0.99). The protein standards were bradykinin (1.1 kDa), myoglobin III (2.5 kDa), myoglobin II (6.2 kDa), myoglobin I (8.2 kDa), myoglobin I and II (14.4 kDa) and myoglobin (16.9 kDa).
time. After 2 h at 90³C the enzymic activity was s 90% of the original activity. 3.4. Determination of molecular mass
SDS-PAGE analysis of the puri¢ed enzyme presented only one band when stained with silver stain. A high linear correlation (r = 0.99) was found between log Mr of the protein standards and their Rf (Fig. 3). The esterase molecular mass was calculated to be 1.6 kDa. The estimated molecular mass for the esterase using gel ¢ltration was 1.4 kDa. A signi¢cant correlation (r = 0.97) between elution volume over void volume (Ve /Vo ) and the protein standard log Mr
Calculated number of residues 2 1 5 5 2 1 1
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Calculated molecular mass (Da) 230 129 435 285 274 71 101 1543
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155
than the 700 kDa phytase isolated from the same microorganism. The same e¡ect was observed for the
disul¢de
from
bond-forming
Sulfolobus solfataricus
enzyme
of
12.5
kDa
[14]. This observation
is important in the context of thermophilic enzymes, particularly those exposed directly to environmental stress. Decreasing protein size may be a strategy for increasing stability by increasing globularity due to an increase in intramolecular packing and deletion of surface loops [16^18].
Acknowledgments
The authors wish to thank The University of
Fig. 4. MALDI-TOF spectrum trace.
Aberdeen Protein Sequence Facility for the MALDI and amino acid analysis. Financial support from the was obtained. MALDI-TOF analysis indicated that
Brazilian National Council of Research and Devel-
the molecular mass of the esterase was 1566 (Fig. 4).
opment (Bolsista do CNPq-Bras|èlia/Brazil) to D. Si-
Amino acid analysis revealed that the esterase was
moes is also gratefully acknowledged.
composed of 17 amino acids (Table 1) with a minimal molecular mass of 1543 Da. Both MALDI-TOF and amino acid analyses were performed using aliquots from the same sample thus providing an assurance that a protein with higher molecular mass was not present, since it is unlikely that a protein with signi¢cantly higher molecular mass would have only seven di¡erent amino acids. No other peaks were
References
[1] Krisch, K. (1971) Carboxylic ester hydrolases. In : The Enzymes (P.D. Boyer, Ed.), Vol. 5, pp. 43^69, Academic Press, London. [2] Cowan, D.A. (1992) Enzymes from thermophilic archaebacteria : current and future applications in biotechnology. In :
visible in the spectrum obtained.
The Archaebacteria : Biochemistry and Biotechnology (M.J. Danson, D.W. Hough, and G.G. Lunt, Eds.), pp. 149^180, Portland Press, London.
4. Discussion
[3] Matsunaga, A., Koyama, N. and Nosoh, Y. (1974) Puri¢cation and properties of esterase from
The four methods used for determining the molecular mass of the esterase suggest a value of 1570 corresponding to a peptide of 17 amino acids. This
lus.
Bacillus stearothermophi-
Arch. Biochem. Biophys. 160, 505^513.
[4] Adoga, G. and Mattey, M. (1979) Properties of an extracellular peptide with esterase activity produced by
lytica.
Candida lipo-
FEMS Microbiol. Lett. 6, 61^63.
esterase is smaller than any previously isolated mi-
[5] Laxer, S., Pinsky, A. and Bartoov, B. (1976) Partial character-
croenzymes, which have ranged from 5.0 to 12.5 kDa
isation of a thermophilic rennin. In : Enzymes and Proteins
[4^7,13,14].
from Thermophilic Microorganisms (H. Zuber, Ed.), Vol.
reported 1 1 (mg protein) [15] range from 47 mol acid min 1 1 (mg protein) [7], so the to 780 mol acid min
W
Speci¢c
W
activities
3
3
previously
3
3
smaller size is associated with a lower speci¢c activity with the present esterase.
(1995) Extracellular esterase activity from
mophilus.
Bacillus stearother-
Biotechnol. Lett. 17, 953^958.
[7] Chadan, R.C. and Shahani, K.M. (1963) Puri¢cation and
The general tendency for enzymes with lower molecular mass to have higher thermostability than their larger counterparts was also observed in this case. The phytase from
26, pp. 67^75. Birkha ë user Verlag, Basel. [6] Simoes, D.C.M., McNeill, D., Kristiansen, B. and Mattey, M.
