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Journal of Biotechnology 133 (2008) 6572

Purification and characterization of the enantioselective

esterase from Kluyveromyces marxianus CBS 1553
Daniela Monti a, , Erica Elisa Ferrandi a , Monica Righi a ,
Diego Romano b , Francesco Molinari b,
b

a Istituto di Chimica del Riconoscimento Molecolare, CNR, Via Mario Bianco 9, 20131 Milano, Italy
Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Universit`a degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy

Received 22 May 2007; received in revised form 31 August 2007; accepted 10 September 2007

Abstract
An intracellular esterase from the yeast Kluyveromyces marxianus CBS 1553 with interesting enantioselective hydrolytic activity towards racemic
esters of 1,2-O-isopropylidene glycerol (IPG) was purified and characterized. Optimal culture conditions for the obtainment of the enantioselective
esterase on a 5 l-fermentation scale were investigated. Two esterase activities (EST1 and EST2) in the crude cell extract were identified by
native PAGE with specific activity staining and separated from each other by anion-exchange chromatography. EST1 showed higher activity and
enantioselectivity than EST2 in the resolution of racemic IPG acetate and was further purified by hydrophobic interaction chromatography and
preparative electrophoresis (final specific activity
= 300 U mg1 ), showing a main protein band with a molecular mass of 29 kDa. EST1 showed
optimal activity between pH 8.0 and 10.0 and was stable in the pH range 7.010.0. Moreover, it was rather thermostable and active up to 80 C,
and retained most of its activity in the presence of 15% (v/v) of various organic solvents. The enzyme showed similar Vmax in the hydrolysis of
the acetate esters of IPG, whereas the Km value towards (S)-IPG acetate was significantly lower than the one towards the (R)-enantiomer (5.3 and
70 M, respectively). Finally, comparison of EST1 activity in the presence of different glycerol esters and synthetic substrates with different chain
lengths showed a strong preference of this biocatalyst for short-chain substrates.
2007 Elsevier B.V. All rights reserved.
Keywords: Esterase; Kluyveromyces marxianus; 1,2-O-Isopropylidene glycerol; Enzyme purification; Biotransformation

1. Introduction
1,2-O-Isopropylidene glycerol (IPG, also called solketal), in
both enantiomeric forms, is an important chiral building block
for the synthesis of many optically active compounds, such as adrenoreceptor antagonists, prostaglandins, phospholipids and
leukotrienes (Jurczac et al., 1986; Xia and Hui, 1997). Production of enantiomerically pure (R)- and (S)-IPG by standard
synthetic protocols is costly and laborious and biological methods have thus received increasing attention in the last years.
The enantioselective hydrolysis of (R,S)-IPG esters is difficult to
achieve with commercially available esterases and lipases (Liu
et al., 2001; Mezzetti et al., 2003), while interesting results have
been obtained in biotransformations catalyzed by esterases from

Corresponding author. Tel.: +39 02 5031 9148; fax: +39 02 5031 6694.
Corresponding author. Tel.: +39 02 2850 0038; fax: +39 02 2890 1239.
E-mail addresses: daniela.monti@icrm.cnr.it (D. Monti),
Francesco.Molinari@unimi.it (F. Molinari).

0168-1656/$ see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2007.09.004

different Bacillus species. Whole cells of a Bacillus coagulans

strain have been employed for the enantioselective hydrolysis
of different racemic IPG esters with the formation of the (S)alcohol as the major enantiomer, the highest enantiomeric ratio
being observed for the benzoate (Romano et al., 2005), and the
same enantiomer is predominantly formed from its caprylate
ester in the biotransformation catalyzed by a recently isolated
Bacillus subtilis esterase (Droge et al., 2005).
In our previous study, the easily cultivable yeast
Kluyveromyces marxianus CBS 1553 was shown to be able
to enantioselectively hydrolyze racemic IPG acetate with the
formation of (R)-IPG as the major enantiomer (Molinari et
al., 2004) (Scheme 1). The biotransformation was performed
by repeated-batch operation of a membrane reactor containing
lyophilized K. marxianus cells (20 cycles), allowing the recovery
of 19.2 g l1 of enantiomerically pure (R)-IPG acetate starting
from 60 g l1 of racemic mixture.
Here we present detailed information on the production and
purification of the intracellular enantioselective esterase from

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D. Monti et al. / Journal of Biotechnology 133 (2008) 6572

Scheme 1. Enantioselective hydrolysis of racemic 1,2-O-isopropylideneglycerol acetate.

