Influence of dlutaraldehyde cross-linking modes on the recyclability of immobilized lipase b from candida antarctica for transesterification of soy bean oil

Article abstract of DOI:10.3390/molecules23092230

Lipase B from Candida antarctica (CAL-B) is largely employed as a biocatalyst for hydrolysis, esterification, and transesterification reactions. CAL-B is a good model enzyme to study factors affecting the enzymatic structure, activity and/or stability after an immobilization process. In this study, we analyzed the immobilization of CAL-B enzyme on different magnetic nanoparticles, synthesized by the coprecipitation method inside inverse micelles made of zwitterionic surfactants, with distinct carbon chain length: 4 (ImS4), 10 (ImS10) and 18 (ImS18) carbons. Magnetic nanoparticles ImS4 and ImS10 were shown to cross-link to CAL-B enzyme via a Michael-type addition, whereas particles with ImS18 were bond via pyridine formation after glutaraldehyde cross-coupling. Interestingly, the Michael-type cross-linking generated less stable immobilized CAL-B, revealing the influence of a cross-linking mode on the resulting biocatalyst behavior. Curiously, a direct correlation between nanoparticle agglomerate sizes and CAL-B enzyme reuse stability was observed. Moreover, free CAL-B enzyme was not able to catalyze transesterification due to the high methanol concentration; however, the immobilized CAL-B enzyme reached yields from 79.7 to 90% at the same conditions. In addition, the transesterification of lipids isolated from oleaginous yeasts achieved 89% yield, which confirmed the potential of immobilized CAL-B enzyme in microbial production of biodiesel.