Enterobacter aerogenes
of
10^13 kDa [13] had a higher optimum temperature
characterisation of milk lipase. I. Puri¢cation. J. Dairy Sci. 46, 275^283. [8] Guilbault, G.G. and Kramer, D.N. (1964) Fluorometric determination of lipase, acylase, alpha- and gamma-chymotrypsin and inhibitors of these enzymes. Anal. Chem. 36, 409^ 412.
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D. de C.M. Simoes et al. / FEMS Microbiology Letters 147 (1997) 151^156
156
[9] Kramer, D.N. and Guilbault, G.G. (1963) A substrate for the £uorometric determination of lipase activity. Anal. Chem. 35,
la aerogenes :
evidence for unusually small active enzyme pep-
tide. J. Ferment. Bioeng. 77, 23^27. [14] Guagliardi, A., Cerchia, L., De Rosa, M., Rossi, M. and
588^589. [10] Scha ë gger, H. and Von Jagow, G. (1987) Tricine-sodium do-
Bartolucci, S. (1992) Isolation of a thermostable enzyme cata-
decyl sulphate polyacrylamide gel electrophoresis for the sep-
lysing disulphide bond formation from the archaebacterium
aration of proteins in the range from 1 to 100 kDa. Anal.
Sulfolobus solfataricus.
FEBS Lett. 303, 27^30.
[15] Adoga, G. (1980) Secretion of Extracellular Lipolytic Enzyme
Biochem. 166, 368^379. [11] Morrissey, J.H. (1981) Silver stain for proteins in polyacrylamide gels : a modi¢ed procedure with enhanced uniform sen-
by
Candida lipolytica.
Ph.D. Thesis, University of Strathclyde,
Glasgow. [16] Hubbard, S. and Argos, P. (1994) Cavities and packing at
sitivity. Anal. Biochem. 117, 307^310. [12] Sokal, R.R. and Rohlf, F.J. (1995) Biometry : The Principles and Practice of Statistics in Biological Research, 850 pp. W.H.
protein interfaces. Prot. Sci. 3, 2194^2206. [17] Jaenicke, R. (1991) Protein stability and molecular adaptation to extreme conditions. Eur. J. Biochem. 202, 715^728.
Freeman and Company, New York. [13] Tambe, S.M., Kaklij, G.S., Kelkar, S.M. and Parekh, L.J. (1994) Two distinct molecular forms of phytase from
Klebsiel-
[18] Jaenicke, R. (1991) Protein folding : local structure, domains, subunits, and assemblies. Biochemistry 30, 3147^3161.
FEMSLE 7409 13-5-97
Puri¢cation and partial characterisation of a 1.57 kDa thermostable esterase from Bacillus stearothermophilus Davina de C.M. Simoes *, David McNeill, Bjorn Kristiansen, Michael Mattey Department of Bioscience and Biotechnology, The Todd Centre, University of Strathclyde, 31 Taylor Street, Glasgow G4 0NR, UK
Received 11 July 1996; revised 9 December 1996; accepted 9 December 1996
Abstract
The molecular mass of esterases usually falls in the range of 20^160 kDa, although an esterase of 5.7 kDa from Candida has been described. Three other enzymes smaller than 10 kDa have been reported, all of which were more thermostable than their higher molecular mass counterparts. This paper describes the purification of an extracellular esterase hydrolysing fluorescein dibutyrate from Bacillus stearothermophilus NCIMB 13335. The esterase had a molecular mass of 1.57 kDa when analysed by SDS-PAGE, gel filtration and MALDI-TOF spectrometry. This enzyme retained more than 90% of its activity after incubation at 90³C for 2 h. lipolytica
Keywords : Bacillus stearothermophilus
; Low molecular mass enzyme; Thermostability; Esterase
1. Introduction
Esterases are widely distributed enzymes in various kinds of living organisms [1]. In aqueous solution, the esterases catalyse the hydrolytic cleavage of esters, forming the constituent acid and alcohol [1,2]. In contrast to the lipases, their action is generally restricted to short chain fatty acid esters [1,3]. Their molecular mass is usually in the range of 20^160 kDa. One esterase of just 5.7 kDa from Candida lipolytica has been described [4] and at least three other enzymes smaller than 10 kDa (microenzymes) have been mentioned in the literature [5^7]. However, only * Corresponding author. Present address: University of Sunderland, School of Health Sciences, Chester Road, Sunderland SR1 3SD, UK. Tel.: +44 (191) 515 2482; fax: +44 (191) 515 2502; e-mail: [email protected]
the esterase from C. lipolytica and the rennin from a thermophilic actinomycete [4,5] have been characterised to the extent of an amino acid composition. The amino acid composition of these enzymes was characterised by a high content of proline, glutamic acid and glycine. In addition, all of them were more thermostable than their higher molecular mass counterparts. The extracellular esterase of 42^47 kDa described by Matsunaga et al. [3] was not produced by B. stearothermophilus NCIMB 13335 [6]. However, preliminary observations of growth on solid medium with emulsi¢ed tributyrin showed large clearing zones indicative of substantial extracellular esterase activity, although direct assay suggested rather low esterase activity. One possible explanation for this observation was the presence of a very small but stable enzyme, which would have a faster di¡usion