K. marxianus CBS 1553 and its functional characterization.

The specificity and selectivity of the purified enzyme have been
investigated as well.
2. Materials and methods
2.1. Chemicals
Unless otherwise stated, all chemicals were of analytical
grade and were purchased from SigmaAldrich.

at room temperature, the reaction mixture was quenched with

100 ml of 5% (w/v) NaHCO3 solution and the product extracted
with ethyl acetate. The organic extracts were dried over Na2 SO4
and the solvent removed. The crude product was purified (78%
yield) by flash chromatography (ethyl acetate/hexane 1/1) on
silica gel pretreated with triethylamine.
(R,S)-IPG butyrate (C4 ) and caproate (C6 ) were prepared by
the same procedure used for the acetate ester with the corresponding acid chloride derivative.
2.4. Organism and cultivation conditions

2.2. Analytical methods

Thin-layer chromatography (TLC): precoated silica gel
60 F254 plates (Merck), detection was performed with the
molybdate reagent ((NH4 )6 Mo7 O24 4H2 O, 42 g; Ce(SO4 )2 , 2 g;
H2 SO4 conc., 62 ml; made up to 1 l of deionized water).
Flash chromatography: silica gel 60 (70230 mesh, Merck).
Spectrophotometric determinations of protein concentration
and enzymatic activities with chromogenic substrates were
performed using a Jasco V-530 UV/VIS spectrophotometer.
The enantiomeric composition of (R,S)-isopropylideneglycerol
(IPG) esters and IPG and conversions were routinely determined
by gas-chromatographic (GC) analyses on a Dani 6500 gascromatograph equipped with a Merck-Hitachi D2000 integrator
and using a chiral capillary column (DMePeBeta-CDX-PS086,
MEGA, Legnano, Italy), having 0.25 mm-diameter, 25 mlength and 0.25 m-thickness. Column temperature was initially
maintained at 90 C for 10 min and then raised with the
following gradients: 90120 C at 2 C min1 , 120180 C
at 1 C min1 (acetic acid ester); 90110 C at 4 C min1 ,
110180 C at 1 C min1 (butyric acid ester); 90115 C at
5 C min1 , 115180 C at 1 C min1 (caproic acid ester).
Under these conditions, typical retention times were: (R)IPG, 8.09 min; (S)-IPG, 8.69 min; (S)-IPG acetate, 11.37 min;
(R)-IPG acetate, 11.93 min; (S)-IPG butyrate, 20.35 min; (R)IPG butyrate, 20.79 min; (S)-IPG caproate, 31.66 min; (R)-IPG
caproate, 32.02 min. The stereochemical outcome of the transformations was expressed as enantiomeric excess (e.e.) of the
major enantiomer or as enantiomeric ratio (E) (Chen et al., 1982).
2.3. Synthesis of 1,2-O-isopropylideneglycerol (IPG) esters
The synthesis of (R,S)-isopropylideneglycerol (IPG) acetate
was carried out by adding at 4 C anhydrous acetic anhydride
(18 ml) and pyridine (24 ml) to a solution of racemic 1,2-Oisopropylideneglycerol (12 g) in dry benzene (90 ml). After 24 h

K. marxianus CBS 1553 was from CBS (Centraalbureau voor

Schimmelcultures, Baarn, Holland) and routinely maintained on
malt extract slants (malt extract 100 g l1 , agar 15 g l1 , yeast
extract 5 g l1 , pH 5.6) at 4 C. The microorganism was cultured in 1 l Erlenmeyer flasks containing 100 ml of medium and
incubated for 48 h at 28 C on a reciprocal shaker (120 spm).
Unless mentioned otherwise, the standard medium (SM) contained 100 g l1 malt extract and 50 g l1 yeast extract, pH 5.6.
Scale-up of the cultures was performed by inoculating 160 ml
of 24 h-cultures in a 5 l Biolafitte fermentor (initial OD600 = 2).
Fermentation conditions were 200 rpm, 1 vvm of aeration, 28 C
and pH 5.6. Cells were harvested by centrifugation (25 min at
8000 rpm and 4 C) in a Beckmann JS-21 centrifuge equipped
with a JA-20 rotor, washed three times with deionized water and
immediately used in the biotransformation reactions or frozen at
the temperature of 20 C and lyophilized. Dry weight determinations were performed by washing cells samples with deionized
water and drying at 110 C for 24 h.
The screening of the enantioselective esterase induction was
performed by supplementing the standard medium with (R,S)IPG acetate (10 g l1 ) at the inoculum time (t0 ) or at the early
exponential growth phase (7.5 h after the inoculum). Cultures
(100 ml) were kept at 28 C and 120 spm for 24 h, cells were
harvested by centrifugation as previously described and immediately used in the biotransformation reactions.
2.5. Enzyme purication
2.5.1. Enzyme extraction and precipitation
K. marxianus lyophilized cells (25 g dry weight from 2.5 l
of fermentation broth recovered after 24 h of growth) were
resuspended in 200 ml of 10 mM Tris/HCl buffer, pH 8.0,
1 mM EDTA, containing 1 l ml1 Protease Inhibitor Cocktail
(Sigma) and incubated for 24 h at 4 C under mild agitation.
After centrifugation (50 min, 18,000 rpm), the supernatant was