Full text of DOI:10.3390/molecules23092230

molecules
Article
Influence of Glutaraldehyde Cross-Linking Modes on
the Recyclability of Immobilized Lipase B from
Candida antarctica for Transesterification of Soy
Bean Oil
Iago A. Modenez 1, Diego E. Sastre 2, Fernando C. Moraes 3 and Caterina G. C. Marques Netto 1,*
ID
1
Laboratório de Metaloenzimas e Biomiméticos, Departamento de Química, UFSCar,
São Carlos-SP 13565-905, Brazil; iago_modenez@hotmail.com
Departamento de Física e Ciências Interdisciplinares, Instituto de Física, Universidade de São Paulo,
2
São Carlos-SP 13563-120, Brazil; diegosastre82@gmail.com
Laboratório de Analítica, Bioanalítica, Biossensores, Eletroanalítica e Sensores, Departamento de Química,
3
UFSCar, São Carlos-SP 13565-905, Brazil; fcmoraes@ufscar.br
*
Correspondence: caterina@ufscar.br; Tel.: +55-16-3306-6653
Academic Editors: Frank Hollmann, Selin Kara and Caroline E. Paul
Received: 3 August 2018; Accepted: 29 August 2018; Published: 2 September 2018
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Lipase B from Candida antarctica (CAL-B) is largely employed as a biocatalyst for hydrolysis,
esterification, and transesterification reactions. CAL-B is a good model enzyme to study factors
affecting the enzymatic structure, activity and/or stability after an immobilization process. In this study,
we analyzed the immobilization of CAL-B enzyme on different magnetic nanoparticles, synthesized by the
coprecipitation method inside inverse micelles made of zwitterionic surfactants, with distinct carbon chain
length: 4 (ImS4), 10 (ImS10) and 18 (ImS18) carbons. Magnetic nanoparticles ImS4 and ImS10 were shown
to cross-link to CAL-B enzyme via a Michael-type addition, whereas particles with ImS18 were bond
via pyridine formation after glutaraldehyde cross-coupling. Interestingly, the Michael-type cross-linking
generated less stable immobilized CAL-B, revealing the influence of a cross-linking mode on the
resulting biocatalyst behavior. Curiously, a direct correlation between nanoparticle agglomerate sizes
and CAL-B enzyme reuse stability was observed. Moreover, free CAL-B enzyme was not able to catalyze
transesterification due to the high methanol concentration; however, the immobilized CAL-B enzyme
reached yields from 79.7 to 90% at the same conditions. In addition, the transesterification of lipids
isolated from oleaginous yeasts achieved 89% yield, which confirmed the potential of immobilized CAL-B
enzyme in microbial production of biodiesel.
Keywords: lipase; enzyme immobilization; magnetic nanoparticles; cross-linking types;
biodiesel synthesis
1. Introduction
Enzyme immobilization is largely known as an empiric technique to obtain more stable and
active enzymes [
and immobilization methodology will lead to different outcomes [
literature in this area [ ]. Among the several available supports for enzyme immobilization,
1]. It is known that few modifications on the enzyme structure, types of supports
2,3], which explains the vast
4,5
magnetic nanoparticles made of magnetite (Fe3O4) are particularly interesting due to the large global
surface area, high mobility and a strong magnetization effect, which allows them to be recovered and
recycled by applying an external magnetic field [
nanoparticles are randomly oriented at room temperature, but rapidly align their magnetic moments
under the influence of a magnetic field, leading to a strong magnetization and no hysteresis [ ].
6,7]. The single magnetic domains from magnetite
8
Molecules 2018, 23, 2230; doi:10.3390/molecules23092230
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2230
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Moreover, the magnetic field has been described to enhance enzymatic activities and stabilities and
its use is still very limited [ ]. Moreover, despite of the studies describing enzyme immobilization on
9
magnetic nanoparticles, little is known on how the enzyme structure is affected when it is immobilized
on different support types and sizes, and even on how the cross-linking agents affect the binding
between enzyme and support [10], modifying the enzymatic catalysis.
Lipases are well-known versatile enzymes, with numerous uses in biotechnology, organic
synthesis, medicinal chemistry, in the food industry, and for biofuel synthesis. A classic example of a
stable and highly employed enzyme is lipase B from Candida antarctica (CAL-B), which turns CAL-B
into a highly desirable enzyme to study the influence of an immobilization protocol on the enzyme
activity and stability. The CAL-B enzyme has also the advantage of being able to perform a wide variety
of reactions, such as hydrolysis, transesterification, and aldol reactions. This enzyme has also been
used for biofuel synthesis, but since CAL-B has the drawback of being inactivated by methanol due to
the replacement of water molecules to alcohol in the protein surface [11], immobilization protocols
were developed to improve its stability in presence of methanol [12,13]. However, the enzyme stability
was not evaluated against protocol changes, since subtle variations on the immobilization methods
revealed distinct susceptibility towards methanol [13].
Surface activation is normally required for enzyme immobilization. For instance, magnetite
nanoparticles suffer a silanization process carried out by (3-aminopropyl)triethoxylsilane (APTES)
coupling agent [14]. The silanization procedure enables the addition of functional groups on the particle
surface and protects its magnetic core from oxidation processes due to the contact of iron (2+) to the
atmospheric oxygen [15]. Additionally, silanization facilitates the cross-linking between support and
enzyme, enabling an easy and direct covalent immobilization. Undoubtedly one of the most applied
cross-linking agents is glutaraldehyde [16], but its relatively simple chemical structure can be deceiving,
since in aqueous solution it possess several monomeric and polymeric forms in equilibrium [17]. In the
protein immobilization context, glutaraldehyde can react with several functional groups such as amine,
thiol, imidazole and a variety of amino acids (e.g., lysine, arginine, cysteine and serine), despite of that,
it is well known that this reaction tends to occur, mainly, in the
ε-amino group of lysyl residues due
to its higher nucleophilicity [18]. The -amino group nucleophilic attack towards the glutaraldehyde
ε
molecule, usually, leads to the formation of Schiff bases. However, the linkage of glutaraldehyde with
amino groups showed exceptional stability at extreme pHs and temperatures [10], which does not
match to the Schiff base properties [17]. Hence, several alternative mechanisms had been proposed for
glutaraldehyde crosslinking, such as a Michael-type addition involving the conjugate addition of the
protein amino groups to the α,β-unsaturated oligomers ethylenic double bonds of glutaraldehyde [19].
A slightly different mechanism involved the formation of quaternary pyridinium compounds due to
its dimerization in the presence of amino groups [20]. Despite of all reported mechanisms, the reaction
of glutaraldehyde with proteins is still under debate.
Recently, Yi et al. described the immobilization of a lipase from Candida rugosa on the surface of
magnetic nanoparticles inside reverse micelles, and showed an enhanced activity and stability when
compared to a nonmicellar immobilization [21]. Therefore, we attempt to analyze the influence of
different reverse micelle–made magnetic nanoparticles and glutaraldehyde cross-linking on the activity
and stability of CAL-B for oil transesterification reaction in methanol (Scheme 1). Hence, we report the
immobilization of this enzyme on three different magnetic nanoparticles synthesized inside reverse
micelles by the coprecipitation method and evaluated their transesterification potential of soy bean
oil and lipid extracts from oleaginous fungus. Despite no direct correlation of the surfactant size was
observed to influence CAL-B activity/stability, it was found a correlation between the nanoparticle
agglomerate sizes and the enzymatic stability. Moreover, the glutaraldehyde cross-linking mode
influenced the recyclability of immobilized CAL-B, in which Michael-type addition resulted in less
stable enzymes.

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Scheme 1. Immobilized CAL-B catalyzed transesterification reaction of triacylglycerol from soy bean
oil in methanol to obtain fatty acid methyl ester (FAME also called “biodiesel”).
2. Results and Discussion
2.1. Characterization of the Magnetic Nanoparticles
Three different magnetic nanoparticles were synthesized (NP-ImS4, NP-ImS10 and NP-ImS18)
by using inverse micelles, based on Souza et al. protocol [22] in a chloroform/water suspension,
containing zwitterionic surfactants with carbon chains of four, ten and eighteen carbons (Figure 1A,
Figures S1–S3).
Figure 1. (A) Scheme of NP-ImSn synthesis using reverse micelles made of zwitterionic surfactants
ImSn (n = 4, 10 and 18); (B) Typical sizes obtained for the particles using different analyses.
X-ray powder diffraction (XRD) analysis of all synthesized nanoparticles (Figures S4–S6) indicated
that the magnetic core was composed of small grains of magnetite with low crystallinity, as was also
observed by Salviano et al. [23]. The nanoparticles crystal sizes were determined by using Scherrer equation
(Equation (1)), in which B is the peak width; K is the Scherrer constant (0.94 for spherical crystals);
λ is the
X-ray wavelength and L is the crystallite size. Hence, by XRD analysis, the NP-ImS4 size was approximately
of 3.68 nm, whereas for NP-ImS18, the particle size was approximately 6.52 nm. NP-ImS10 could not have
their size determined, due to their apparent low crystallinity.
Kλ
L cosθ
B (2θ ) =
(1)

