Asymmetric hydrolysis of dimethyl-3-phenylglutarate in sequential batch reactor operation catalyzed by immobilized Geobacillus thermocatenulatus lipase

Article abstract of DOI:10.1016/j.cattod.2014.12.039

Abstract The main goal of this work was to study the stereoselective behavior of immobilized Geobacillus thermocatenulatus lipase (BTL2) in a sequential batch reactor using the partial and asymmetric hydrolysis of dimethyl-3 phenylglutarate (DMFG) as a mo

Full text of DOI:10.1016/j.cattod.2014.12.039

G Model
CATTOD-9422; No. of Pages6
ARTICLE IN PRESS
Catalysis Today xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Catalysis Today
journal homepage: www.elsevier.com/locate/cattod
Asymmetric hydrolysis of dimethyl-3-phenylglutarate in sequential
batch reactor operation catalyzed by immobilized Geobacillus
thermocatenulatus lipase
Nadia Guajardo, Claudia Bernal, Lorena Wilson, Zaida Cabrera∗
Escuela de Ingeniería Bioquímica, Pontificia Universidad Católica de Valparaíso, Avda. Brasil, 2147 Valparaíso, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
The main goal of this work was to study the stereoselective behavior of immobilized Geobacillus ther-
mocatenulatus lipase (BTL2) in a sequential batch reactor using the partial and asymmetric hydrolysis of
dimethyl-3 phenylglutarate (DMFG) as a model reaction. To reach this goal, BTL2 lipase was immobilized
on Sepharose and silica supports with cyanogen bromide and octyl groups (monofunctional supports)
and undecanol-glyoxyl and octyl-epoxides groups (heterofunctional supports), to determine the effect of
the enzyme orientation during the immobilization process on their catalytic properties. In the hydrolysis
of DMFG, the biocatalyst obtained with undecanol-glyoxyl Sepharose proved to be the most stereolec-
tive with an enantiomeric excess (e.e.) value of 90% in aqueous media. This behavior can be attributed to
differences in the orientation of the lipase on the support. In sequential batch reactor operation, the e.e.
remained constant in the first two batches; however, from the third batch on the e.e. decreased slightly
maybe due to a change in the conformation of the enzyme at the reaction conditions. Finally, the high
purity S-methyl-3-phenyl glutarate produced in sequential batch reactor operation shows that the bio-
catalyst can be reused at least twice without losing stereoselectivity, favoring a reduction in the process
cost.
Received 29 September 2014
Received in revised form
23 December 2014
Accepted 25 December 2014
Available online xxx
Keywords:
Asymmetric hydrolysis
Dimethyl-3-phenylglutarate
Stereoselectivity
Silica supports
Sequential batch reactor
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
lipase is in the presence of a hydrophobic interface, the lid flips
back, and the hydrophilic outside portion of the lid comes into
Lipases are the most used enzymes in biocatalysis and organic
chemistry. They recognize a wide variety of substrates while
exhibiting high stereoselectivity in many instances [1]. Lipase from
Geobacillus thermocatenulatus (BTL2) is a non-commercial intra-
cellular enzyme presenting two “lids” covering the active site, a
feature also reported for lipases from Pseudomonas stutzeri [2],
Pseudomonas aeruginosa [3], Staphylococcus simulans [4], Serratia
marcenses [5], Pseudomonas sp MIS38 [6] and T1 from Geobacillus
zalihae [7]. The interest in this lipase is based on its stability at high
pH and temperature and in the presence of organic solvents, and
the ability to carry out selective reactions [8,9].
contact with other hydrophilic pocket “receptor” located in the
vicinity of the active site of the lipase; thus, the active site is
exposed to the reaction medium, leading to the fixation of the open
conformation on said interface, a phenomenon known as interfa-
cial activation mechanism [13]. This opening and closing lid may
involve changes in amino acid residues located even far from the
active site of the lipase, which can modify its catalytic properties
[14,15].
The development of new biocatalysts is very important for the
application of enzymes in industrial processes and in this con-
text immobilization techniques are a simple strategy to solve the
enzyme solubility problems [16]. Along with this, it is possible to
modulate the catalytic properties of the enzyme by immobilization,
as is the case with lipases [17].
During catalysis, most lipases bearing a lid suffer a quite com-
plex conformational change from a closed form to an open form,
with significant variations around their active site [10–12]. When a
The heterofunctional supports are a good alternative for the
immobilization of enzymes, because it combines hydrophobic
adsorption through the interfacial activation mechanism of lipase
[18,19] and multipoint covalent attachment between the sup-
port and the enzyme, benefiting from the advantages of both
∗
Corresponding author. Tel.: +56 322372021; fax: +56 322273803.
E-mail address: zaida.cabrera@ucv.cl (Z. Cabrera).
http://dx.doi.org/10.1016/j.cattod.2014.12.039
0920-5861/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: N. Guajardo, et al., Asymmetric hydrolysis of dimethyl-3-phenylglutarate in
sequential batch reactor operation catalyzed by immobilized Geobacillus thermocatenulatus lipase, Catal. Today (2015),
http://dx.doi.org/10.1016/j.cattod.2014.12.039

G Model
CATTOD-9422; No. of Pages6
ARTICLE IN PRESS
2
N. Guajardo et al. / Catalysis Today xxx (2015) xxx–xxx
immobilization strategies [20,21]. Immobilization in heterofunc-
tional supports requires that the process be carried out in two steps,
with the aim to favor an adequate interaction between the enzyme
and the support in every step [20] (adsorption or covalent bonding).