D. Monti et al. / Journal of Biotechnology 133 (2008) 6572

filtered on a 0.45 m filter and crude protein extracts were

fractionated by ammonium sulfate precipitation (45 and 85%
(NH4 )2 SO4 saturation). The 45% sat. ammonium sulfate precipitate showed negligible activity and was discarded.
2.5.2. Anion-exchange chromatography
Protein suspension obtained by ammonium sulfate precipitation (85% saturation) was centrifuged (30 min at 6000 rpm
and 4 C), dissolved in 10 mM Tris/HCl buffer, pH 8.0, 1 mM
EDTA, 1 mM DTT, 5% (v/v) glycerol (buffer A) and dialyzed
against the same buffer (2 5 l). After centrifugation (20 min,
13,000 rpm), the enzyme solution (46 ml) was applied to a Fractogel EMD DEAE-650 (S) (Merck) column (16 mm 110 mm),
equilibrated with buffer A, at a flow rate of 1 ml min1 . After
loading, the column was washed with 200 ml of the equilibration
buffer and bound proteins were eluted by a linear gradient from 0
to 0.15 M NaCl in the same buffer within 3 h at 1 ml min1 flow
rate. At the end of the gradient, the column was washed with
buffer containing 1 M NaCl until complete protein elution. Fractions (3 ml) were assayed for esterase activity using pNP-acetate
as a substrate. Protein concentration was monitored at 280 nm.
2.5.3. Hydrophobic interaction chromatography
After slow addition of solid ammonium sulfate (25% saturation), the main esterase activity obtained after DEAE
chromatography (EST1) was applied to a Phenyl Sepharose Fast
Flow (Amersham Pharmacia) column (16 mm 100 mm) equilibrated with buffer A containing 25% (NH4 )2 SO4 . Following
washing of the column with the equilibration buffer, esterase
activity retained on the resin was eluted by a linear decrease
of the concentration of (NH4 )2 SO4 to 5% saturation in 3 h at
1 ml min1 flow rate. EST1 was eluted from the column as a
single activity peak at about 10% saturation (NH4 )2 SO4 and
concentrated by ultrafiltration to 1 ml final volume.
2.5.4. Preparative electrophoresis and electroelution
Preparative electrophoresis in native conditions was performed according to Ornstein (1964) with a 8% running gel
(20 cm 16 cm 1 mm) on a PROTEAN II xi cell (Bio-Rad).
One hundred microliters of concentrated EST1 solution from
the previous step was loaded into each well and the gel was
run under constant current conditions (1 h at 16 mA, then 5 h at
24 mA) at 15 C. After electrophoresis, two gel lanes (0.5 cm
width) were stained with Coomassie Brilliant Blue and esterase
activity staining, respectively. A 1.5-cm section of gel containing EST1 was excised, cut in 0.5 cm pieces and transferred to
a dialysis tube (4 cm diameter) filled with buffer A. Electroelution was performed at constant voltage (2 h at 50 V, then 1 h
at 100 V) in a horizontal electrophoresis chamber (Bio-Rad)
filled with buffer A so that it just covered the dialysis tube. The
electroeluted material was removed from the dialysis tube and
immediately used for the determination of the esterase activity
with the IPG acetate assay. The remaining sample was concentrated by ultrafiltration, extensively dialyzed against deionized
water and lyophilized. The solid residue was redissolved in
100 l of water for determination of protein content and analyzed by SDS-PAGE.