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Due to resolution issues, scanning electron microscopy (SEM) of these nanoparticles only revealed
the mean size of NP-ImS18 particles (9.6 nm), since NP-ImS4 and NP-ImS10 were smaller than
NP-ImS18 (Figures S7–S9). However, the dynamic light scattering (DLS) measurements of the silanized
particles at pH 7 indicated a uniform nanoparticle agglomeration since NP-ImS4, NP-ImS10 and
NP-ImS18 (Figures S10–S12) showed a mean diameter of 54, 37, and 80 nm, respectively. In fact,
the agglomerated nanoparticles (corresponding to 8–12 units in the case of NP-ImS18) were used
for the immobilization protocol (Figure 1B). The agglomeration of nanoparticles might be due to
different concentra−ti1ons used for each analyses, since for SEM a 0.5 mg
·
L−1 was employed, whereas a
concentration was used for DLS analysis [24]. Following this premise, if a much
suspension of 1g
·
L
higher concentration (10g
(Figure S13).
·
L
−1) is employed for DLS analysis, micron-sized agglomerates are observed
In addition, infrared analysis of the magnetic nanoparticles after silanization indicated the
presence of residual zwitterionic surfactant on the particles surface even after several washing
procedures (Figure S14). Microanalysis assays revealed a 4%, 0.6% and 1.5% of surfactant impurity on
the surface of NP-ImS4, NP-ImS10 and NP-ImS18 particles, respectively and it was already shown that
the presence of detergents might increase lipases activity upon immobilization procedures [25].
2.2. Improvement of CAL-B Immobilization Protocol
The immobilization protocol was optimized by finding the best enzyme/nanoparticles ratio,
glutaraldehyde concentration and immobilization time. The optimization was based on the yield
1
of soy bean oil transesterification reaction, Scheme 1, which was verified by H-NMR analyses.
For instance, the areas corresponding to the singlet of methoxyl groups from the methyl esters at
3.7 ppm (A1) (Figure S14) and the triplet of the carbonyl methylene groups at 2.3 ppm (A2) (Figure S15)
were correlated using Equation (2).
2A1
Y (%) = 100.
(2)
3A2
Firstly, the amount of magnetic nanoparticles used in the immobilization protocol was evaluated
by keeping constant the lipase (10 mg
mL−1) and glutaraldehyde (0.028 mol −1) concentrations,
·
·
L
as well as the immobilization period of 60 min. As observed in Figure 2B, higher concentrations
of nanoparticles naturally lead to a higher lipase loading, which never achieved 100%, possibly be
due to the quantification method used (Bradford method). Classical Bradford method for CAL-B
quantification can mistakenly minimize the amount of immobilized CAL-B up to 10 times, as was
observed by Nicolás et al. [26]. Hence, possibly, at 10 mg of magnetic nanoparticles used, 100%
of CAL-B was already immobilized, which was associated to higher reaction yields (Figure 2A).
The increase in yield upon increasing amounts of support reached a maximum at 10 mg of magnetic
support, which is probably due to steric hindering effects on the immobilized enzyme.

Molecules 2018, 23, 2230
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Figure 2. Analysis of the parameters evaluated in the immobilization protocol. (
of nanoparticles used; ( ) Load of immobilized CAL-B on the amount of magnetic nanoparticles used;
) Effect of the glutaraldehyde concentration used in the reaction yield (left axis) and in CAL-B loading
(right axis- dotted lines) and ( ) effect of the immobilization time. NP-ImS4/CAL-B (black line, ),
A) Effect of the amount
B
(C
D
ꢀ
NP-ImS10/CAL-B (red line, #) and NP-ImS18/CAL-B (blue line, 4).
Different concentrations of the cross-linker were also evaluated (Figure 2C), keeping the
lipase concentration (10 mg
mL−1) and immobilization time (60 min) constants and using the best
·
nanoparticle amount previously determined (10 mg). The reaction yield was enhanced up to a
maximum of 76% for NP-ImS4/CAL-B when 0.083 mol. L−1 of glutaraldehyde was used, whereas
particles NP-ImS10/CAL-B and NP-ImS18/CAL-B had their maximum conversions of 75 and 66%,
respectively, at lower concentrations of glutaraldehyde (0.055 mol. L−1). It was seen that excess
of glutraldehyde led to the diminishment of CAL-B immobilization, possibly due to intra-CAL-B
crosslinking in detriment of immobilization (Figure 2C, dotted lines). However, it was described that
high concentrations of cross-linking agent increased the enzyme rigidity, which might have influenced
in the active site mobility, leading to a decrease in the reaction yield [27]. Moreover, excessive
glutaraldehyde concentrations might affect the tridimensional structure of the enzyme [28].
The immobilization period was also evaluated due to its direct influence on the lipase loading
and possibly on the enzyme conformation upon immobilization, since different researchers related a
maximum activity in a specific period of time with decrease of the enzyme activity after this period
eg [29–34]. Some of the reasons for such behavior are possibly: the change of enzyme conformation
and denaturation [29], the formation of new bonds between enzyme and support [35], mass transfer
problems [36], extensive aggregation of the support [37] or due to steric hindrance against immobilized
enzymes [38]. Hence, the amelioration of immobilization time was obtained employing 10 mg of magnetic
nanoparticles and 0.083 mol. L−1 of glutaraldehyde for NP-ImS4 and 0.055 mol −1 of glutaraldehyde
L
·
for NP-ImS10 and NPImS18. In Figure 2D it is seen that the immobilization period has a deep influence
on the transesterification yield of all the studied systems. Naturally, longer periods of immobilization