0378-1097 / 97 / $17.00 Copyright ß 1997 Published by Elsevier Science B.V. All rights reserved PII S 0 3 7 8 - 1 0 9 7 ( 9 6 ) 0 0 5 2 0 - 4
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rate in solid medium than a conventional sized enzyme and hence give a large clearing zone. The purpose of this study was therefore to purify and determine the molecular mass of the extracellular esterase from B. stearothermophilus NCIMB 13335. 2. Materials and methods
2.1. Bacteria and culture conditions
Stock cultures of B. stearothermophilus NCIMB 13335 were grown under the conditions described previously [6], except that yeast extract in the medium was replaced by an amino acids supplement (ICN Biomedicals Inc.) at a ¢nal concentration of 150 WM of each essential and non-essential amino acid. The fermentation was interrupted when the level of enzyme activity was maximal, after 22 h of growth. 2.2. Puri¢cation of esterase
Cells were removed from the medium by centrifugation at 15 000Ug for 15 min at 4³C. The supernatant was ¢ltered through Whatman No. 1 paper to remove excess tributyrin. Subsequently the medium was ¢ltered through a tangential £ow ¢lter (Miniultrasette, Filtron Corporation) with a nominal cuto¡ of 10 kDa. The ¢ltrate was loaded on to a BioGel P-6 column (1.1U91 cm, Bio-Rad Laboratories) for size exclusion chromatography. The column was pre-equilibrated with 100 mM ammonium bicarbonate (pH 8.5), and eluted with the same bu¡er at a £ow rate of 0.07 ml min31 . All the fractions (1 ml) were analysed for esterase activity (£uorimetric assay) and protein content. Fractions which showed esterase activity were pooled, freeze dried, and their composition was analysed by HPLC using a Hamilton PRP-3 reverse-phase column (10 Wm, 300 Aî, 150U4.1 mm, Phenomenex). The detection was at 214 nm, the mobile phase was 0.1% tri£uoroacetic acid, and the £ow rate was 0.25 ml min31 . 2.3. Enzyme assays
A £uorimetric assay [8,9] was used for esterase activity. Fluorescein dibutyrate was dissolved in 2-
methoxyethanol at a concentration of 5U1034 M. 30 Wl of this substrate solution was added to 3.0 ml of 0.1 M Tris-HCl bu¡er at 40³C (pH 8.0) giving a ¢nal concentration of 5U1036 M. The substrate showed appreciable spontaneous decomposition under the assay conditions, so the mixture without added enzyme was used to measure the non-catalysed rate of breakdown. The enzymic hydrolysis of tributyrin was measured by titration of the acids liberated with sodium hydroxide. The substrate was prepared by emulsifying 10 g of tributyrin in 90 ml of 10% gum acacia in water, using a top-drive homogeniser at maximum speed for 5 min. Substrate (0.5 ml) and 0.5 ml of 0.1 M phosphate bu¡er (pH 8.0) were added to 1.0 ml of 6 10 kDa ¢ltrate (containing 37.5 mg of protein) and adjusted to pH 8.0 with NaOH. The mixture was incubated at 40³C for 2 h in a shaking water bath. The reaction was stopped by the addition of 10 ml of an acetone:ethanol (1:1) mixture. The liberated butyric acid was then determined by titration with 0.1 M NaOH. 2.4. Estimation of protein content
Protein content, unless otherwise stated, was determined by the Lowry method, using lysozyme as standard. Detection of peptides after gel ¢ltration was carried out by mixing the gel ¢ltration samples (50 Wl) with 950 Wl of 0.2% ninhydrin solution in ethanol and incubating for 15 min at 90³C. The absorbance was monitored at 540 nm using a Pye Unicam SP8-100 UV spectrophotometer. Calibration was performed using leucine. 2.5. Estimation of molecular mass by SDS-PAGE
Discontinuous SDS-PAGE was performed using a 16.5% total monomer concentration and 6% crosslinker (bisacrylamide) acrylamide slab gel [10]. Samples after puri¢cation were freeze dried and subsequently resuspended in distilled water. Protein bands were located by silver staining [11]. The molecular mass was estimated using the following standards: bradykinin (1.1 kDa), myoglobin III (2.5 kDa), myoglobin II (6.2 kDa), myoglobin I (8.2 kDa), myoglobin I and II (14.4 kDa), myoglobin (16.9 kDa). The myoglobin peptides were in a calibration kit for PAGE, from BDH.