67

2.6. Polyacrylamide gel electrophoresis and zymogram

analysis
Enzyme purity was monitored by SDS-PAGE (10% T, 4%
C) according to the method of Laemmli (1970). The gels were
stained with Coomassie Brilliant Blue and molecular mass under
denaturing conditions was determined by comparison with standard markers (Bio-Rad).
Esterase activity staining (zymogram) was performed after
non-denaturing PAGE (8% T, 4% C) by incubation of the gel
in a solution of 50 mg -naphthyl acetate and 30 mg Fast Blue
RR Salt in 100 ml of 25 mM Tris/HCl buffer, pH 7.4, 1% (v/v)
acetone.
2.7. Enzyme activity and protein assay
2.7.1. IPG acetate assay
Biotransformations were performed with 15 g l1 (dry
weight) of fresh K. marxianus whole cells or with 10 mU EST1
solution (determined using p-nitrophenyl acetate as a substrate)
in 2 ml of 10 mM Tris/HCl buffer, pH 8.0, 5% (v/v) glycerol,
1 mM EDTA, 1 mM DTT, containing 2.5 g l1 of (R,S)-IPG
acetate in 10 ml screw capped test tubes, and incubation under
stirring at 28 C. At scheduled times, samples (100 l) were
extracted with an equal volume of ethyl acetate and analyzed
by GC analyses as previously described. One unit (U) of the
esterase activity was defined as the amount of cells (dry weight)
or enzyme releasing 1 mol of IPG per minute.
2.7.2. p-Nitrophenyl (pNP) acetate assay
Three microliters of a 1 mg ml1 acetone solution of pNPacetate (5.7 mM) were diluted with 50 mM phosphate buffer,
pH 7.0, to a final volume of 1 ml. Production of p-nitrophenol
after addition of enzyme solution (10100 l) was monitored
at 400 nm and 20 C (400 nm = 15,000 M1 cm1 ) and one unit
was defined as the amount of enzyme releasing 1 mol of pnitrophenol per minute.
Protein concentration was determined according to the
method of Bradford (1976) (Bio-Rad Protein Assay), using
bovine serum albumin as a standard.
2.8. Substrate specicity assays
Substrate specificity of EST1 was estimated using IPGderivatives, pNP-esters and triglycerol esters with different chain
lengths.
Biotransformations of different IPG-esters (acetate, butyrate
or caproate) catalyzed by EST1 were performed and analyzed
according to the IPG acetate assay previously described.
Determination of Km and Vmax of EST1 for (R)- and (S)-IPG
acetate was performed with optically pure substrates obtained
from the corresponding alcohols using the same procedure previously described for the racemic mixture. Initial reaction rates
with (R)-IPG acetate (00.4 M) or (S)-IPG acetate (00.08 M)
as substrates were estimated using the test-kit for the determination of acetic acid (R-Biopharm GmbH, Darmstadt, Germany)
in accord with the manufacturers protocol (see also Baumann

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D. Monti et al. / Journal of Biotechnology 133 (2008) 6572