Molecules 2018, 23, 2230
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results in higher lipase loading on the nanoparticles, in which 180 min was the optimum immobilization
time, achieving 79.7, 90 and 83% yield for NP-ImS4/CAL-B, NP-ImS10/CAL-B and NP-ImS18/CAL-B,
respectively. Longer immobilization times resulted in the stabilization of the value of transesterification
yield (Figure 2D).
2.3. Immobilized CAL-B Characterization
After achieving the best immobilization protocol for each of the supports, an infrared analysis
was performed in order to provide some insights into CAL-B structure. Interestingly, NP-ImS4/CAL-B
and NP-ImS10/CAL-B presented bands at 1715 cm−1 in the infrared spectra (Figure 3), which is the
region for carbonyl stretching [39], whereas NP-ImS18/CAL-B presented strong bands at 1560 and
1494 cm−1 instead, which could be from heterocyclic ring stretching modes [40]. Even though the
carbonyl stretching region might include aldehyde, acids, amides, esters, and ketones, it should
be noted that Monsan et al. observed a similar band when cross-linked lysine to α,β-unsaturaded
polyglutaraldehyde, via a Michael-type addition. In their study, control reactions using crotonaldehyde
and lysine also presented a band a 1720 cm−1, corroborating to their hypothesis. Moreover, they
observed similar stabilities for the glutaraldehyde–lysine bond formed both at pH 7 and 10.3, which
would result from the same cross-linking modes formation [41]. Hence, based on this previous study,
we attribute the band at 1715 cm−1 (Figure 3) to an aldehyde moiety of the cross-link.
It should be noted that NP-ImS10/CAL-B also present the bands at 1560 and 1494 cm−1 to some
extent, indicating that it is possibly an intermediate between NP-ImS4/CAL-B and NP-ImS18/CAL-B
systems. Moreover, although it is not possible to exclude the formation of an imine bond upon cross-linking,
due to the presence of a band at 1632 cm−1 (Figure 3) before CAL-B immobilization, it is possible to infer
that other glutaraldehyde cross-linking structures were also present in the NP/CAL-B systems.
As previously stated, glutaraldehyde has several equilibrium structures in solution, which might
interfere on the cross-linking mode for enzyme immobilization [42]. For example, the formation
of a quaternary pyridinium moiety has been described when approximately 3000 equivalents of
glutaraldehyde were added to an ovalbumin solution at pH 4.2 [20]. Even though a neutral pH and
30 times less excess of glutaraldehyde were employed in our experiments, in order to analyze if
the pH is relevant to the cross-linking mode, we performed CAL-B immobilization on NP-ImS18 at
three different pHs (4, 7 and 11) followed by FTIR assays (Figure S17). We observed that at pH 4 as
well as pH 7, two bands centered at 1560 cm−1 and 1413 cm−1 were evident, probably from the
formation of a pyridinium bond, whereas the aldehyde band (1715 cm−1) was more pronounced at pH
11 than at neutral pH. Hence, noticing that our immobilization conditions are different from the one
described to form pyridinium, possibly, the zwitterionic surfactants might affect on the local pH of the
nanoparticles as was previously described [43,44], in which ImS18 would have a larger impact than
ImS10 and ImS4. The local pH change would influence on the formation of a pyridinium/hetorocyclic
moiety, and as a proof of concept, we performed CAL-B immobilization on NP-ImS4 in the presence
of surfactant ImS18 as a contaminant and surprisingly, the band at 1715 cm−1 was not observed
(Figure S18), indicating a possible effect of ImS18 on the local pH of the nanoparticle surface. On the
other hand, a Michael-type addition [19] was achieved when amine was in a concentration equal
to or higher than 2:1 per glutaraldehyde [45]. Moreover, an arginine-lysine cyclic structure was
observed when 0.5 mM glutaraldehyde was employed at pH 7.5 [46–48]. When considering these
facts, NP-ImS4/CAL-B and NP-ImS10/CAL-B systems are possibly cross-linked through a Michael
addition, whereas NP-ImS18/CAL-B is possibly cross-linked through the formation of a pyridine
ring. The presence of an aldehydic band was also observed in other glutaraldehyde-lipase studies
after surface activation [49], and in our FTIR spectra, this band could also be due to the presence
of unreacted aldehydic groups. Although, we believe it would be more plausible to come from the
cross-linking mode, since the immobilization took long periods to reach completion (up to 2 h) and
that glutaraldehyde cross-linking normally occur rapidly under standard conditions [50].