Hierarchical macro-mesoporous silicas and Sepharoses are
porous and highly functionalizable supports. Both carriers have
been used for the immobilization of enzymes, however, silica sup-
ports have a greater surface than Sepharose reducing in this way
the incidence of diffusional restrictions [22,23].
and Sepharose CL-6B beads were purchased from GE Healthcare
(Sweden). BTL2 lipase was produced by fermentation with the
recombinant microorganism Escherichia coli BL21 (DE3) [8].
2.2. Synthesis and functionalization of supports
Synthesis of silica support was carried out according to previous
works by Bernal et al. [21,27].
In general, lipases have been immobilized on hydrophobic
siliceous mesoporous and Sepharose supports [24,25], but they
have also been immobilized on Sepharose [26] and silica het-
erofunctional supports [21,27], being promising supports for the
design of biocatalysts for reactions in organic synthesis.
2.2.1. Functionalization of octyl silica support
The silica support was functionalized with octyl chains accord-
ing to the procedure reported by Bernal et al. [21]. One gram of
support is contacted with 30 mL of toluene at 10% of triethoxy-
octylsilane under reflux at 105 ◦C for 5 h. The obtained silica octyl
(Silica-O) support was then washed, filtered and stored at 25 ◦C.
The preparation of chiral derivatives of 3-phenylglutaric acid
is of great interest for the industry since, having a chiral glu-
taric fragment in their structure, these enantiopure isomers are
important intermediates in the synthesis of drugs. Among them,
(−)-paroxetine (antidepressant) [28], spiro-substituted piperidine
(antiasthmatic) [29], SCH 54016 (cholesterol inhibitor) [30] and an
antagonist of tachykinins receptors NK1 and NK2, compounds with
potential activity in the treatment of asthma, arthritis and migraine
[31]. A common route toward optically active 3-arylglutaric acid
monoesters is the enzymatic hydrolysis of the respective prochiral
diesters. If the asymmetric reaction stops at the monoester and the
stereoselectivity value of the process is very high, 100% yield of an
enantiomerically pure compound will be produced [25]. In many
cases, the characteristic of the enzyme may allow stopping the
reaction at the monoester, but some lipases may recognize these
monoesters even better than the diester, generating the diacid. In
this case, it will be necessary to develop strategies to modify the
lipase specificity [32].
Fryszkowska et al. [33], found that Novozym 435 catalyzed the
desymmetrization of 3-arylglutaric acid anhydrides with moder-
ate to good enantiopurities. However, their study on the effect
of the enzyme amount and re-use of the lipase was not satisfac-
tory. A larger scale process was developed by Homman et al. [34],
where about 200 kg of S-monoester with e.e. > 99% were produced
in three batches (in parallel) with an 80% yield. This process has
been employed by the company Schering-Plough. However, they do
not consider the reuse of the enzyme, due to the partial inactivation
of the catalyst.
2.2.2. Functionalization of heterofunctional silica support
Silica heterofunctional (Silica-HE) support was functionalized
in a similar way than Silica-O support, except that a mixture of
GPTMS and triethoxyoctylsilane was used. 1 g of chemically synthe-
sized silica was contacted with a toluene solution with 8% of GPTMS
and 2% triethoxyoctylsilane under reflux at 105 ◦C for 5 h. The sup-
port obtained was washed, filtered and stored at 25 ◦C. The epoxide
groups were quantified by back-titration with NaHCO3/KI [27]. The
synthesized Silica-HE support exhibited a surface of 443 m2/g and
70 ␮moleEpoxides/g [27].
2.2.3. Functionalization of heterofunctional Sepharose support
Sepharose heterofunctional support (Seph-HG) was prepared
in two steps. The first step involved the incorporation of epoxide
groups to the Sepharose matrix. The formation of epoxide groups
was performed with epichlorohydrin; details of this procedure can
be found elsewhere [35].
In the second step, the resulting support was washed with
distilled water and filtered under vacuum. Subsequently,
a
hydrophobic chain of 11-mercapto-1-undecanol in 20 mL of
100 mM sodium bicarbonate and 30 mL of 1,4-dioxane at pH 9.5
was added to the support. This suspension was maintained under
mechanical stirring for 24 h at 25 ◦C. Later on, the resulting support
was washed with a 50% (v/v) solution of 1,4-dioxane and filtered
under vacuum. Subsequently, diols groups on the support were oxi-
dized to aldehydes with NaIO4, as previously described [35]. Finally
the support obtained was washed with distilled water, filtered and
stored at 4 ◦C.
Base on the above exposed, the main goal of this work was to
evaluate the stereoselectivity of the lipase biocatalysts in aqueous
medium, using the asymmetric hydrolysis of dimethyl-3 phenyl-
glutarate (DMFG) (Scheme 1) as model reaction, in a sequential
batch reactor. To reach this goal we worked with BTL2 lipase immo-
bilized on Sepharose support with cyanogen bromide, octyl and
undecanol-glyoxyl groups and with silica supports with octyl and
octyl-epoxides groups, to modulate the catalytic properties of the
lipase.
