Purification and improvement of the functional properties of Rhizopus oryzae lipase using immobilization techniques

Article abstract of DOI:10.1016/j.molcatb.2014.09.012

The adsorption/immobilization of Rhizopus oryzae lipase (ROL) has permitted the development of several strategies to improve the properties of this industrial enzyme. The enzyme can be purified, immobilized, hyperactivated and stabilized, though its enantioselectivity could still be improved. A moderately hydrophobic support (e.g., phenyl-Toyopearl) allows the almost-quantitative immobilization of the enzyme via selective hydrophobic adsorption, while any contaminant proteins are not adsorbed. A further desorption of the adsorbed enzyme with surfactants (e.g., 0.5% sucrose laurate) allows the complete purification of the enzyme in only one step, with a 90% purification yield and an 8-fold purification factor. By using a more hydrophobic support, the ROL can be immobilized and hyperactivated. The enzyme is completely adsorbed on octyl-Sepharose and C18-Sepabeads. The pure enzyme (when adsorbed on these supports) becomes hyperactivated to approximately 250% of the activity of the soluble enzyme. In this study, two protocols for the covalent immobilization of ROL were developed: a one-point immobilization on CNBr-activated Sepharose and a multipoint covalent immobilization on highly activated glyoxyl-agarose supports. The multipoint attached enzyme decreased in activity to 50% of the activity of the soluble enzyme, but the derivatives were 300-fold more stable than the soluble enzyme. The hydrolysis of racemic 2-O-butyryl-2-phenylacetic acid was modulated by the different immobilized derivatives. In fact, the most active and selective preparation was demonstrated to be the C18-SB-ROL derivative (0.05 UI/mg and E = 22) when compared with the CNBr-ROL enzyme preparation (0.0073 UI/mg and E = 3.5).

Full text of DOI:10.1016/j.molcatb.2014.09.012

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Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
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Purification and improvement of the functional properties of Rhizopus
oryzae lipase using immobilization techniques
N. Ghattasa,b, M. Filiceb, F. Abidia, J.M. Guisanb,∗, A. Ben Salaha,∗∗
a Laboratory of Protein Engineering and Bioactive Molecules (LIP-MB), National Institute of Applied Sciences and Technology, University of Carthage,
B.P. 676, 1080 Tunis Cedex, Tunisia
b Departamento de Biocatálisis, Instituto de Catálisis (CSIC), Campus UAM Cantoblanco, 28049 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The adsorption/immobilization of Rhizopus oryzae lipase (ROL) has permitted the development of several
strategies to improve the properties of this industrial enzyme. The enzyme can be purified, immobi-
lized, hyperactivated and stabilized, though its enantioselectivity could still be improved. A moderately
hydrophobic support (e.g., phenyl-Toyopearl) allows the almost-quantitative immobilization of the
enzyme via selective hydrophobic adsorption, while any contaminant proteins are not adsorbed. A fur-
ther desorption of the adsorbed enzyme with surfactants (e.g., 0.5% sucrose laurate) allows the complete
purification of the enzyme in only one step, with a 90% purification yield and an 8-fold purification factor.
By using a more hydrophobic support, the ROL can be immobilized and hyperactivated. The enzyme is
completely adsorbed on octyl-Sepharose and C18-Sepabeads. The pure enzyme (when adsorbed on these
supports) becomes hyperactivated to approximately 250% of the activity of the soluble enzyme. In this
study, two protocols for the covalent immobilization of ROL were developed: a one-point immobilization
on CNBr-activated Sepharose and a multipoint covalent immobilization on highly activated glyoxyl-
agarose supports. The multipoint attached enzyme decreased in activity to 50% of the activity of the
soluble enzyme, but the derivatives were 300-fold more stable than the soluble enzyme. The hydrolysis of
racemic 2-O-butyryl-2-phenylacetic acid was modulated by the different immobilized derivatives. In fact,
the most active and selective preparation was demonstrated to be the C18-SB-ROL derivative (0.05 UI/mg
and E = 22) when compared with the CNBr-ROL enzyme preparation (0.0073 UI/mg and E = 3.5).
© 2014 Elsevier B.V. All rights reserved.