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It should be noted that of C–N–H in amides, scissoring/bending of N-H in amines/imines and
stretching of C=C in arenes are observed around 1480–1550 cm−1, however, NP-ImS10/CAL-B and
NP-ImS18/CAL-B systems present other bands that might also be related to pyridine formation,
such as the ones located at 3200–3000 cm−1 (Figure 3B, region I), 895 cm−1, 794 cm−1 and
689 cm−1
[51] (Figure 3B, region II), again confirming that NP-ImS10/CAL-B system has similar
bands to NP-ImS4/CAL-B and NP-ImS18/CAL-B systems.
Figure 3. Infrared spectra of free CAL-B (black line), NP-ImS4/CAL-B (red line), NP-ImS10/CAL-B
−1 −1
(blue line), NP-ImS18/CAL-B (green line). (A) Region from 4000 cm to 400 cm and (B) region from
−1
−1
.
1850 cm to 400 cm

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As a matter of fact, our immobilization protocol initiates with the activation of the surface of the
magnetic nanoparticles with glutaraldehyde prior to CAL-B addition. Considering that CAL-B has
a higher concentration of amine groups at the opposite direction from the active site (Figure 4) [50],
we assume that the immobilization took place at this region, letting the active site far away from steric
hindrances from the particle. Interestingly, this region is rich on lysines and arginines (Figure 3), two of
the most reactive aminoacids towards aldehyde [47,52].
Figure 4. Surface representation of the crystal structure of CAL-B enzyme from Candida antartica (PDB
ID: 4K6G) showing the location of the exposed lysines (in blue)/arginines (in orange) residues and the
catalytic site (in red).
In order to verify the effects of the immobilization on CAL-B transesterification reaction,
a comparison based on the energy and entropy of activation was performed.◦Free CAL-B enzy◦me
◦
showed very low rate constants for each of the reactions temperatures (27 C, 32 C and 37 C)
and consequently, the reaction energy of activation (Ea) was the highest among all the samples
(29.4 kJ
mol−1). These results implicate on the inactive behavior of CAL-B free enzyme on the reaction
·
conditions, with yields around 2% (Table 1). However, an increase up to 45 times on the reaction
yield was observed upon CAL-B immobilization, which was accompanied to an increase in the
reaction constant and on the decrease on the activation energy. It is important to notice that free
CAL-B in presence of ImS10 surfactant did not present any activation behavior (reaction yield: 1.5%,
Table 1), indicating that CAL-B activation was due to the immobilization. The observed increase on the
activation entropy of immobilized CAL-B might be related to a more mobile enzyme on the transition
state, which seemed to aid on the enhancement of the reaction yield.
Table 1. Thermodynamic and kinetic parameters of the activation energy and entropy associated to the
transesterification reaction yield for free and immobilized CAL-B.
−1
−1
)
a
Yield (%) b
=
−1
)
Biocatalyst
Ea (kJ mol
)
k (µMs
∆S (J mol
Free CAL-B
29.4
25.6
18.2
27.8
−73
−138
−142
−145
0.048
2/1.5 c
79.7
90
NP-ImS4/CAL-B
NP-ImS10/CAL-B
NP-ImS18/CAL-B
33.2
21.7
14.0
83
a- Determined at 32 ◦C; b- determined at at 32 ◦C and 24 hours.; c- reaction in presence of ImS10 surfactant.
2.4. Activity and Reuse Stability of Immobilized CAL-B for Biodiesel Synthesis
The transesterification reaction by immobilized CAL-B was performed at different reaction times
◦
◦
◦
(0, 2, 4, 6, 8 and 24 h) and temperatures (27 C, 32 C and 37 C) for a proper evaluation of each
immobilized system. As shown in Figure 5, the increase on reaction time and temperature resulted
in higher yields for all of immobilized CAL-B, achieving the better transesterification yield (around
80–90%) at 37 ◦C after 24 h of reaction.