et al., 2001). Spectrophotometric determination of NADH concentration was performed continuously at 340 nm and 20 C in a
1-ml scale and kinetic parameters Km and Vmax were calculated
from Michaelis-Menten plots using the Solver tool of Microsoft
Excel.
Enzyme activity against pNP-esters, specifically acetate (C2 ),
butyrate (C4 ), caproate (C6 ), caprylate (C8 ), laurate (C12 ), palmitate (C16 ), were performed by incubating the enzyme with
0.5 mM substrate at 20 C in 100 mM Tris/HCl buffer, pH 7.4,
0.5% (w/v) Triton X-100, 0.125% (w/v) arabic gum. Spectrophotometric determination of absorbance changes at 400 nm
were recorded continuously.
EST1 activity on the substrates triacetin, tributyrin, tricaprylin and triolein was determined using a titration assay with
a Metrohm 718 STAT Titrino potentiometric titrator (Herisau,
Switzerland). Tests were performed in the presence of 10 mM
substrate at 20 C in 2.5 mM Tris/HCl buffer, pH 8.0, 100 mM
NaCl, 0.5% (w/v) Triton X-100, 0.125% (w/v) arabic gum and
by using 10 mM NaOH for titration.
2.9. Inuence of pH, temperature, salts and solvents on
EST1 activity and stability
EST1 functional properties in different conditions were
investigated using a partially purified enzyme sample obtained
after anion-exchange chromatography and void of contaminant
esterase activities, as assessed by zymogram analysis. All the
experiments were run in at least duplicate and corrections were
made for spontaneous hydrolysis of the substrate (pNP-acetate)
when needed.
Determination of the pH dependence of EST1 activity was
performed over the pH range from 2 to 10 using three different
buffer systems (pH 2.08.0, McIlvaine (0.2 M disodium hydrogen phosphate, 0.1 M citric acid) buffer; pH 8.09.0, 0.1 M
Tris/HCl buffer; pH 9.010.0, 0.1 M glycine/NaOH buffer).
Immediately after dilution, esterase activity was measured at
20 C by adding 3 l of a 1 mg ml1 acetone solution of
pNP-acetate and monitoring absorbance changes at 348 nm,
the pH independent isosbestic point of p-nitrophenol and
the p-nitrophenoxide ion (348 p-nitrophenol = 5300 M1 cm1 )
(Manco et al., 1998).
For the determination of temperature dependence of EST1
activity, esterase solutions (50 l) were diluted with 950 l of
50 mM potassium phosphate buffer, pH 7.0, at temperatures
ranging from 20 and 90 C. Three microliters of a 1 mg ml1
acetone solution of pNP-acetate were added and the release of
p-nitrophenol was monitored at 400 nm after 3 min of incubation.
The pH stability profile of EST1 was determined after incubation at different pH values (2.011.0) for 4 h at 20 C. Residual
activity was measured by diluting 10 l-aliquots of enzyme
solution into 1 ml assay mixture containing 50 mM potassium
phosphate buffer, pH 7.0, and 3 l of a 1 mg ml1 acetone
solution of pNP-acetate. Absorbance changes at 400 nm were
monitored.
To assay the thermostability of the enzyme, EST1 solutions
(50 l) were incubated for 1 h at temperatures ranging from 20

and 90 C and then diluted into 1 ml of 50 mM potassium phosphate buffer, pH 7.0, at 20 C. Residual activity was immediately
measured by adding 3 l of a 1 mg ml1 acetone solution of
pNP-acetate in the spectrophotometric assay at 400 nm.
The effect of different metal ions, detergents, EDTA and
organic solvents on EST1 activity was investigated using pNPacetate as a substrate in the standard assay conditions. The
following metal ions were tested at 10 mM: Ca2+ , Co2+ , Cu2+ ,
Hg2+ , K+ , Mg2+ , Mn2+ , Zn2+ , whereas Fe2+ , Fe3+ and Sn2+
were used at 0.1 mM. EDTA was tested at 10 mM and SDS at
1% (w/v).
The activity of the enzyme was determined in the presence of the following water-miscible organic solvents: acetone,
methanol, ethanol, dimethyl sulfoxide (DMSO) and dimethylformamide (DMF). Increasing solvents concentrations up to
15% (v/v) were tested.
3. Results and discussion
3.1. Expression and purication of the enantioselective K.
marxianus esterase
During our previous studies on the expression of esterases
active in the enantioselective hydrolysis of racemic IPG acetate,
several K. marxianus strains grown on a standard rich medium
(100 g l1 malt extract and 50 g l1 yeast extract, pH 5.6) showed
the ability to perform the rapid hydrolysis of this substrate with
enantiomeric ratio in the range of 1216 (Molinari et al., 2004).
In order to obtain a suitable amount of the K. marxianus
esterase for subsequent characterization, a possible induction
effect of substrate addition into culture medium on esterase specific activity of K. marxianus CBS 1553 was investigated, but
only a slight effect (about threefold improvement of esterase
specific activity) was observed when supplementing the standard medium with (R,S)-IPG acetate (10 g l1 ) at the inoculum
time without any significant improvement of biomass growth or
biocatalyst enantioselectivity. Taking into account these quite
modest results, we considered much more convenient scalingup the enzyme production to a 5-l fermentor in the standard

Fig. 1. Expression of the enantioselective esterase from K. marxianus CBS

1553time course of cell growth expressed as total biomass dry weight ()
and esterase specific activity using (R,S)-IPG acetate as a substrate ().