Received 30 January 2014
Received in revised form
17 September 2014
Accepted 18 September 2014
Available online xxx
Keywords:
Improvement of lipase properties
Kinetic resolution of racemic mixtures
One-step purification of lipases
1. Introduction
properties of Rhizopus oryzae lipase (ROL) are discussed. Immobi-
lization techniques were used [3] as the primary protocols for the
Enzymes exhibit many excellent properties that can be used
for possible industrial implementation: very high catalytic activ-
ity under very mild conditions, high specificity and high selectivity
[1,2]. However, because of their biological origin, enzymes have
some important limitations: (A) They are the minimal portion of a
crude extract, and their purification is complex. (B) They are soluble
and unstable. (C) They have minimal activity and are not selec-
tive towards non-natural substrates. For these reasons, many of
the functional properties of enzymes need to be greatly improved
to increase the possibility of industrial implementation. In this
manuscript, the purification and improvement of the functional
enzyme biotechnology.
The immobilization of lipases on hydrophobic supports, by
means of interfacial adsorption (e.g., on octyl-Sepharose or
octadecyl-Sepabeads) [4,5], represents
a simple and elegant
methodology for obtaining highly active lipase catalysts, where
the hydrophobic nature of the support plays a critical role [5]. In
addition, several interesting stabilizations of the enzyme may be
achieved. For example, the open form of the enzyme that is sta-
bilized on a hydrophobic support can often be much more stable
than the native enzyme [5]. In addition, the process of adsorption
is sometimes very selective, and thus, an additional desorption of
the adsorbed lipases with surfactants allows a very simple protocol
for lipase purification [6,7].
Another relevant method for improving enzyme stability is a
multipoint covalent immobilization on highly activated glyoxyl
supports [6]. For this, the enzyme is oriented on the activated
support via the surface region with the highest number of Lys
∗
Corresponding author. Tel.: +34 915854809; fax: +34 915854760.
Corresponding author.
E-mail addresses: jmguisan@icp.csic.es (J.M. Guisan), bensalahraouf@yahoo.fr
∗∗
(A. Ben Salah).
http://dx.doi.org/10.1016/j.molcatb.2014.09.012
1381-1177/© 2014 Elsevier B.V. All rights reserved.
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2
residues. When using this method, an intense multipoint covalent
immobilization is favored [7]. This immobilization process must be
performed at pH 10 (4 or 25 ◦C), but unfortunately, many enzymes
become unstable under these alkaline conditions. In these cases,
soluble stabilizing additives (e.g., glycerol, polyethyleneglycol and
trehalose) can be used during the immobilization process [8].
Conversely, the simplest protocol for enzyme immobilization
involves the immobilization of lipases or other enzymes on CNBr-
activated Sepharose at a neutral pH, which takes place via the amino
terminus of the enzyme (pK = 7.5) rather than the Lys residues
on its surface (pK = 10.5). In addition, the use of neutral pH val-
ues, short immobilization times and low temperatures cause this
method to be favored over multipoint covalent immobilization. In
general, these immobilized derivatives exhibit the same activity as
the pure and diluted enzyme solutions. However, these derivatives
can be prepared by using impure enzyme extracts, can be used in
organic cosolvents and can be stirred without the issue of enzyme
aggregation, which can mask the actual properties of the soluble
enzymes. In addition, the immobilization of lipases in the pres-
ence of surfactants prevents dimerization and allows the one-point
immobilization of any lipase monomers. The most suitable blank for
testing the properties of different lipase derivatives is a one-point
derivative immobilized on CNBr-activated Sepharose. This blank is
much more accurate than soluble lipases [11].
20 mM NaCl and 2 mM benzamidine. The solution was then cen-
trifuged at 10,000 rpm for 10 min, and the supernatant containing
the lipase was used for the subsequent purification studies.
2.2.2. Enzymatic activity assay
The activities of the soluble lipases and their immobilized prepa-
rations were prepared and evaluated in a 3-mL quartz reaction
cell (cuvette) equipped with magnetic stirring and analyzed spec-
trophotometrically by measuring the increase in absorbance at
348 nm (ε = 5150 M−1 cm−1). The activity is produced by the release
of p-nitrophenol (pNP) during the hydrolysis of 0.4 mM pNPB in
25 mM sodium phosphate buffer (pH 7) and 1% acetonitrile at 25 ◦C.
To initialize the reaction, 0.02–0.1 ml of the lipase solution (blank
or supernatant) or a suspension was added to 2.5 ml of a sub-
strate solution. The enzymatic activity is given as ␮mol of pNP
produced per minute per mg of enzyme (IU) under the condi-
tions described above. A wavelength of 348 nm was chosen as the
standard absorbance because at this wavelength the absorbance of
pNP is independent of the pH [10].