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Figure 5. Influence of the transesterification reaction time on the yield of FAMEs production at different
temperatures: 27 ◦C (black line), 32 ◦C (red line) and 37 ◦C (blue line) for each of the immobilized
systems: (A) NP-ImS4-CALB, (B) NP-ImS10-CAL-B and (C) NP-ImS18-CALB.
The stability of immobilized CAL-B was evaluated for its reuse ability (Figure 6).
Interestingly, the NP-ImS10/CAL-B system presented the highest catalytic conversion which
might be associated to its lower activation energy (Ea = 18.2 KJ
mol−1), since lower Ea leads
·
to higher rate constants (k) and consequently, on higher turnover frequency of a catalyst [53].
However, the NP-ImS18/CAL-B system was the most stable one, being able to retain 68% of its
original activity up to five cycles (Figure 5), whereas NP-ImS4/CAL-B system retained 51% of its
original activity up to four cycles and NP-ImS10/CAL-B was the least stable (28% of activity retention
up to the third cycle). Surprisingly, the highest stability of NP-ImS18/CAL-B, seems to be associated
to a higher activation energy [54] and lower k value, combined to the higher entropy of activation than
free CAL-B (Table 1). A large negative entropy of activation might be due to the ordering of solvent
molecules in the transition state [55], but also from protein surface mobility [56]. Therefore, although
the transesterification reaction with NP-ImS18/CAL-B seems to undergo slower (Table 1 and Figure 5)
than the other systems; as the k value indicates, the highest entropy of activation and more mobile
enzymatic surface seems to not interfere on the active site conformation during successive cycles.
More importantly, NP-ImS18/CAL-B system was the only immobilized system which presented
strong pyridine stretching bands in the infrared spectrum (Figure 3). Hence, we assume that the
Michael-type cross-linking between CAL-B and NP-ImS4 and NP-ImS10 resulted in the formation of
free aldehyde. Michael-type addition reaction involves the nucleophilic attack of the amine (or other
nucleophile) to an α,β-unsaturated glutaraldehyde molecule, generating an aminated glutaraldehyde,
in which the aldehyde portion remains unreacted. Thus, the presence of free aldehyde on the particle
surface might not interfere at the first reaction cycle, but would be able to keep reacting with free amine
groups from CAL-B, unfolding it or hindering its active site, which inhibits the catalytic reaction on the
successive cycles. In order to verify this possibility, we performed an infrared assay of NP-ImS4/CAL-B
before and after three transesterification cycles (Figure 6B) and it was evident that the aldehyde band
located at 1715 cm−1 vanished after the recycle, corroborating to the hypothesis of a Michael-type
addition occurring in NP-ImS4 system. Moreover, other cross-linking reactions, that does not generate
aldehydic portions, have been described to produce a high methanol tolerance [57].
In our experiment, it was also evident that the largest agglomerates of nanoparticles measured
prior to the immobilization protocol (by DLS experiments) had the highest enzymatic stability,
suggesting a direct correlation between nanoparticle sizes (agglomerates) and stability, in which
NPImS18/CAL-B (agglomerate size = 80 nm; 5 reuse cycles) > NPImS4/CAL-B (agglomerate
size = 54 nm; three reuse cycles) > NPImS10/CAL-B (agglomerate size = 37 nm; two reuse cycles).
Support size has been described to have a deep influence in the resulting biomaterial [58],
achieving a faster saturation of lipase adsorption with smaller beads [59]. Also, it was observed a
6.3-fold higher catalytic efficiency for pectinase immobilization onto nano-silica support in comparison
to the micron-sized silica beads [60]. However, considering that we were working with aggregated

Molecules 2018, 23, 2230
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particles, this trend might not be observed, since larger aggregates gave more stable immobilized
CAL-B than smaller ones (Figure 6), which was also found in Wang et al.’s work [61], but not discussed
by the authors. Curiously, in their report, they described the influence of silane chain length on the
stability of an immobilized lipase, in which they found out that the larger the chain length, the more
stable was the immobilized lipase. Therefore, we suggest that the immobilized CAL-B stability would
be influenced by aggregate sizes, glutaraldehyde cross-linking and/or impurity of surfactant.
In terms of productivity over eight successive cycles, the trend follows NPImS18/CAL-B (337,8%
yield) > NPImS10/CAL-B (275,5% yield) > NPImS4/CAL-B (266% yield), which would be in agreement to
the FTIR analysis, since NPImS18/CAL-B did not present the 1715 cm−1 band, whereas NPImS10/CAL-B
was an intermediate between NPImS4/CAL-B and NPImS18/CAL-B systems (Figure 3).
Figure 6.
yield for each system is represented in colored bars: NP-ImS4/CAL-B (black bars), NP-ImS10/CAL-B
(red bars) and NP-ImS18/CAL-B (blue bars). ( ) Infrared spectra of NP-ImS4/CAL-B before (black
line) and after (red line) the three reaction cycles.
(A) Recycling of immobilized CAL-B for transterification reaction. The reaction percentage
B
Since the immobilization of CAL-B on NP-ImS10 achieved 45 times more activity than free CAL-B,
we employed this system in the transesterification reaction of lipids extracted from the black yeast
Exophiala oligosperma and from the bacteria Bacillus subtilis. The reaction yield with E. oligosperma
oils were of 89%, whereas the yield using the bacteria oils could not be determined due to its low
conversion might be due to the high loads of phospholipids which could act as lipase inhibitors [62].
3. Materials and Methods
3.1. Materials
Lipase from Candida Antarctica B (CALB) (powder), imidazole, KOH, K2CO3, MgSO4, FeCl3.6H2O,
FeCl2.4H2O, NaOH, n-alkylbromide *, 1,3-propane sultone, (3-aminopropyl)triethoxysilane and
glutaraldehyde were obtained from Sigma-Aldrich (Saint Louis, Missouri, MO, USA). The solvent
solutions (acetonitrile, ethyl acetate, acetone, hexane, dichloromethane, chloroform, methanol, toluene)
were purchased from Synth (Diadema, São Paulo, Brazil) and used without previous purification. *
Corresponding to n = 4, 10 and 18.
3.2. Characterization Techniques
Nuclear magnetic resonance (1H–NMR) analysis were performed using a Bruker 400 MHz
spectrometer (Bruker, Fällanden, Switzerland). Scanning electron microscopy (SEM) images were
obtained using a FEI Inspect F50 equipment (FEI, Hillsboro, OR, USA). Infrared spectra were recorded
on a BOMEM spectrophotometer (ABB BOMEM, Quebec, QC, Canada), MB102 model, using KBr
pellets. The X-ray diffraction analyses were carried out on a Bruker Nanostar (Karlsruhe, Germany).