2.3. Lipase activity determination
The activity of the biocatalysts was assayed by following the
increase of absorbance at 348 nm produced by the release of pNP
by the hydrolysis of 0.4 mM pNPB in 25 mM sodium phosphate
buffer at pH 7.0 and 30 ◦C. One international unit of activity (IU)
was defined as the amount of enzyme that hydrolyzes 1 ␮mol of
pNPB per minute under the conditions described above.
2. Materials and methods
2.1. Materials
2.4. Immobilization procedures
Sodium silicate (25–29% SiO2 and 7.5–9.5% Na2O), ethylace-
tate (EtAc), sulfuric acid (98%), cetyltrimethylammonium bromide
(CTAB), sodium bicarbonate, 1,4 dioxane and tert-butanol were
purchased from Merck (Darmstadt, Germany). 4-Nitrophenyl buti-
rate (pNPB), 4-nitrophenol (pNP), glycidyloxypropyltrimethoxysi-
lane (GPTMS 99.7%), epichlorohydrin, 11-mercapto-1-undecanol,
sodium borohydride, dimethyl-3-phenylbutirate and diglyme were
purchased from Sigma–Aldrich (St. Louis, MO, USA). Cyanogen
bromide 4B Sepharose (Seph-CNBr), Octyl Sepharose (Seph-O)
2.4.1. Immobilization of BTL2 on Silica-O and Seph-O supports
The immobilization of BTL2 on Silica-O (Silica-O-BTL2) and
Seph-O (Seph-O-BTL2) was carried out as follows: 1 g of support
was added to 30 mL of lipase solution (0.2 mg protein/mL) in 25 mM
sodium phosphate pH 7 at 25 ◦C under mild stirring. Aliquots from
the suspension and the supernatant were withdrawn at 10 and
30 min to assay their hydrolytic activities and protein contents.
Immobilization proceeded until no activity, or constant activity,
was obtained in the supernatant. The activity was determined as
Please cite this article in press as: N. Guajardo, et al., Asymmetric hydrolysis of dimethyl-3-phenylglutarate in
sequential batch reactor operation catalyzed by immobilized Geobacillus thermocatenulatus lipase, Catal. Today (2015),
http://dx.doi.org/10.1016/j.cattod.2014.12.039

G Model
CATTOD-9422; No. of Pages6
ARTICLE IN PRESS
N. Guajardo et al. / Catalysis Today xxx (2015) xxx–xxx
3
Scheme 1. Asymmetric hydrolysis of dimethyl-3-phenylglutarate catalyzed by immobilized BTL2.
Table 1
described previously and the biocatalyst was collected by filtration
and stored at 4 ◦C.
Immobilization parameters of the BTL2 biocatalysts.
a
b
Biocatalysts
Yp (%)
Ya (%)
Activity expressed (IU/g)
2.4.2. Immobilization of BTL2 on Silica-HE support
The immobilization of BTL2 on Silica-HE (Silica-HE-BTL2) was
carried out in the same manner as Silica-O, but after hydrophobic
adsorption, pH was raised to 9 for 1 h, to allow for covalent attach-
ment. Finally, the biocatalyst was collected by filtration and stored
at 4 ◦C.
Seph-CNBr-BTL2
Seph-O-BTL2
Seph-HG-BTL2
Silica-O-BTL2
Silica-HE-BTL2
31
25
45
28
35
48
80
98
54
89
16
38
56
33
68
2
1
4
1
1
a
Yp = ((P0 − PL)/P0) × 100, where P0 is the offered protein and PL is the amount of
protein in the supernatant.
b
Ya = (A/A0) × 100, where A0 is the offered activity and A is the expressed activity
in the biocatalyst.
2.4.3. Immobilization of BTL2 on Seph-HG support
The immobilization of BTL2 on Seph-HG (Seph-HG-BTL2) sup-
port was carried out as follows: 1 g of support was added to 30 mL
of lipase solution (0.2 mg protein/mL) in 5 mM sodium phosphate
buffer pH 7 at 25 ◦C under mild stirring. Aliquots from suspen-
sion and supernatant were withdrawn at 10 and 30 min to assay
their hydrolytic activities and protein contents. Immobilization
proceeded until no activity, or constant activity, was obtained in
the supernatant. Then, the biocatalyst was filtered and incubated
at pH 10 with 1 M bicarbonate buffer for 1 h to allow for covalent
attachment. Later on, the Schiff bases formed were reduced with
sodium borohydride gently stirring for 20 min at 25 ◦C. Finally, the
biocatalyst was washed with distilled water, filtered under vacuum
and stored at 4 ◦C.
reaction medium consisting in 1 mM of substrate (DMFG) in 25 mM
of phosphate buffer pH 7.
The hydrolysis rate of DMFG was determined by measuring the
product concentration in the linear product vs. time zone.
Conversion and enantiomeric excess (e.e.) of the reaction were
calculated using the following equations:
[MFG]Final
[DMFG]Initial
Conversion (%) =
× 100
× 100
(3)
(4)
ꢀ
ꢁ
[MFG]S − [MFG]R
[MFG]S + [MFG]R
e.e. (%) =
where [MFG]Final is the final concentration of MFG (mM),
[DMFG]Initial is the initial concentration of DMFG (mM), [MFG]S is
the concentration of S-isomer and [MFG]R is the concentration of
R-isomer.