2.2.3. Immobilization of the R. oryzae lipase
The enzymatic solution was added to 1 g of the support, and
the mixture was incubated under mild mechanical stirring at 25 ◦C
under the conditions described below. To verify the immobiliza-
tion, samples of the suspensions were withdrawn using cut pipette
tips, and samples of the supernatants were taken using a filtered
pipette tip. As a control, the activity of a reference solution of the
enzymatic solution without any support and incubated under the
same conditions was measured in a parallel experiment.
Rhizopus oryzae lipase (ROL) is an interesting industrial enzyme
capable of catalyzing a number of different biotransformations,
including biodiesel production [12], citronellol ester synthesis [13],
enantioselective resolution reactions [14], transesterification of
phosphatidyl choline [15], and the synthesis of palm stearin [16].
Because of this, there have been several attempts to improve the
properties of the enzyme [17,18].
In this paper, we discuss the purification and improvement of
the functional properties of ROL using different adsorption/
immobilization protocols on different supports (primarily
hydrophobic supports). Covalent immobilization on CNBr-
activated Sepharose and on glyoxyl-agarose supports was also
studied. The thermal stability of the different derivatives was
evaluated, and the hydrolysis of racemic 2-O-butyryl-phenylacetic
acid was also examined.
2.2.4. Immobilization of the ROL onto different hydrophobic
supports
A 1-ml volume of the R. oryzae-produced lipase (2 mg total
protein/ml as determined by Bradford assay [20]) was mixed
with 9 ml of 10 mM sodium phosphate at pH 7.0, and 1 g of the
hydrophobic support was added. Different hydrophobic supports
were used to test different immobilization procedures (butyl-
Sepharose, phenyl-Sepharose, butyl-Toyopearl, phenyl-Toyopearl,
hexyl-Toyopearl, C18 Sepabeads and octyl-Sepharose). The activ-
ities of the supernatant and suspension of each immobilization
mixture were periodically checked via the method described above.
At the end of the immobilization, each enzyme derivative was
recovered by filtration under a vacuum and washed thoroughly
with distilled water.
2. Materials and methods
2.1. Materials
Sodium dodecyl sulfate (SDS), p-nitrophenyl butyrate (p-
NPB), hexadecyltrimethylammonium bromide (CTAB) and sucrose
monolaurate were purchased from Sigma Chemical Co. (St. Louis,
USA). Octyl-SepharoseTM, CNBr-activated Sepharose 4B (CNBr-
Sepharose), butyl-Sepharose, phenyl-Sepharose, butyl-Toyopearl,
phenyl-Toyopearl, hexyl-Toyopearl and LMW (low molecular
weight electrophoresis markers) were purchased from GE Health-
care (Uppsala, Sweden). The C18 Sepabeads were purchased from
Resindion Srl (Milan, Italy), and the sodium borohydride (NaBH4)
was obtained from Sigma Chemical Co. (St. Louis, USA). All of the
other reagents and solvents used were of analytical or HPLC grade.
2.2.5. SDS-PAGE of the different preparations
The SDS-PAGE electrophoresis was performed according to
Laemmli’s method [21] in an SE 250-Mighty Small II electrophoretic
unit (Hoefer Co.) using 12% polyacrylamide gels with a separation
zone of 9 cm × 6 cm and a concentration zone of 5% polyacrylamide.
The gels were stained with Coomassie Blue. LMW molecular weight
markers were used.
2.2.6. Enzyme desorption from the hydrophobic support
2.2. Methods
A total of 1 g of washed phenyl-Toyopearl-ROL derivative was
incubated for 1 h at 25 ◦C with 10 ml of 10 mM sodium phosphate
(pH 7) and increasing concentrations of different detergents (Triton
X100, Lauryl sucrose, CTAB and SDS). To check the desorption yield,
the activities of the supernatant and suspension were checked by
the method described above. The enzyme desorption was consid-
ered quantitative when both activities were identical. To recover
the pure enzyme solution, the supernatant was separated from the
support by filtration under a vacuum.
