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
lipolytica 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.
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
D. de C.M. Simoes et al. / FEMS Microbiology Letters 147 (1997) 151^156
152
U
1034 M.
rate in solid medium than a conventional sized en-
methoxyethanol at a concentration of 5
zyme and hence give a large clearing zone. The pur-
30
pose of this study was therefore to purify and deter-
ml of 0.1 M Tris-HCl bu¡er at 40³C (pH 8.0) giving
mine the molecular mass of the extracellular esterase
a ¢nal concentration of 5
from
showed
B. stearothermophilus NCIMB 13335.
Wl
of this substrate solution was added to 3.0
appreciable
U
1036 M. The substrate
spontaneous
decomposition
under the assay conditions, so the mixture without
added enzyme was used to measure the non-cata-
2. Materials and methods
lysed rate of breakdown. The enzymic hydrolysis of
tributyrin was measured by titration of the acids
2.1. Bacteria and culture conditions
B. stearothermophilus
liberated with sodium hydroxide. The substrate was
prepared by emulsifying 10 g of tributyrin in 90 ml
NCIMB
of 10% gum acacia in water, using a top-drive ho-
13335 were grown under the conditions described
mogeniser at maximum speed for 5 min. Substrate
previously [6], except that yeast extract in the me-
(0.5 ml) and 0.5 ml of 0.1 M phosphate bu¡er (pH
Stock cultures of
6 10 kDa ¢ltrate (con-
dium was replaced by an amino acids supplement
8.0) were added to 1.0 ml of
(ICN Biomedicals Inc.) at a ¢nal concentration of
taining 37.5 mg of protein) and adjusted to pH 8.0
150
WM
of each essential and non-essential amino
with NaOH. The mixture was incubated at 40³C for
acid. The fermentation was interrupted when the lev-
2 h in a shaking water bath. The reaction was
el of enzyme activity was maximal, after 22 h of
stopped by the addition of 10 ml of an acetone :etha-
growth.
nol (1 :1) mixture. The liberated butyric acid was
2.2. Puri¢cation of esterase
then determined by titration with 0.1 M NaOH.
2.4. Estimation of protein content
Cells were removed from the medium by centrifu-
Ug
for 15 min at 4³C. The super-
Protein content, unless otherwise stated, was de-
natant was ¢ltered through Whatman No. 1 paper to
termined by the Lowry method, using lysozyme as
remove excess tributyrin. Subsequently the medium
standard. Detection of peptides after gel ¢ltration
was ¢ltered through a tangential £ow ¢lter (Mini-
was carried out by mixing the gel ¢ltration samples
ultrasette, Filtron Corporation) with a nominal cut-
(50
o¡ of 10 kDa. The ¢ltrate was loaded on to a Bio-
ethanol and incubating for 15 min at 90³C. The ab-
Gel P-6 column (1.1
gation at 15 000
U
Wl)
with 950
Wl
of 0.2% ninhydrin solution in
91 cm, Bio-Rad Laboratories)
sorbance was monitored at 540 nm using a Pye Uni-
for size exclusion chromatography. The column was
cam SP8-100 UV spectrophotometer. Calibration
pre-equilibrated with 100 mM ammonium bicarbon-
was performed using leucine.
ate (pH 8.5), and eluted with the same bu¡er at a
£ow rate of 0.07 ml min31 . All the fractions (1 ml)
2.5. Estimation of molecular mass by SDS-PAGE
were analysed for esterase activity (£uorimetric assay) and protein content. Fractions which showed
Discontinuous SDS-PAGE was performed using a
esterase activity were pooled, freeze dried, and their
16.5% total monomer concentration and 6% cross-
composition was analysed by HPLC using a Hamilî,
ton PRP-3 reverse-phase column (10 Wm, 300 A
linker (bisacrylamide) acrylamide slab gel [10]. Sam-
150
4.1 mm, Phenomenex). The detection was at
quently resuspended in distilled water. Protein bands
214 nm, the mobile phase was 0.1% tri£uoroacetic
were located by silver staining [11]. The molecular
U
acid, and the £ow rate was 0.25 ml min31 .
2.3. Enzyme assays
ples after puri¢cation were freeze dried and subse-
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).
A £uorimetric assay [8,9] was used for esterase
activity. Fluorescein dibutyrate was dissolved in 2-
The myoglobin peptides were in a calibration kit for
PAGE, from BDH.
D. de C.M. Simoes et al. / FEMS Microbiology Letters 147 (1997) 151^156
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%.
154
D. de C.M. Simoes et al. / FEMS Microbiology Letters 147 (1997) 151^156
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
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 bond-forming enzyme of 12.5 kDa
from Sulfolobus solfataricus [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
Fig. 4. MALDI-TOF spectrum trace.
was obtained. MALDI-TOF analysis indicated that
the molecular mass of the esterase was 1566 (Fig. 4).
Amino acid analysis revealed that the esterase was
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
visible in the spectrum obtained.
4. Discussion
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
esterase is smaller than any previously isolated microenzymes, which have ranged from 5.0 to 12.5 kDa
[4^7,13,14]. Speci¢c activities previously reported
range from 47 Wmol acid min31 (mg protein)31 [15]
to 780 Wmol acid min31 (mg protein)31 [7], so the
smaller size is associated with a lower speci¢c activity
with the present esterase.
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 Enterobacter aerogenes of
10^13 kDa [13] had a higher optimum temperature
The authors wish to thank The University of
Aberdeen Protein Sequence Facility for the MALDI
and amino acid analysis. Financial support from the
Brazilian National Council of Research and Development (Bolsista do CNPq-Bras|èlia/Brazil) to D. Simoes is also gratefully acknowledged.
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