2.4.4. Immobilization of BTL2 on Seph-CNBr support
The immobilization of BTL2 on Seph-CNBr (Seph-CNBr-BTL2)
was carried out as follows: 1 g of support was added to 30 mL of
lipase solution (0.2 mg protein/mL) in 100 mM sodium phosphate
buffer pH 7 at 4 ◦C under mild stirring. Aliquots from suspension
and supernatant were withdrawn at 5 min to assay their hydrolytic
activities and protein contents. Immobilization proceeded until no
activity, or constant activity, was obtained in the supernatant. The
unreacted groups on the support were blocked incubating the bio-
catalyst in 1 M ethanolamine at pH 8 for 2 h. Finally, the biocatalyst
was washed, filtered under vacuum and stored at 4 ◦C.
The determination of e.e. in sequential batch reaction was car-
ried out with the same amount of biocatalyst per volume of reaction
medium in each batch. After each batch, the biocatalyst was filtered
and washed several times with distilled water and 25 mM phos-
phate buffer and then the reactor was reloaded with the washed
biocatalyst and fresh reaction medium for the next batch. To com-
pare the selectivities among biocatalysts, the e.e. was determinate
between 20 and 25% of conversion.
2.6. Inactivation of BTL2 biocatalysts
2.4.5. Immobilization parameters
The inactivation of the biocatalysts was studied under non-
reactive conditions. The biocatalysts were incubated at 25 ◦C
in phosphate buffer at pH 7. At different times, samples were
withdrawn under stirring to have a homogeneous biocatalyst sus-
pension. Inactivation was modeled according to the theory of
enzyme deactivation [36]; the residual activity of the biocatalysts
was measured as described previously, and half-life (period of time
at which the enzyme have lost one half of its initial activity) was
calculated.
Immobilization yields in terms of protein (Yp) and activity (Ya)
were calculated according to Eqs. (1) and (2) respectively:
P0 − PL
Yp
Ya
=
=
× 100
(1)
(2)
P0
A
× 100
A0
where P0 is the offered protein, PL is the amount of protein in the
supernatant, A0 is the offered activity and A is the expressed activity
in the biocatalyst.
3. Results and discussion
2.5. Asymmetric hydrolysis of DMFG
3.1. Lipase immobilization
The asymmetric hydrolysis of DMFG was carried out as follows:
Results of immobilization presented in Table 1 show that the
28 IU (pNPB hydrolysis) of biocatalysts were contacted with 3 mL of
lipase was immobilized in all functionalized supports. Yp ranged
Please cite this article in press as: N. Guajardo, et al., Asymmetric hydrolysis of dimethyl-3-phenylglutarate in
sequential batch reactor operation catalyzed by immobilized Geobacillus thermocatenulatus lipase, Catal. Today (2015),
http://dx.doi.org/10.1016/j.cattod.2014.12.039

G Model
CATTOD-9422; No. of Pages6
ARTICLE IN PRESS
4
N. Guajardo et al. / Catalysis Today xxx (2015) xxx–xxx
Table 2
Selectivity of BTL2 biocatalysts in the asymmetric hydrolysis of DMFG in 25 mM phosphate buffer pH 7 at 25 ◦C, 28 IU (pNPB hydrolysis) of biocatalyst and 3 mL of reaction
volume.
Biocatalysts
Conversion (%)
Reaction rate (␮moleProduct/gbiocatalyst/h)
e.e. (%)a
Seph-CNBr-BTL2
Seph-O-BTL2
Seph-HG-BTL2
Silica-O-BTL2
Silica-HE-BTL2
21
21
20
22
25
1.2 × 10−4
2.8 × 10−4
3.6 × 10−4
1.6 × 10−4
5.0 × 10−5
71
22
82
32
23
a
The e.e. was calculated at 20–25% conversion.
1
0.8
0.6
0.4
0.2
0
0
10
20
30
40
50
60
70
80
Time (days)
Fig. 2. Inactivation kinetics of BTL2 biocatalysts at 25 ◦C. Circles: Seph-CNBr-BTL2,
triangles: Seph-HG-BTL2. Line: monophasic without residual activity inactivation
model.
Fig. 1. Viewing the BTL2 lipase structure. (1) White surface: alpha helix structure,
(2) black surface: lysine residues.
highest value was obtained with Seph-HG-BTL2 (e.e. = 82%). The
stereoselective behavior of Seph-HG-BTL2 can be attributed to the
combination of the functionalization of the support with covalent
and hydrophobic groups affecting the orientation and three dimen-
sional conformation of the enzyme. The low e.e. obtained with silica
supports can be attributed to the characteristic of the material that,
having a greater amount of octyl groups, can lead to increased
lipase hyperactivation, which may have affected the conformation
of the enzyme causing a loss of selectivity [39]. Considering these
results the biocatalyst Seph-HG-BTL2 was selected for the following
experiments.
from 25 to 45% (20 mg of protein/g of support was offered to all
carriers), being the heterofunctional supports the ones that show
higher yields (45 and 35% for Sepharose and silica respectively). In
some cases immobilization yields (Yp, Ya) were low because a high
amount of protein was loaded on the supports in order to obtain
more active biocatalysts for the asymmetric hydrolysis reaction.