2.2.1. Production of R. oryzae lipase (ROL)
The R. oryzae lipase was produced as described by Ben Salah
et al. [19]. Briefly, after 72 h of growth, the cells were removed via
filtration, and the lipase in the supernatant was precipitated by the
addition of ammonium sulfate up to 60% saturation, which was then
followed by centrifugation at 8000 rpm at 4 ◦C for 30 min. The pellet
was dissolved in 20 mM sodium acetate buffer, pH 5.2, containing
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2.2.7. Immobilization of the ROL on a CNBr-activated support
A commercial CNBr-Sepharose support was activated prior to
use by suspending in an acidic aqueous solution (pH 2–3) for 1 h.
The support was then dried via filtration under a vacuum. Once
activated, 1 g of the CNBr-Sepharose was added to 10 ml of the
lipase solution (0.2 mg/ml) from the previous desorption studies.
The mixture was then stirred at 4 ◦C and 250 rpm for 20 min. Next,
the solution was removed via filtration, and the supported lipase
was washed twice with 100 mM NaHCO3 at pH 8 and then re-
suspended in 10 ml of 1 M ethanolamine at pH 8 for 90 min to block
the unreacted imidocarbonate reactive groups. Subsequently, the
reaction mixture was filtered and washed with an abundant vol-
ume of distilled water. The immobilization yield is the percentage of
enzyme that becomes immobilized. This is calculated by comparing
the hydrolytic activity of the supernatant before and after immo-
bilization by using the enzymatic activity assay described above.
The immobilization yield was 56% with a protein loading amount
of approximately 1.12 mg/g support [11].
Fig. 1. The adsorption of Rhizopus oryzae lipase on different hydrophobic supports.
10 ml of an enzyme solution was added to 1 ml of different hydrophobic supports
at pH 7.0 and 25 ◦C. ꢀ: percentage of the enzyme adsorbed on the support. ꢁ: per-
centage of activity of the adsorbed enzyme relative to the soluble enzyme that was
adsorbed. Supports: (1) butyl-Sepharose, (2) phenyl-Sepharose, (3) butyl-Toyopearl,
(4) hexyl-Toyopearl, (5) phenyl-Toyopearl, (7) octyl-Sepharose and (8) decaoctyl
(C18)-Sepabeads. The experiments were performed as described in Section 3.
2.2.8. Stability of the soluble ROL at alkaline pH
The stability of the pure soluble ROL in a sodium bicarbonate
buffer at pH 10 was studied during a 24-h period at 25 ◦C and 4 ◦C
in the presence of the desorption detergent (0.5% sucrose laurate)
and 50% trehalose (w/v). The samples were periodically withdrawn
and their activities were measured using the pNPB activity assay
described previously.
8450). For these assays, a Kromasil C18 (250 mm × 4.6 mm, 5-␮m
particle diameter) column was used, and the mobile phase was 20%
acetonitrile/80% 10 mM ammonium dihydroxy phosphate (v/v), pH
2.9, at a flow rate of 1.5 mL/min. The UV detection was performed
at 225 nm for all of the substrates.
2.2.9. Immobilization of the ROL on glyoxyl-Sepharose
2.2.12. Determination of the enantiomeric excess and
enantioselectivity
A highly activated glyoxyl-agarose support was prepared as pre-
viously described [9]. Next, 3 g of the activated support were added
to 30 ml of the pure ROL solution containing 50% trehalose (w/v).
The immobilization was performed at pH 10 over a 24-h period
at 25 ◦C under very gentle stirring. To finalize the immobilization
reaction, solid NaBH4 was added to the suspension at a 1-mg/ml
concentration, and the suspension was gently stirred for 30 min.
Borohydride reduces the Schiff bases formed between the enzyme
and the support, and the unreacted aldehyde groups remain in the
support. Subsequently, the suspension was filtered, and the glyoxyl
derivative was washed with an abundant volume of distilled water.
The immobilization progress was followed by the enzymatic assay
described above.
The enantiomeric excess of the released acid (ee) (at conver-
sions between 10 and 15%) was analyzed via chiral reverse-phase
HPLC. The column used was a Chiracel OD-R. The mobile phase was
an isocratic mixture of acetonitrile:10 mM ammonium dihydroxy
phosphate (30:70, v/v). The final pH of the three mobile phases was
adjusted to 2.3 with HCl, and the flow rate was 0.5 mL/min. The UV
detection was performed at 210 nm.
The enantiomeric ratio (E) was calculated in all of the
cases using the equation reported by Chen et al. [23]:
E = ln[1 − c(1 + eep)]/ln[1 − c(1 − eep)], where c is the conversion
ratio of the reaction, and eep is the enantiomeric excess of the
formed enantiomer.