FEMSLE 7409 13-5-97
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153
acids generated were identi¢ed on-line employing a C18 reverse phase narrow bore cartridge. The system was calibrated using Pierce Standard H with norleucine as the internal standard (250 pmol of each amino acid) for derivatisation and 24 pmol (420 Wg) myoglobin standard for hydrolysis. 2.9. Determination of thermostability
Fig. 1. Elution pro¢le of the 6 10 kDa ¢ltrate, containing 20 mg protein, on a Bio-Gel P-6 column. Calibration of the column (1.1U91 cm) was performed at 0.07 ml min31 with ammonium bicarbonate (pH 8.5). The protein standards were bradykinin (1.1 kDa), insulin chain A (2.5 kDa), insulin chain B (3.5 kDa) and insulin (5.7 kDa). The ninhydrin reaction was calibrated with 10 mM leucine. Esterase activity: solid line; ninhydrin reaction: dotted line.
150 Wl of 0.1 mg pure enzyme per ml of 20 mM Tris-HCl bu¡er (pH 8.0) were placed in sealed glass tubes and incubated at 70, 80 or 90³C. Every 12 h, three tubes were removed from each incubator, left to cool at room temperature and subsequently stored at 320³C. At the end of the incubation period (4 days), samples were assayed for enzyme activity using the £uorimetric assay. Activities were expressed as a percentage of the activity at time 0 h. 2.10. Statistical analysis
2.6. Estimation of molecular mass by gel ¢ltration
Gel ¢ltration was carried out using the same BioGel P-6 column described in Section 2.2. For molecular mass determination, the column was calibrated with reference proteins. The proteins were bradykinin (1.1 kDa), insulin chain A (2.5 kDa), insulin chain B (3.5 kDa) and insulin (5.7 kDa). The void volume (Vo ) was measured with blue dextran (2000 kDa).
The probability plot correlation coe¤cient test for normality was used to determine whether data were normally distributed. Pearson's product moment correlation coe¤cient [12] was applied to express correlations between the following variables: relative mobility (Rf) and log Mr ; elution volume (Ve ) over Vo and log Mr. Statistical signi¢cance for all statistical procedures was established at P 6 0.05. The data are presented as means þ S.D. Analysis was carried out with the MINITAB statistical package (release 8.0).
2.7. Estimation of molecular mass by MALDI-TOF analysis
The molecular mass was determined using a Vestec Laser-desorption Mass Spectrometer at the University of Aberdeen Protein Sequence Facility. The time of £ight data acquired were converted to mass charge ratio by calibrating with insulin using the modi¢ed Galatica Grams/386 software. 2.8. Amino acid analysis
The analysis was carried out on an Applied Biosystems 420H amino acid analyser with automatic hydrolysis and derivatisation at the University of Aberdeen Protein Sequence Facility. The PTC amino
Fig. 2. Thermostability of esterase activity measured using the £uorimetric assay. Activity at 0 h was taken to be 100%.