The expressed activity shows a similar trend to the protein loading,
as can be seen in Table 1. It can be observed that the expressed
activities of the immobilized BTL2 lipase on Sepharose and silica
heterofunctional supports were higher than in Sepharose and silica
octyl supports. This behavior can be attributed to the presence of
lysine residues on the lid of the enzyme that may increase rigidity
and lead to conformation changes in the lipase, as shown in Fig. 1
[37]
In this case, the heterofunctional silica biocatalysts were more
active than the heterofunctional Sepharose biocatalysts with 68 IU
(pNPB hydrolysis) per gram of support. This behavior can be
attributed to the higher surface area in silica support than in com-
mercial Sepharose. For this reason, it is possible to functionalize
silica with a greater amount of octyl groups achieving a higher
hyperactivation of the lipase or a higher immobilization selectivity
with respect to other proteins in the enzymatic solution [38].
3.3. Stability of BTL2 immobilized on Seph-HG support
In order to determine the stability of the selected biocatalyst,
inactivation kinetics were performed with Seph-HG-BTL2 and com-
pared with Seph-CNBr-BTL2. Seph-CNBr-BTL2 was used to mimic
the behavior of the soluble enzyme, since in this case there is
no rigidification, nor the drawbacks of aggregation or precipita-
tion [40]. We studied the stability of Seph-HG-BTL2 biocatalyst at
25 ◦C in phosphate buffer under non-reactive conditions (Fig. 2).
The Seph-HG-BTL2 biocatalyst was 2.2 times more stable than the
Seph-CNBr-BTL2, half-lives being 86 days and 39 days respectively.
This behavior indicates that the immobilization of BTL2 lipase on
Seph-HG support stabilized the open conformation of the enzyme
by the hydrophobic groups of the support and the multipoint cova-
lent attachment between the glyoxyl groups of the support and
the lysine residues of the lipase increased its rigidity, consequently
increasing its stability. The inactivation kinetics of Seph-CNBr-
BTL2 and Seph-HG-BTL2 were adjusted to monophasic inactivation
kinetics without residual activity.
3.2. Biocatalysts selection
The conversion, reaction rate and the e.e. obtained with the dif-
ferent immobilized BTL2 preparations in the hydrolysis of DMFG
in phosphate buffer at pH 7 and 25 ◦C are shown in Table 2.
The chromatographic analysis showed that the (S)-monomethyl
was produced in greater amount, and the presence of diacid was
not evidenced. Reaction rates were low, with values between
5.0 × 10−5 and 3.6 × 10−4 (␮moleProduct/gbiocatalysts/h). The e.e. for
(S)-monomethyl ester is dependent on changes in the selectiv-
ity of the lipase to be immobilized in the different supports. The
3.4. Temperature selection
With the aim of studying the effect of temperature on the
stereoselectivity of the biocatalyst and the initial reaction rate,
Please cite this article in press as: N. Guajardo, et al., Asymmetric hydrolysis of dimethyl-3-phenylglutarate in
sequential batch reactor operation catalyzed by immobilized Geobacillus thermocatenulatus lipase, Catal. Today (2015),
http://dx.doi.org/10.1016/j.cattod.2014.12.039

G Model
CATTOD-9422; No. of Pages6
ARTICLE IN PRESS
N. Guajardo et al. / Catalysis Today xxx (2015) xxx–xxx
5
Table 3
that may indicate changes in the conformation of the enzyme that
ultimately will be responsible for the decrease in stereoselectivity.
From the point of view of a green process, if the substrate can be
converted almost 100%, the biocatalyst will represent the largest
waste; however, according to the results obtained it can be reused
at least twice without losing stereoselectivity, rising a compro-
mise between loss of stereoselectivity and biocatalyst reuse, an
aspect seldom studied for the reaction of DMFG hydrolysis. Cabrera
and Palomo [42] reported that the stereoselectivity of biocatalysts
obtained with a lipase from Rhizopus oryzae decreased after two
reuse cycles; however, according to the best of our knowledge,
there are no works reported for the sequential batch reactor opera-
tion with BTL2 immobilized on Sepharose supports functionalized
with undecanol-glyoxyl groups.
Effect of temperature on selectivity of Seph-HG-BTL2 and initial reaction rate in the
asymmetric hydrolysis of DMFG with 28 IU (pNPB hydrolysis) of biocatalyst and
3 mL of reaction volume in 25 mM phosphate buffer pH 7.
Parameters
Temperature (◦C)
10
25
80
35
75
e.e. (%)a
90
Reaction rateb (␮moleProduct/gbioc/min)
2 × 10−5
8.7 × 10−4 1.5 × 10−4
a
The e.e. was calculated at 20–25% conversion.
Initial reaction rate.
b
100
80
60
40
20
0
4. Conclusion
Stereoselectivity of lipase from Geobacillus thermocatenulatus
immobilized on mono and heterofunctional supports was studied,
showing that the chemical nature of the support and the lipase ori-
entation significantly influence the e.e. achieved and the possibility
of biocatalyst reuse.