2.2.10. Thermal stability of the different ROL-immobilized
derivatives
2.2.12.1. Reproducibility. All of the experiments were performed in
triplicate, and the experimental error was consistently less than 5%.
One gram of the different ROL derivatives was incubated in 10 ml
of 25 mM sodium phosphate at pH 7.0 at different temperatures.
The samples were periodically withdrawn using a pipette with a
cut-tip and placed under vigorous stirring to maintain a homoge-
neous biocatalyst suspension. The activity was measured using the
pNPB assay described above. The experiments were conducted in
triplicate, and the plotted results are the mean of these repetitions.
The experimental errors were less than 5%. Stabilization is defined
as the ratio of the half-life of the problem derivative divided by that
of the preparations being compared to it. The thermal stability of
the glyoxyl derivatives was studied with an enzymatic hydrolytic
assay that was modified by adding 0.5% SDS to the cuvette.
3. Results and discussion
3.1. The adsorption of ROL on hydrophobic supports:
hyperactivation and purification
The adsorption yields were lower when using less-hydrophobic
supports. In addition, the recovered activity was less than 100%
(Fig. 1). When using medium-hydrophobic supports (phenyl-
Toyopearl and phenyl-Sepharose), the adsorption yield improved
(very close to 100%), and the recovered activity was higher
(very close to 100%). Complete adsorption was observed on the
most-hydrophobic supports (C8-Sepharose and C18-Sepabeads),
and an interesting hyperactivation of the immobilized enzyme
was observed (approximately 250%). These highly hydropho-
bic supports allowed the development of protocols for the
immobilization-hyperactivation of the ROL.
2.2.11. Enzymatic hydrolysis of ( )-2-O-butyryl-2-phenylacetic
acid
The activities of the different immobilized preparations of the
ROL were analyzed in a hydrolysis reaction with ( )-2-O-butyryl-2-
phenylacetic acid. The substrate was dissolved to a concentration of
1 mM in 2 mL of 25 mM phosphate buffer at pH 7 at 25 ◦C. Next, 0.1 g
of the immobilized ROL preparation was added to the ester solution
[22]. The degree of hydrolysis was analyzed by reverse-phase HPLC
(Spectra Physic SP 100 coupled with a UV detector Spectra Physic SP
Following the selective adsorption, the enzyme desorption with
surfactants was studied (Fig. 2). Lauryl sucrose and TRITON X-100
were found to completely desorb the enzyme at high concentra-
tions (0.5% lauryl sucrose and 0.7% TRITON X-100). In both cases,
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Fig. 2. Desorption study of R. oryzae lipase adsorbed on phenyl-Toyopearl deriva-
tive in the presence of different concentrations of different surfactants. Legend: (ꢂ)
Triton X100; (᭹) Lauryl sucrose; (ꢃ) Sodium dodecyl sulfate; (ꢁ) Cetyl trimethyl
ammonium bromide. One milliliter of the ROL derivative was incubated in 10 ml of
25 mM phosphate buffer, pH 7, at 25 ◦C. Different concentrations of the surfactants
were added, and the suspension and the supernatant were assayed according to the
standard assay with p-nitrophenyl butyrate.
Fig. 3. Inactivation time-courses of the soluble ROL at pH 10.0 in presence of polyols
as preservatives. Legend: (ꢀ) 25 ◦C; (x) 4 ◦C; (᭹) Lauryl Sucrose, 25 ◦C; (ꢂ) Lauryl
Sucrose, 25 ◦C and Trehalose 50% (w/v); (ꢃ) Lauryl Sucrose, 4 ◦C; (ꢁ) Lauryl Sucrose,
4
◦C and Trehalose 50% (w/v). The experiments were performed at pH 10.0 in the
presence of 0.5% sucrose laurate after desorption of the enzyme from the phenyl-
Toyopearl.
activity to the immobilized soluble enzyme. Therefore, we were
able to obtain a suitable blank for the immobilized ROL.
the enzyme retained 100% of its catalytic activity. Ionic surfactants
(such as SDS and CTAB) promoted a complete inactivation of the
enzyme in the supernatant and the suspension. Therefore, a selec-
tive adsorption on phenyl-Toyopearl and the additional desorption
with lauryl sucrose appears to be a very promising method for a
one-step purification of ROL. In fact, the crude extract exhibited
a specific activity of 23 U/mg of protein, and the purified enzyme
exhibited a specific activity of 190 U/mg of electrophoretically pure
enzyme. In only one step, a 90% purification yield and an 8-fold
purification factor for the extracellular lipase was obtained (e.g.,
the ratio of the specific activity of the pure enzyme to the crude
extract).