D. Monti et al. / Journal of Biotechnology 133 (2008) 6572

69

Fig. 2. (a) Purification of esterase activities from K. marxianus CBS 1553 (dotted line) by DEAE chromatography from total cell extract proteins (solid line) in
relation to NaCl gradient (dashed line). Activity assays were performed using pNP-acetate as a substrate. (b) Zymogram analysis after native PAGE of purified
esterases: (1) cell extract; (2) EST1 after DEAE; (3) EST2 after DEAE.

culture medium. Samples were withdrawn at scheduled times to

monitor biomass production and esterase specific activity using
(R,S)-IPG acetate as a substrate.
As shown in Fig. 1, the highest specific activity was observed
after 7.5 h of growth, then it decreased in the following hours.
The enantiomeric ratio values of these biotransformations were
similar (E
= 15), thus confirming that the activity profile was
mainly related to the expression of the enantioselective esterase
of interest. Nevertheless, taking into account the biomass production profile, recovery of the cells after 24 h of growth was
considered a good compromise between the obtainment of a
sufficient amount of biomass and its esterase specific activity.
Therefore, the fermentation was repeated in the same conditions and cells were harvested by centrifugation after 24 h of
growth, washed with deionized water and lyophilized.
Purification by anion-exchange chromatography of the cell
extract obtained from 25 g (dry weight) of lyophilized K. marxianus cells revealed two main esterase activities, a major sharp
peak eluting at about 70 mM NaCl (EST1) and a minor broad
peak at 90 mM NaCl (EST2) (Fig. 2A), which resulted in two
clearly distinct bands on activity-stained non-denaturing PAGE
(Fig. 2B).
Hydrolysis of racemic IPG acetate catalyzed by crude cell
extract or by purified EST1 and EST2 after DEAE chromatography were compared (Table 1). EST1 was clearly identified as
the esterase of interest, showing higher activity and enantioselectivity than EST2, and was further purified by hydrophobic
interaction chromatography.
Although this purification step allowed us to improve the
esterase specific activity of about 10 times (Table 2, line 3),
zymogram analysis of native PAGE of the EST1 sample (Fig. 3A,

Table 1
Hydrolysis of racemic IPG acetate catalyzed by crude cell extract or by purified
EST1 and EST2 after DEAE chromatography
Catalysta

Time (h)

Conversion (%)

Crude cell extract

EST1
EST2

1
1
3

38
57
21

13
17
4

Biotransformations catalyzed by crude or purified esterase were performed

by addition of 10 mU enzyme solution (determined using p-nitrophenyl acetate
as a substrate) to 2 ml of 10 mM Tris/HCl buffer, pH 8.0, 5% (v/v) glycerol,
1 mM EDTA, 1 mM DTT, containing 2.5 g l1 of (R,S)-IPG acetate in 10 ml
screw capped test tubes, and incubation at 28 C.

lanes 1 and 3) showed the concurrent purification of a contaminating protein band with markedly different electrophoretic
mobility in the respect of the active band. Unsuccessful attempts
of separating the two proteins by standard chromatographic
methods (data not shown) prompted us in loading the whole partially purified sample (about 1 mg of protein) on a preparative
electrophoresis polyacrylamide gel under native conditions with
subsequent electroelution of the active band (Fig. 3A, lanes 2 and
4). Samples from each purification step were analyzed by SDSPAGE (Fig. 3B) and the purified enzyme preparation clearly
showed the presence of a major protein band with a molecular mass of 29 kDa (lane 7). After the final separation step, the
specific activity of purified EST1 was 324 U mg1 , which corresponded to a 3560-fold increase of specific activity and to a
6.6% purification yield (Table 2, line 4).
To the best of our knowledge, the K. marxianus esterase
described in this work is one of the most active esterases for
the enantioselective hydrolysis of racemic IPG esters reported

Table 2
Purification of EST1 from K. marxianus CBS 1553
Purification step

Volume (ml)

Total proteina (mg)

Activityb (U)

Specific activity (U mg1 )

Yield (%)

Purification factor

Cell extract
Fractogel DEAE
Phenyl Sepharose
Preparative electrophoresis

46
9
30
75

535
30
1
0.01

48.76
41.54
12.5
3.24

0.09
1.38
12.5
324

100
85
26
6.6

1
15
137
3560

Protein content was determined according to Bradford, using bovine serum albumine as a standard.
Activity was determined at 28 C using (R,S)-IPG acetate (2.5 g l1 ) as a substrate in 10 mM Tris/HCl buffer, pH 8.0, 5% (v/v) glycerol, 1 mM EDTA, 1 mM
DTT. Biotransformations were analyzed by GC analyses as described in Section 2.
b

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D. Monti et al. / Journal of Biotechnology 133 (2008) 6572