Biocatalyst obtained with the heterofunctional support
Sepharose activated with undecanol-glyoxyl groups exhibited
the best results in terms of stereoselectivity. The asymmetric
hydrolysis of DMFG in sequential batch reactor operation cat-
alyzed by this biocatalyst reached 90% of e.e. for (S)-monomethyl
ester, and its reuse in batch reactors was possible at least twice
without losing stereoselectivity. These results suggest that the
enzyme orientation by hydrophobic adsorption and the multipoint
covalent attachment between lipase and support is very important
for determining the final biocatalyst characteristics, allowing its
reuse and also indicate that the biocatalyst can be used more than
once, which will reduce the costs of the process.
1
2
3
4
5
Batch
Fig. 3. Asymmetric hydrolysis of DMFG in sequential batch reactor operation at
10 ◦C, 25 mM phosphate buffer pH 7, 28 IU (pNPB hydrolysis) of biocatalyst and 3 mL
of reaction volume. The e.e. was calculated at 20–25% conversion.
experiments were conducted at three temperatures (Table 3). The
results show that low temperatures favored the stereoselectivity of
the enzyme. For example, at 10 ◦C the e.e. (%) was 10% higher than
at 25 ◦C and 15% higher than at 35 ◦C; however, the initial reaction
rate at 10 ◦C decreased by 97% compared with 25 ◦C. It has been
reported that a decrease in the reaction temperature can improve
the selectivity of the enzyme, a situation that was also observed
in this work [41]. Although an increase in temperature led to an
increase in the initial reaction rate, e.e. was severely reduced; for
this reason a temperature of 10 ◦C (e.e. = 90%) was selected to carry
out the asymmetric hydrolysis in successive batch reactor opera-
tion. To the best of our knowledge, this is the highest e.e. reported
in aqueous medium [42,43].
Acknowledgments
This work was supported by Fondecyt, Chile (Project 1130981).
Ms. Guajardo thanks Conicyt for her PhD scholarship.
The authors also thank Prof. Andrés Illanes for reviewing the
manuscript.
References
[1] M.T. Reetz, Lipases as practical biocatalysis, Curr. Opin. Chem. Biol. 6 (2002)
145–150.
[2] A. Maraitea, P. Hoyos, J.D. Carballeira, C.A. Cabrera, M.B. Ansorge-Schumacher,
A.R. Alcántara, Lipase from Pseudomonas stutzeri: purification, homology mod-
elling and rational explanation of the substrate binding mode, J. Mol. Catal. B:
Enzym. 87 (2013) 88–98.
3.5. Sequential batch reactor operation
[3] S.L. Cherukuvada, A.S. Seshasayee, K. Raghunathan, S. Anishetty, G. Pennathur,
Evidence of a double-lid movement in Pseudomonas aeruginosa lipase: insights
from molecular dynamics simulations, PLoS Comput. Biol. 1 (2005) 182–189.
[4] F. Frikha, M. Ladjimi, Y. Gargouri, N. Miled, 3-D structure modelling of the
Staphylococcus simulans lipase: conformational changes, substrate specificity
and novel structural features, FEMS Microbiol. Lett. 286 (2008) 207–221.
[5] R. Meier, T. Drepper, V. Svensson, K. Jaeger, U. Baumann, A calcium-gated lid
and a large-roll sandwich are revealed by the crystal structure of extracellular
lipase from Serratia marcescens, J. Biol. Chem. 282 (2007) 31477–31483.
[6] C. Angkawidjaja, H. Matsumura, Y. Koga, K. Takano, S. Kanaya, X-ray crystallo-
graphic and MD simulation studies on the mechanism of interfacial activation
of a family I.3 lipase with two Lids, J. Mol. Biol. 400 (2010) 82–95.
[7] Y. Wang, D.Q. Wei, J.F. Wang, Molecular dynamics studies on T1 lipase: insight
into a double-flap mechanism, J. Chem. Inf. Model. 50 (2010) 875–878.
[8] C. Carrasco-López, C. Godoy, B. De las Rivas, G. Fernández-Lorente, J.M. Palomo,
J.M. Guisán, R. Fernández-Lafuente, M. Martínez-Ripoll, J. Hermoso, Activa-
tion of bacterial thermoalkalophilic lipases is spurred by dramatic structural
rearrangements, J. Biol. Chem. 289 (2009) 4365–4372.
Fig. 3 shows the e.e. of the product obtained in the asymmet-
ric hydrolysis of DMFG in sequential batch reactor operation. The
results show that e.e. decreased 21% from the first to the last batch,
indicating that the stereoselectivity of the lipase is affected. As
shown in Table 4, although a decrease batch after batch in initial
reaction rate was not observed, small variations were produced
Table 4
Initial reaction rate of the asymmetric hydrolysis of DMFG in sequential batch
reactor operation at 10 ◦C, 25 mM phosphate buffer pH 7, 28 IU (pNPB hydrolysis)
biocatalyst and 3 mL of reaction volume.
Batch of reaction
Reaction rate (␮moleProduct/gbiocatalysts/h)
1
2
3
4
5
0.0035
0.0056
0.0052
0.0055
0.0049
[9] C. Godoy, O. Romero, B. de las Rivas, C. Mateo, G. Fernandez-Lorente, J. Guisan,
J. Palomo, Changes on enantioselectivity of a genetically modified thermophilic
lipase by site-directed oriented immobilization, J. Mol. Catal. B: Enzym. 87
(2013) 121–127.