Molecules 2018, 23, 2230
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Bradford assays were performed using a HP-8452A—Hewlett Packard spectrophotometer (Hewlett
Packard, Palo Alto, CA, USA). DLS analysis were performed on a Malvern Zetasizer V (Malvern,
Malvern, UK) using suspensions of 1 g
L−1 and 10 g L−1 concentrations, and the obtained sizes were
µ
·
·
plotted by number of particle size distribution. Microanalysis were performed by Microanalytical
Laboratory of Universidade Federal de São Carlos, São Carlos (SP-Brazil), using a Fisions CHN, mod.
EA 1108 elemental analyzer (Thermo Scientific, Waltham, MA, USA).
3.3. Synthesis of Alkylimidazoles
The zwitterionic surfactants synthesis was performed by adaptation of a literature procedure [22],
which consists of two steps: the synthesis of an akyl imidazole molecule followed an alkylation
utilizing 1,3-propane sultone.
An imidazole solution (1.30 g, 0.0191 mol) in acetonitrile (10 mL) was added to a round-bottom
flask containing potassium hydroxide (0.76 g, 0.013 mol) and potassium carbonate (0.50 g, 0.0036 mol).
Then, a solution of n-alkyl bromide (0.012 mol) was added dropwise to the mixture, and the reaction
proceeded for 16 h at 80 ◦C under continuous stirring. After this period, the solids were removed by
filtration and the solution was diluted in 100 mL of ethyl acetate and washed three times with 50 mL
of distilled water. The organic phase was dried with anhydrous magnesium sulfate, filtrated and the
solvent was evaporated under vacuum, giving a yellow residue. The given product was characterized
by 1H-NMR in CDCl3.
3.4. Synthesis of ImS-n Sufactants
A solution of 1,3-propane sultone (1.34 g, 0.011◦mol) in acetone (12 mL) was slowly added to the
flask containing the akyl imidazole (0.012 mol) at 0 C. The reaction mixture was heated to the room
temperature and, then, stirred for 5 days. After that, the reaction solution was filtrated giving the product
as a solid. The solid was washed two times with a mixture of 1:1 hexane/dichloromethane and dried
under vacuum, giving the zwitterionic surfactant in 51% yield. The given product was characterized by
1H-NMR in CDCl3.
3.5. Magnetic Nanoparticles Synthesis
The magnetic nanoparticles were synthesized by the coprecipitation method previously described [63
]
using the zwitterionic surfactants for reverse micelle synthesis. A round-bottom flask containing a solution
of zwitterionic surfactant (ImS-n) (1.0 g) in chloroform (50 mL) under inert atmosphere was mechanically
stirred for 30 min. In parallel, a mixture 1:2 of FeCl3
·
6H2O (0.13 g, 4.81
×
10−4 mol) and FeCl2
4H2O
·
(0.0062 g, 3.12
×
10−5 mol) was added to deoxygenated distilled water (4 mL). The solution containing iron
ions was added dropwise to the surfactant solution under stirring, giving a colloidal suspension of reverse
micelles. Finally, an aqueous solution of sodium hydroxide (0.0038 mol, 1 mL) was quickly added to the
suspension. The reaction was allowed to proceed for 30 min at room temperature. Subsequently, the black
powder (Fe3O4) was magnetically separated and washed with deoxygenated water until pH 7. The same
procedure was applied using the three ImS-n molecules, obtaining three different sizes of nanoparticles
(NP-ImS4, NP-ImS10 and NP-ImS18).
3.6. Nanoparticles Functionalization with Silanol Groups
The magnetic nanoparticles were washed two times with deoxygenated methanol and dispersed
in 50 mL of a mixture 1:1 of methanol/toluene under inert atmosphere. Then, (3-aminoprop◦yl)
triethoxysilane (1 mL, 0.0043 mol) was added to the mixture and the system refluxed for 12 h at 110 C.
The solvent was removed by evaporation under vacuum, the black powder (Fe3O4@APTES) was
washed with distilled water and dried under vacuum. The obtained particles were characterized
by scanning electron microscope (SEM), infrared spectroscopy (IR), dynamic light scattering (DLS),
X-Ray diffraction Powder (XRD) and microanalysis. CHN for NP-ImS4 (3.16/1.70/0.90), NP-ImS10
(5.56/1.64/2.04) and NP-ImS18 (17.12/4.23/5.83).