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3. Results
3.1. Puri¢cation of esterase activity
Esterase activity was detected only in the 6 10 kDa ¢ltrate after ¢ltration using the tangential £ow ¢lter Mini-ultrasette. Bio-Gel P-6 resolved peptides in the range 1.1^5.7 kDa. Size exclusion chromatography of this ¢ltrate presented a single peak of esterase activity from fractions 78^82 (Fig. 1). Fractions 79^81 were pooled and HPLC analysis con¢rmed that the enzyme was homogeneous. 3.2. Enzyme activity
After puri¢cation, the esterase activity, using £uorescein dibutyrate as a substrate, was 0.11 nmol £uorescein released min31 (mg protein)31 . Increasing concentrations of £uorescein dibutyrate revealed that the esterase was saturated at a substrate concentration of 5 WM. The Km and Vmax determined from a Lineweaver-Burk plot were respectively 0.91 WM and 35 ng £uorescein released min31 (mg protein)31 . The esterase also hydrolysed tributyrin. The speci¢c activity of the enzyme using the titration assay was 15.6 Wmol acid released min31 (mg protein)31 . These results con¢rmed the presence of a catalyst for the hydrolysis of esters. 3.3. Determination of thermostability
The puri¢ed esterase was stable at elevated temperatures for periods up to 96 h (Fig. 2). More than 90% of the original activity was retained after 96 h at 70³C. At 80³C the activity decreased linearly with Table 1 Amino acid composition of esterase Amino acid Residue weight Aspartic acid 115 Glutamic acid 129 Serine 87 Glycine 57 Histidine 137 Alanine 71 Threonine 101 Total
Fig. 3. Standard curve for SDS-PAGE using 16.5% total monomer concentration and 6% cross-linker (bisacrylamide) acrylamide slab gel [10] (r = 0.99). The protein standards were bradykinin (1.1 kDa), myoglobin III (2.5 kDa), myoglobin II (6.2 kDa), myoglobin I (8.2 kDa), myoglobin I and II (14.4 kDa) and myoglobin (16.9 kDa).
time. After 2 h at 90³C the enzymic activity was s 90% of the original activity. 3.4. Determination of molecular mass
SDS-PAGE analysis of the puri¢ed enzyme presented only one band when stained with silver stain. A high linear correlation (r = 0.99) was found between log Mr of the protein standards and their Rf (Fig. 3). The esterase molecular mass was calculated to be 1.6 kDa. The estimated molecular mass for the esterase using gel ¢ltration was 1.4 kDa. A signi¢cant correlation (r = 0.97) between elution volume over void volume (Ve /Vo ) and the protein standard log Mr
Calculated number of residues 2 1 5 5 2 1 1
FEMSLE 7409 13-5-97
Calculated molecular mass (Da) 230 129 435 285 274 71 101 1543
D. de C.M. Simoes et al. / FEMS Microbiology Letters 147 (1997) 151^156
155
than the 700 kDa phytase isolated from the same microorganism. The same e¡ect was observed for the
disul¢de
from
bond-forming
Sulfolobus solfataricus
enzyme
of
12.5
kDa
[14]. This observation
is important in the context of thermophilic enzymes, particularly those exposed directly to environmental stress. Decreasing protein size may be a strategy for increasing stability by increasing globularity due to an increase in intramolecular packing and deletion of surface loops [16^18].
Acknowledgments
The authors wish to thank The University of
Fig. 4. MALDI-TOF spectrum trace.
Aberdeen Protein Sequence Facility for the MALDI and amino acid analysis. Financial support from the was obtained. MALDI-TOF analysis indicated that
Brazilian National Council of Research and Devel-
the molecular mass of the esterase was 1566 (Fig. 4).
opment (Bolsista do CNPq-Bras|èlia/Brazil) to D. Si-
Amino acid analysis revealed that the esterase was
moes is also gratefully acknowledged.
composed of 17 amino acids (Table 1) with a minimal molecular mass of 1543 Da. Both MALDI-TOF and amino acid analyses were performed using aliquots from the same sample thus providing an assurance that a protein with higher molecular mass was not present, since it is unlikely that a protein with signi¢cantly higher molecular mass would have only seven di¡erent amino acids. No other peaks were
References
[1] Krisch, K. (1971) Carboxylic ester hydrolases. In : The Enzymes (P.D. Boyer, Ed.), Vol. 5, pp. 43^69, Academic Press, London. [2] Cowan, D.A. (1992) Enzymes from thermophilic archaebacteria : current and future applications in biotechnology. In :
visible in the spectrum obtained.
The Archaebacteria : Biochemistry and Biotechnology (M.J. Danson, D.W. Hough, and G.G. Lunt, Eds.), pp. 149^180, Portland Press, London.