Please cite this article in press as: N. Guajardo, et al., Asymmetric hydrolysis of dimethyl-3-phenylglutarate in
sequential batch reactor operation catalyzed by immobilized Geobacillus thermocatenulatus lipase, Catal. Today (2015),
http://dx.doi.org/10.1016/j.cattod.2014.12.039

G Model
CATTOD-9422; No. of Pages6
ARTICLE IN PRESS
6
N. Guajardo et al. / Catalysis Today xxx (2015) xxx–xxx
[10] M. Guncheva, D. Zhiryakova, Catalytic properties and potential applications of
Bacillus lipases, J. Mol. Catal. B: Enzym. 68 (2011) 1–21.
[11] F.J. Contesini, D.B. Lopez, G.A. MacEdo, M.D.G. Nascimento, P.D.O. Carvalho,
Asperguillus sp. lipase: potential biocatalyst for industrial use, J. Mol. Catal. B:
Enzym. 67 (2010) 163–171.
[27] C. Bernal, P. Urrutia, A. Illanes, L. Wilson, Hierarchical meso-macroporous sil-
ica grafted with glyoxyl groups opportunities for covalent immobilization of
enzymes, New Biotechnol. 30 (2013) 1–7.
[28] M.S. Yu, I. Lantos, Z.Q. Peng, J. Lu, T. Cacchio, Asymmetric synthesis of (−)-
paroxetine using PLE hydrolysis, Tetrahedron 41 (2000) 5647–5651.
[29] T.L. Lee, P.C. Hong, H.L. Huang, S.F. Chen, C.L. Wang, Y.S. Wen, Asymmetric syn-
theses of trans-3,4-disubstituted 2-piperidinones and piperidines, Tetrahedron
Asymmetry 12 (2001) 419–426.
[30] Y. Chen, A. Zaks, S. Chackalamannil, S. Dugar, Asymmetric synthesis of sub-
stituted 2-azaspiro-3.5-nonan-1-ones: an enantioselective synthesis of the
cholesterol absorption inhibitor (+)-SCH 54016, J. Org. Chem. 61 (1996)
8341–8343.
[12] N. Miled, F. Beisson, J. De Caro, A. De Caro, V. Arondel, R. Verger, Interfacial
catalysis by lipases, J. Mol. Catal. B: Enzym. 11 (2001) 165–171.
[13] F. Secundo, G. Carrea, C. Tarabiono, P. Gatti-Lafranconi, S. Brocca, M. Lotti, K.
Jaeger, M. Puls, T. Eggert, The lid as a structural and functional determinant of
lipase activity and selectivity, J. Mol. Catal. B: Enzym. 39 (2006) 166–170.
[14] J.M. Palomo, R.L. Segura, C. Mateo, M. Terreni, J.M. Guisán, R. Fernandez-
Lafuente, Synthesis of enantiomerically pure glycidol via a fully enantioselec-
tive lipase-catalyzed resolution, Tetrahedron Asymmetry 16 (2005) 869–874.
[15] J.M. Palomo, G. Fernández-Lorente, C. Mateo, M. Fuentes, R. Fernández-
Lafuente, J.M. Guisan, Modulation of the enantioselectivity of Candida antarctica
B lipase via conformational engineering: kinetic resolution of ( )-␣-hydroxy-
phenylacetic acid derivatives, Tetrahedron Asymmetry 13 (2002) 1337–1345.
[16] E. Katchalski-Katzir, Immobilized enzymes – learning from past successes and
failures, Trends Biotechnol. 11 (1993) 471–478.
[31] N. Kucharczyk, Section review-central
& peripheral nervous systems:
tachykinin antagonists in development, Exp. Opin. Invest. Drugs. 4 (1995)
299–311.
[32] Z. Cabrera, J.M. Palomo, G. Fernandez-Lorente, R. Fernandez-Lafuente, J.M.
Guisan, Partial and enantioselective hydrolysis of diethyl phenylmalonate by
immobilized preparations of lipase from Thermomyces lanuginose, Enzyme
Microb. Technol. 40 (2007) 1280–1285.
[17] A. Bastida, P. Sabuquillo, P. Armisen, R. Fernández-Lafuente, J. Huguet, J. Guisán,
A single step purification, immobilization and hyperactivation of lipases via
interfacial adsorption on strongly hydrophobic supports, Biotechnol. Bioeng.
58 (1998) 486–493.
[18] G. Fernandez-Lorente, Z. Cabrera, C. Godoy, R. Fernandez-Lafuente, J.M. Palomo,
J.M. Guisan, Interfacially activated lipases against hydrophobic supports: effect
of the support nature on the biocatalytic properties, Process Biochem. 43 (2008)
1061–1067.
[19] J. Palomo, G. Mun˜oz, G. Fernández-Lorente, C. Mateo, R. Fernández-Lafuente,
J.M. Guisan, Interfacial adsorption of lipases on very hydrophobic support
(octadecyl-Sepabeads): immobilization, hyperactivation and stabilization of
the open form of lipases, J. Mol. Catal. B: Enzym. 19–20 (2002) 279–286.
[20] O. Barbosa, R. Torres, C. Ortiz, A. Berenguer-Murcia, R.C. Rodrigues, R.
Fernandez-Lafuente, Heterofunctional supports in enzyme immobilization:
from traditional immobilization protocols to opportunities in tuning enzyme
properties, Biomacromolecules 14 (2013) 2433–2462.