Molecules 2018, 23, 2230
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3.7. Immobilization of Lipase B from Candida antarctica on Fe3O4@APTES
For lipase immobilization, a modified literature procedure [64] was employed. A small amount
(0.01 g) of silanized nanoparticles (Fe3O4@APTES) were dispersed in a phosphate buffer solution
pH 7 (0.8 mL). Then, 10
µ
L of a glutaraldehyde solution (2.76 mol
×
L
−1) was added, followed by
200 L of a lipase solution (10 mg/mL, 300
µ
µmol
×
L
−1). The mixture was stirred for 1 h at room
temperature. The black powder (Fe3O4@APTES-CALB) was magnetically separated, washed with
distilled water and dried. The enzyme load was determined by Bradford method, quantifying the
difference in absorbance before and after the immobilization procedures.
3.8. Lipase Catalytic Activity Optimization
Catalytic activity optimization experiments were carried out using soybean oil (0.5 mL), methanol
(60
immobilization process: amount of the nanoparticles (3, 5, 7 and 10 mg), glutaraldehyde concentration
(0.028 mol
−1, 0.055 mol −1, 0.083 mol x L−1 and 0.110 mol −1) and immobilization time
µL) and the biocatalyst Fe3O4@APTES-CALB. Three parameters were evaluated during the
×
L
×
L
×
L
(30, 60, 120, 180 and 240 min.). After the immobilization, immobilized CAL-B was subjected to a
transesterification reaction using soy bean oil and methanol without using any additional solvents.
The total content of Fe3O4@APTES-CALB was added to 250 µL of soy bean oil with 20 µL of methanol,
then, 2 equivalents of methanol was added in three steps (after 150 and 300 min of the reaction) and the
system was stirred at 37 ◦C for 24 h. After that period, the nanoparticles were magnetically separated,
washed with hexane and dried. The reaction product was characterized by 1H-NMR, quantifying
FAMEs production.
The same procedure was carried out using the three different sizes of magnetic nanoparticles
obtained thought the application of the ImS-4, ImS-10 and ImS-18 surfactants molecules.
3.9. Microbial Lipid Extraction Process
Total lipids from B. subtilis were extracted using 1 L of cultures in LB medium with optical density
of ≈1, employing the Bligh and Dyer method [65].
Total lipids from E. oligosperma were obtained after placing the yeast extract in an Erlenmeyer
flask with 500 mL of ethyl acetate under ultrasonic bath for one hour (3 ). Then, the mixture was
×
filtrated in a Buchner funnel, dried with anhydrous magnesium sulfate (MgSO4), and the solvent was
evaporated under vacuum. The given substrate was fractioned using silica column chromatography
applying a solvent mixture of hexane/ethylacetate 1:1, achieving 0.315 g of a yellowish oil.
3.10. Biodiesel Synthesis Catalyzed by Lipase B from Candida antarctica Immobilized on Magnetic Nanoparticles
The transesterification reaction was carried out using a lipid fungus extraction and methanol
without using any additional solvents. A little amount of Fe3O4@APTES-CALB (10 mg) was added to
250 µL of extracted lipid (containing triacylglyerols) with 20 µL of methanol, then, 2 equivalents of
methanol was added in three steps (after 150 and 300 min of the reaction) and the system was stirred
at 37 ◦C for 24 h. After that, the nanoparticles were magnetically separated, washed with hexane and
dried. The reaction product was characterized by 1H-NMR.
3.11. Determination of the Transesterification Reaction Activation Energy
The transesterification reaction activation energy was also determined using soybean oil (0.5 mL),
methanol (60
µ
L) and the biocatalyst Fe3O4@APTES-CALB (10 mg). The system was stirred at three
different temperatures (27 ◦C, 32◦C and 37 ◦C) and every two hours 20
µL of the reaction mixture
were taken out, dried in the vacuum and characterized by 1H-NMR. The same procedure was carried
out using the three different sizes magnetic nanoparticles obtained thought the application of the
ImS-4, ImS-10 and ImS-18 surfactants molecules and using the free lipase for comparison. Constant k
was determined by the reaction yields at different temperatures, using an adapted method from

Molecules 2018, 23, 2230
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O´Connor et al. [66]. An Ahrrenius plot (k vs. 1/T(K)) gave the results of energy of activation, whereas
the entropy of activation was calculated using the Eyring equation [67].
4. Conclusions
Lipase B from Candida antarctica was used as a model enzyme to study the effects of immobilization
on the structure, activity, and stability. Three different magnetic nanoparticles were synthesized by the
coprecipitation method using zwitterionic surfactants. It was seen that although the same conditions
were used in the immobilization and catalytic procedure, infrared analysis showed that NP-ImS4 and
NP-ImS10 cross-linked to CAL-B via a Michael-type addition, which generated less stable immobilized
CAL-B when compared to NP-ImS18, which was bonded to the enzyme via a pyridine formation.
The highest stability observed for NP-ImS18/CAL-B system might also be associated to the initial size
of particle agglomerates, since the larger agglomerate size, the higher immobilized CAL-B stability
was observed. In relation to CAL-B activity, transesterification reactions of soy bean reached up
to 90% of conversion into biodiesel using the immobilized CAL-B, whereas the free enzyme only
achieved a yield of 2% due to its denaturation/unfolding process in the presence of high methanol
concentrations. Finally, the transesterification reaction of lipids extracted from a fungi achieved a yield
of 89%, confirming the potential use of immobilized CAL-B in biodiesel production from renewable
oil sources.
Supplementary Materials: The Supplementary materials are available online
Author Contributions: I.A.M. designed and performed the research. C.G.C.M.N. designed the research.
D.E.S. performed the research. I.A.M., D.E.S. and C.G.C.M.N. analyzed data, interpreted the results and wrote the
manuscript. F.C.M. performed the SEM and XRD experiments. All authors read and approved the final manuscript.
Funding: This research was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grant
numbers 2016/01622-3 and 2014/13411-1. This research was funded by Conselho Nacional de Desenvolvimento
Científico e Pesquisas (CNPq) grant number 121249/2017-4. The APC was funded by Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP).
Acknowledgments: We kindly thank Alzir Batista for the shared facilities
Conflicts of Interest: The authors declare that they have no conflicts of interest.
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