3.4. Thermal stability of the different immobilized derivatives
Initially, the thermal inactivation at 45 ◦C was studied (Fig. 5).
The soluble enzyme and the mildly immobilized derivative (on
CNBr-activated Sepharose) inactivated very quickly and with very
similar inactivation rates (half-life of approximately 1 h). The solu-
ble enzyme and the mildly immobilized derivative showed similar
results (same activity and stability). The derivatives that were
adsorbed on the hydrophobic supports were found to be more
stable (e.g., the C8-Sepharose derivative was 25-fold more stable,
with a half-life time of 25 h), and the derivative adsorbed on the
C18 Sepabeads was especially stable and retained 100% of its activ-
ity even after 25 h. Similarly, a high stabilization was observed for
the derivative that was immobilized by multipoint covalent immo-
bilization on the glyoxyl-agarose. It was found that multipoint
covalent immobilization and adsorption on highly hydrophobic
supports promotes very interesting stabilizing effects.
3.2. Covalent immobilization on highly activated glyoxyl-agarose
supports
We first studied the stability of the soluble enzyme at pH 10,
as this pH was necessary to perform the immobilization protocol.
The soluble enzyme becomes inactive in the presence of 0.5% lauryl
sucrose, which was used for the desorption of the enzyme from the
phenyl-Toyopearl. Additionally, the enzyme was inactivated at pH
10 at both 4 and 25 ◦C and in the presence or absence of a stabilizing
poly-ol (50% trehalose). In the absence of trehalose, the enzyme was
very unstable at both temperatures (though it was more stable at
4 ◦C) (Fig. 3). However, in the presence of trehalose, the enzyme
became very stable, even at 25 ◦C, and it retained 100% of its activity
after more than 10 h. Therefore, these conditions were selected to
perform the immobilization at pH 10.
To compare the most stable derivatives, they were inactivated at
60 ◦C (Fig. 6). The C18-Sepabead derivative was 5-fold more stable
than the octyl-agarose, and the glyoxyl derivative was also very sta-
ble (12-fold more stable) and even exhibited a second very-stable
The enzyme was found to immobilize very quickly (Fig. 4). Over-
all, 80% of the enzyme was immobilized in less than 5 h, but the
immobilization was allowed to proceed for 24 h in order to improve
the multipoint covalent attachment [10]. Over the extended time
period, the activity of the suspension decreased to approximately
50%, but this decrease was negligible as the new derivative was
highly stable.
3.3. The immobilization of ROL on CNBr-activated sepharose
Fig. 4. The immobilization time-course of the ROL on highly activated glyoxyl-
agarose. The experiments were carried out at pH 10 and 25 ◦C in the presence of
0.5% sucrose laurate and 50% trehalose. Ten milliliters of the enzyme solution (10
IU) were mixed with 1 ml of the activated support. The suspension was stirred very
gently. ꢂ: activity of the soluble enzyme under the immobilization conditions in the
absence of the activated support. ꢃ: activity of the supernatant of the immobiliza-
tion suspension in the presence of the activated support. ꢁ: activity of the entire
immobilization suspension.
The immobilization was performed under very mild exper-
imental conditions to prevent any type of multipoint covalent
attachment, with the conditions set at pH 7.0 (as only the amino
terminus is reactive) at 4 ◦C for 15 min. The immobilization yield
was low (30%), but the immobilized derivative exhibited an equal
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Table 1
Kinetic resolution of racemic 2-O-butyril-mandelic acid.
O
O
O
O
OH
OH
OH
OH
*
+
O
O
O
rac-2-O-butyrylmandelic acid
S-(-)-2-O-butyryl mandelic acid
R-(+)-mandelic acid
.
Derivative
Time
Conversion
ee (%)
UI/mg (×10−3
)
E
CNBr
C18
Gx
96
48
96
2.5
10.3
4.9
55
91
80.5
7.3
50
12
3.5
22
10.4
(not only lipases). This stabilization is due to the formation of a
network between a number of enzyme residues and a number of
active groups on the support. Now, the relative distances among all
residues involved in the network have to remain unaltered during
any conformational change promoted by any distorting agent.