[21] C. Bernal, A. Illanes, L. Wilson, Heterofunctional hydrophilic-hydrophobic
porous silica as support for multipoint covalent immobilization of lipases:
application to palmitate synthesis, Langmuir 30 (2014) 3557–3566.
[22] J. Zhao, Y. Wang, L. Guangsheng, S. Zhu, Immobilization of penicillin G acylase
on macro-mesoporous silica spheres, Bioresour. Technol. 102 (2011) 529–535.
[23] C. Bernal, M. Mesa, L. Sierra, Improvement of thermal stability of ␤-
galactosidase from Bacillus circulans by multipoint covalent immobilization
in hierarchical macro–mesoporous silica, J. Mol. Catal. B: Enzym. 84 (2012)
166–172.
[24] M. Shakeri, K. Kawakami, Enhancement of Rhizopus oryzae lipase activity immo-
bilized on alkyl-functionalized spherical mesocellular foam: influence of alkyl
chain length, Microporous Mesoporous Mater. 118 (2009) 115–120.
[25] G. Fernandez-Lorente, J.M. Palomo, Z. Cabrera, J.M. Guisan, R. Fernandez-
Lafuente, Specificity enhancement towards hydrophobic substrates by
immobilization of lipases by interfacial activation on hydrophobic supports,
Enzyme Microb. Technol. 41 (2007) 565–569.
[26] J.M. Bolivar, C. Mateo, C. Godoy, B.C. Pessela, D.S. Rodrigues, R.L. Giordano,
R. Fernández-Lafuente, J.M. Guisan, The co-operative effect of physical and
covalent protein adsorption on heterofunctional supports, Process Biochem.
44 (2009) 757–763.
[33] A. Fryszkowska, M. Komar, D. Koszelewskib, R. Ostaszewskia, Studies on
enzymatic synthesis of chiral non-racemic 3-arylglutaric acid monoesters,
Tetrahedron Asymmetry 17 (2006) 961–966.
[34] M. Homman, R. Vail, B. Morgan, V. Sabesan, C. Levy, D. Dodds, A. Zaks, Enzymatic
hydrolysis of a prochiral 3-subtituted glutarate ester, an intermediate in the
synthesis of an Nk1/Nk2 dual antagonist, Adv. Synth. Catal. 343 (2001) 744–749.
[35] C. Mateo, J. Bolívar, C. Godoy, J. Rocha-Martín, B. Pessela, J. Curiel, R.
Mun˜oz, J.M. Guisan, G. Fernandez-Lorente, Improvement of enzyme properties
with a two-step immobilizaton process on novel heterofunctional supports,
Biomacromolecules 11 (2010) 3112–3117.
[36] J.P. Henley, A. Sadana, Deactivation theory, Biotechnol. Bioeng. 28 (1986)
1277–1285.
[37] C.A. Godoy, B. de las Rivas, M. José, Guisán, Site-directing an intense multipoint
covalent attachment (MCA) of mutants of the Geobacillus thermocatenulatus
lipase 2 (BTL2): genetic and chemical amination plus immobilization on a
tailor-made support, Process Biochem. 49 (2014) 1324–1331.
[38] K. Wannerberger, S. Welin-Klintstro, T. Arnebrant, Activity and adsorption of
lipase from Humicola lanuginosa on surfaces with different wettabilities, Lang-
muir 13 (1997) 784–790.
[39] C. García-Galán, A. Berenguer-Murcia, R. Fernández-Lafuente, R.C. Rodrigues,
Potential of different enzyme immobilization strategies to improve enzyme
performance, Adv. Synth. Catal. 353 (2011) 2885–2904.
[40] C. Mateo, O. Abian, M. Bernedo, E. Cuenca, E. Fuentes, G. Fernandez-Lorente,
J.M. Palomo, V. Grazu, B.C.C. Pessela, C. Giacomini, G. Irazoqui, A. Villarino, K.
Ovsejevi, F. Batista-Viera, R. Fernandez-Lafuente, J.M. Guisán, Some special fea-
tures of glyoxyl supports to immobilize proteins, Enzyme Microb. Technol. 37
(2005) 456–462.
[41] P. Overbeeke, C. Govardhan, N. Khalaf, J. Jongejan, J. Heijnen, Influence of lid
conformation on lipase enantioselectivity, J. Mol. Catal. B: Enzym. 10 (2000)
385–393.
[42] Z. Cabrera, J.M. Palomo, Enantioselective desymmetrization of prochiral
diesters catalyzed by immobilized Rhizopus oryzae lipase, Tetrahedron Asym-
metry 22 (2011) 2080–2084.
[43] W. Liu, Y. HU, L. Jiang, B. Zou, H. Huang, Synthesis of methyl (R)-3-(4-
fluorophenyl) glutarate via enzymatic desymmetrization of a prochiral diester,
Process Biochem. 47 (2012) 1037–1041.
Please cite this article in press as: N. Guajardo, et al., Asymmetric hydrolysis of dimethyl-3-phenylglutarate in
sequential batch reactor operation catalyzed by immobilized Geobacillus thermocatenulatus lipase, Catal. Today (2015),
http://dx.doi.org/10.1016/j.cattod.2014.12.039