Table 3
Hydrolysis of different racemic IPG esters catalyzed by K. marxianus whole
cells or by purified EST1
Catalysta

Substrate

Activity (U l1 )

Whole cells

(R,S)-IPG acetate
(R,S)-IPG butyrate
(R,S)-IPG caproate

29
24
40

15
2.5
2.8

EST1

(R,S)-IPG acetate
(R,S)-IPG butyrate
(R,S)-IPG caproate

20
4.6
0.7

20
2.5
1

a Biotransformations were performed with 15 g l1 (dry weight) of fresh whole

cells or with 10 mU EST1 solution (determined using p-nitrophenyl acetate as
a substrate) in 2 ml of 10 mM Tris/HCl buffer, pH 8.0, 5% (v/v) glycerol, 1 mM
EDTA, 1 mM DTT, containing 2.5 g l1 of each racemic IPG ester in 10 ml screw
capped test tubes, and incubation at 28 C.

Fig. 3. Native PAGE (a) and SDS-PAGE (b) analyses of purified EST1 from K.
marxianus CBS 1553. (1) EST1 after hydrophobic interaction chromatography,
protein staining; (2) EST1 after preparative electrophoresis, protein staining;
(3) EST1 after hydrophobic interaction chromatography, activity staining; (4)
EST1 after preparative electrophoresis, activity staining; (M), molecular mass
markers (mass in thousands); (5) EST1 after DEAE chromatography; (6) EST1
after hydrophobic interaction chromatography; (7) EST1 after preparative electrophoresis.

up to now. In fact, concerning the different B. subtilis esterases,

CesA (B. subtilis 168) and carboxylesterase NP (B. subtilis Thai
I-8) showed specific activities ranging from 0.002 U mg1 (IPG
acetate) to 0.051 U mg1 (IPG caprylate) with very low enantioselectivity (E = 1.01.9), whereas CesB (B. subtilis 168) was
highly enantioselective towards IPG caprylate (E > 200), but
with a quite low specific activity (0.022 U mg1 ). Moreover, this
esterase was unable to hydrolyze racemic IPG butyrate, whereas
(R)- and (S)-IPG acetate were slowly hydrolyzed but without
selectivity (E = 1.3, specific activity < 0.005 U mg1 ) (Droge et
al., 2001, 2005).
Among other lipases and esterases screened for hydrolytic
activity towards IPG esters, only the lipase from the filamentous fungus Ophiostoma piliferum showed a higher specific
activity than the K. marxianus esterase, i.e., 8400/6000 U mg1
for (R)-/(S)-IPG octanoate and 150/52 U mg1 for (R)-/(S)-IPG
butyrate, respectively, but in both cases the estimated enantioselectivity was very low (IPG octanoate, E = 1.4; IPG butyrate,
E = 2.9). The Bacillus thermocatenulatus lipase was moderately enantioselective but poorly active (IPG butyrate, E = 7.9,
0.18 U mg1 ; IPG octanoate, E = 4.9, 2.6 U mg1 ) and the bacterial esterases from Pseudomonas uorescens and Streptomyces
diastatochromogenes accepted the same substrates with low
activity (specific activity ranging from 0.2 to 1.6 U mg1 ), but
were not enantioselective (Liu et al., 2001).
3.2. Substrate specicity and enantioselectivity of K.
marxianus EST1

EST1 showed a quite narrow specificity towards the acetate

(C2 ) ester, its specific activity towards the butyrate (C4 ) and
caproate (C6 ) esters being almost 5 and 30 times lower,
respectively. Moreover, the enzyme showed only modest enantioselectivity towards IPG butyrate (E = 2.5), while, in the case
of IPG caproate, no enantiopreference was observed.
Interestingly, when using K. marxianus whole cells in
the biotransformations reactions, maximum hydrolytic activity
was observed towards IPG-caproate with low enantioselectivity (E = 2.8), possibly due to the presence of contaminating
esterases, i.e., EST2.
The narrow substrate specificity of EST1 is even more marked
in the hydrolysis of p-nitrophenyl esters, varying from a C2
to a C16 alkyl chain length, since EST1 only hydrolyzed the
acetate ester and was completely inactive in the presence of
the other substrates. Further titration tests using triglycerides
with different chain length as substrates confirmed the observation that only short-chain substrates are converted by EST1,
specifically triacetin and tributyrin (4.06 and 2.8 U ml1 , respectively).
The results obtained during these substrate specificity assays
indicate that EST1 belongs to the esterase class of enzymes
rather than to lipases.
Finally, the Michaelis constant (Km ) and maximum velocity
(Vmax ) of EST1 towards pure enantiomers of (R)- and (S)-IPG
acetate were determined by measuring initial rates of acetic
acid release at various substrate concentrations with a coupled
enzymatic conversion (Baumann et al., 2001). The obtained
results are in agreement with EST1 enantiopreference shown
in biotransformation reactions with racemic IPG acetate, since
the enzyme showed similar Vmax in the hydrolysis of these
substrates ((S)-IPG acetate, 1.2 mol min1 ; (R)-IPG acetate,
1.0 mol min1 ), but a Km value towards (S)-IPG acetate significantly lower than the one towards the (R)-enantiomer (5.3
and 70 M, respectively).
3.3. Catalytic properties of K. marxianus EST1