3.5. The hydrolysis of racemic 2-O-butyryl-2-phenylacetic acid
by different immobilized derivatives
In the hydrolysis of racemic 2-O-butyryl-2-phenylacetic acid,
the activity and the enantioselectivity of the R. oryzae lipase
was strongly modulated by the immobilization protocol that was
applied, suggesting that this may be a good option to further
improve its performance in target reactions. In fact, the most
active and selective preparation was determined to be the C18-
Sepabead-ROL derivative (0.05 UI/mg and E = 22), when compared
with the CNBr-ROL enzyme preparation (0.0073 UI/mg and E = 3.5).
The glyoxyl derivative exhibited intermediate selectivity (E = 10.4)
(Table 1). Overall, the activities showed the same pattern as those
obtained with the synthetic substrate, with the C18 Sepabeads
derivative being the most active.
Fig. 5. Time-course of the thermal inactivation (45 ◦C) of the different ROL deriva-
tives. Immobilized or soluble ROL were incubated at 45 ◦C and pH 7.0, and at different
time points, aliquots of the suspensions/solution were withdrawn and assayed at
25 ◦C. ꢃ: soluble ROL. ꢁ: ROL immobilized on CNBr-activated-Sepharose. ᭹: ROL
immobilized on phenyl-Toyopearl. ꢃ: ROL immobilized on octyl-Sepharose. ꢀ: ROL
immobilized on C18-Sepabeads. *: ROL immobilized on glyoxyl-agarose.
When considered together, all of the results clearly demonstrate
that the catalytic properties of this lipase may be greatly modulated
and improved by performing immobilization and/or rigidification
on different regions of the enzyme’s surface [24].
inactivation phase. Under the assumption that the stabilization
factors are additive, the glyoxyl derivative would therefore be 300-
fold more stable than the soluble enzyme and the CNBr-activated
Sepharose derivatives.
The stabilization of enzyme by multipoint covalent immobiliza-
tion was developed successfully to a number of industrial enzymes
4. Conclusions
By using Rhizopus oryzae lipase (ROL) and different solid
supports, several strategies to improve the properties of an indus-
trial enzyme were developed. The enzyme could be purified,
immobilized, hyperactivated and stabilized. Moreover, its enan-
tioselectivity was also greatly improved. The most relevant results
were as follows: a hyperactivation of 250%, a 300-fold thermal sta-
bilization and an increase in the enantioselectivity from 3.5 to 22.
Throughout the research, different interaction processes
between the enzymes and different supports were used to prepare
optimal and cost-effective enzyme biocatalysts. Based on our
results, we determined the following: (a) Purification is necessary
for the preparation of high-loading enzyme derivatives as well as
for the prevention of undesirable side reactions caused by con-
taminating enzymes. This can be performed via chromatographic
protocols (interaction between lipases and hydrophobic supports).
(b) Immobilization is necessary for the re-use of an enzyme. This
can be performed by attaching the enzyme to a solid support.
(c) Stabilization is necessary for very long periods of re-use of an
Fig. 6. Time-course of the thermal inactivation (60 ◦C) of the different ROL deriva-
tives. Immobilized ROL derivatives were incubated at 60 ◦C and pH 7.0, and at
different time points, aliquots of the suspension were withdrawn and assayed
at 25 ◦C. ꢂ: ROL immobilized on octyl-Sepharose. ꢃ: ROL immobilized on C18-
Sepabeads. ꢁ: ROL immobilized on glyoxyl-agarose.
Please cite this article in press as: N. Ghattas, et al., J. Mol. Catal. B: Enzym. (2014), http://dx.doi.org/10.1016/j.molcatb.2014.09.012

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immobilized enzyme. This can be performed by selecting suitable
immobilization protocols. (d) To modulate selectivity, it is neces-
sary to improve the catalytic properties of the lipases when acting
on non-natural substrates. This can be achieved by using different
immobilization protocols in addition to altering the exact shape of
the open form of the lipase.
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This work was sponsored by the Spanish Ministry of Science
and Innovation (projects AGL-2009-07526 and BIO-2012-36861).
Nesrine Ghattas was the recipient of help from the Government
of Tunisia. Marco Filice thanks CSIC for the JAE-Doc contract ref
JAEDOC092 (“Junta para la Ampliacion de estudios”) cofounded by
ESF (European Social Fund).
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Please cite this article in press as: N. Ghattas, et al., J. Mol. Catal. B: Enzym. (2014), http://dx.doi.org/10.1016/j.molcatb.2014.09.012