4. Discussion
[3] Matsunaga, A., Koyama, N. and Nosoh, Y. (1974) Puri¢cation and properties of esterase from
The four methods used for determining the molecular mass of the esterase suggest a value of 1570 corresponding to a peptide of 17 amino acids. This
lus.
Bacillus stearothermophi-
Arch. Biochem. Biophys. 160, 505^513.
[4] Adoga, G. and Mattey, M. (1979) Properties of an extracellular peptide with esterase activity produced by
lytica.
Candida lipo-
FEMS Microbiol. Lett. 6, 61^63.
esterase is smaller than any previously isolated mi-
[5] Laxer, S., Pinsky, A. and Bartoov, B. (1976) Partial character-
croenzymes, which have ranged from 5.0 to 12.5 kDa
isation of a thermophilic rennin. In : Enzymes and Proteins
[4^7,13,14].
from Thermophilic Microorganisms (H. Zuber, Ed.), Vol.
reported 1 1 (mg protein) [15] range from 47 mol acid min 1 1 (mg protein) [7], so the to 780 mol acid min
W
Speci¢c
W
activities
3
3
previously
3
3
smaller size is associated with a lower speci¢c activity with the present esterase.
(1995) Extracellular esterase activity from
mophilus.
Bacillus stearother-
Biotechnol. Lett. 17, 953^958.
[7] Chadan, R.C. and Shahani, K.M. (1963) Puri¢cation and
The general tendency for enzymes with lower molecular mass to have higher thermostability than their larger counterparts was also observed in this case. The phytase from
26, pp. 67^75. Birkha ë user Verlag, Basel. [6] Simoes, D.C.M., McNeill, D., Kristiansen, B. and Mattey, M.
Enterobacter aerogenes
of
10^13 kDa [13] had a higher optimum temperature
characterisation of milk lipase. I. Puri¢cation. J. Dairy Sci. 46, 275^283. [8] Guilbault, G.G. and Kramer, D.N. (1964) Fluorometric determination of lipase, acylase, alpha- and gamma-chymotrypsin and inhibitors of these enzymes. Anal. Chem. 36, 409^ 412.
FEMSLE 7409 13-5-97
D. de C.M. Simoes et al. / FEMS Microbiology Letters 147 (1997) 151^156
156
[9] Kramer, D.N. and Guilbault, G.G. (1963) A substrate for the £uorometric determination of lipase activity. Anal. Chem. 35,
la aerogenes :
evidence for unusually small active enzyme pep-
tide. J. Ferment. Bioeng. 77, 23^27. [14] Guagliardi, A., Cerchia, L., De Rosa, M., Rossi, M. and
588^589. [10] Scha ë gger, H. and Von Jagow, G. (1987) Tricine-sodium do-
Bartolucci, S. (1992) Isolation of a thermostable enzyme cata-
decyl sulphate polyacrylamide gel electrophoresis for the sep-
lysing disulphide bond formation from the archaebacterium
aration of proteins in the range from 1 to 100 kDa. Anal.
Sulfolobus solfataricus.
FEBS Lett. 303, 27^30.
[15] Adoga, G. (1980) Secretion of Extracellular Lipolytic Enzyme
Biochem. 166, 368^379. [11] Morrissey, J.H. (1981) Silver stain for proteins in polyacrylamide gels : a modi¢ed procedure with enhanced uniform sen-
by
Candida lipolytica.
Ph.D. Thesis, University of Strathclyde,
Glasgow. [16] Hubbard, S. and Argos, P. (1994) Cavities and packing at
sitivity. Anal. Biochem. 117, 307^310. [12] Sokal, R.R. and Rohlf, F.J. (1995) Biometry : The Principles and Practice of Statistics in Biological Research, 850 pp. W.H.
protein interfaces. Prot. Sci. 3, 2194^2206. [17] Jaenicke, R. (1991) Protein stability and molecular adaptation to extreme conditions. Eur. J. Biochem. 202, 715^728.
Freeman and Company, New York. [13] Tambe, S.M., Kaklij, G.S., Kelkar, S.M. and Parekh, L.J. (1994) Two distinct molecular forms of phytase from
Klebsiel-
[18] Jaenicke, R. (1991) Protein folding : local structure, domains, subunits, and assemblies. Biochemistry 30, 3147^3161.
FEMSLE 7409 13-5-97