The substrate specificity and enantioselectivity of purified

EST1 towards IPG esters with different aliphatic side chains
were determined and compared with those observed in K. marxianus whole cells-catalyzed biotransformations (Table 3).

The activity of EST1 was evaluated at pH values ranging

from 2.0 to 10.0. As shown in Fig. 4A, the enzyme displayed
high levels of activity under neutral to alkalophilic conditions,

D. Monti et al. / Journal of Biotechnology 133 (2008) 6572

71

Fig. 4. Catalytic properties of K. marxianus EST1. (a) Influence of pH on EST1 activity. Hydrolysis of pNP-acetate at different pH values was monitored at 348 nm
(348 p-nitrophenol = 5300 M1 cm1 ); () McIlvaine buffer (0.2 M disodium hydrogen phosphate, 0.1 M citric acid); () Tris/HCl buffer; () glycine/NaOH buffer.
(b) Influence of temperature on EST1 activity. Initial reaction rates at different temperatures were determined using pNP-acetate as a substrate and the release of
p-nitrophenol was measured at 400 nm after 3 min of incubation. (c) and (d) Influence of pH and temperature on EST1 stability. Activity assays were performed
using pNP-acetate as a substrate after 4 h of incubation at various pH values (2.011.0) and 1 h of incubation at different temperatures (20100 C).

with no activity below pH 5.0. EST1 was quite stable over the
pH range of 7.010.0 (Fig. 4C), with no loss of activity after
4 h of incubation in these conditions, while it was completely
inactivated at pH 2.0 and retained only 16% residual activity at
pH 11.0.
The enzyme showed a rather thermophilic character, being
active up to 80 C (Fig. 4B). Moreover, it was relatively stable
at temperature below 40 C and was able to retain 75% of the
initial activity after 1 h at 50 C (Fig. 4D).
The influence of cations and EDTA on EST1 activity was
evaluated by measuring the residual enzyme activity in their
presence. It was slightly inhibited by the presence of Mg2+
(80%), Mn2+ (60%), Co2+ (56%), Fe3+ (93%), Fe2+ (70%), Ca2+
(92%), K+ (70%) and Sn2+ (93%), strongly inhibited by Hg2+
(9%) and completely inhibited by Cu2+ and Zn2+ . In the presence of 10 mM EDTA, the residual activity was 18%. Moreover,
short-term incubation with SDS (1%, w/v) resulted in a complete
inactivation of the enzyme.
Finally, EST1 was moderately stable in presence of various water-miscible organic solvents at concentration up to 15%
(v/v). In fact, it retained 82% and 76% residual activity in
the presence of methanol and ethanol, respectively, whereas a
stronger inhibition effect was observed in the presence of acetone (48%), dimethylformamide (34%) and dimethyl sulfoxide
(39%).

In conclusion, production and purification of the enantioselective esterase EST1 from K. marxianus have been studied
in detail and the biocatalyst specificity and enantioselectivity
have been described. The overall activity and stability displayed
by EST1 over a wide range of conditions indicate that it has
the potential to be exploited in biotechnological applications.
The identification and characterization of the enantioselective
esterase present in the cell extract of this yeast will be followed
by a future work finalized to the cloning and sequencing of the
entire gene coding for the enzyme. Moreover, enzyme overexpression in a suitable host will provide an adequate amount of
this very active biocatalyst for the chiral resolution of IPG esters
at an industrial level.
Acknowledgements
This work was supported by Regione Lombardia, Italian
Ministry of Work and Fondo Sociale Europeo (Sovvenzione
Globale Ingenio grant to M.R.).
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