Sol-gel immobilisation of lipases: Towards active and stable biocatalysts for the esterification of valeric acid

Article abstract of DOI:10.3390/molecules23092283

Alkyl esters are high added value products useful in a wide range of industrial sectors. A methodology based on a simple sol-gel approach (biosilicification) is herein proposed to encapsulate enzymes in order to design highly active and stable biocatalysts. Their performance was assessed through the optimization of valeric acid esterification evaluating the effect of different parameters (biocatalyst load, presence of water, reaction temperature and stirring rate) in different alcoholic media, and comparing two different methodologies: conventional heating and microwave irradiation. Ethyl valerate yields were in the 80–85% range under optimum conditions (15 min, 12% m/v biocatalyst, molar ratio 1:2 of valeric acid to alcohol). Comparatively, the biocatalysts were slightly deactivated under microwave irradiation due to enzyme denaturalisation. Biocatalyst reuse was attempted to prove that good reusability of these sol-gel immobilised enzymes could be achieved under conventional heating.

Full text of DOI:10.3390/molecules23092283

molecules
Article
Sol-Gel Immobilisation of Lipases: Towards Active
and Stable Biocatalysts for the Esterification of
Valeric Acid
Soledad Cebrián-García , Alina M. Balu 1 , Araceli García 1 and Rafael Luque 1,2,
1
*
1
Organic Chemistry Department, University of Cordoba, Campus de Rabanales, Edificio Marie Curie (C-3),
Ctra. Nacional IV-A, Km 396, E14014 Cordoba, Spain; b02cegas@uco.es (S.C.-G.); qo2balua@uco.es (A.M.B.);
qo2ganua@uco.es (A.G.)
Scientific Centre for Molecular Design and Synthesis of Innovative Compounds for Medicine, Peoples
Friendship University of Russia (RUDN University), 6 Miklukho-Maklaya str., 117198 Moscow, Russia
Correspondence: rafael.luque@uco.es or q62alsor@uco.es; Tel.: +34-957-211050
2
*
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 31 July 2018; Accepted: 4 September 2018; Published: 6 September 2018
Abstract: Alkyl esters are high added value products useful in a wide range of industrial sectors.
A methodology based on a simple sol-gel approach (biosilicification) is herein proposed to encapsulate
enzymes in order to design highly active and stable biocatalysts. Their performance was assessed
through the optimization of valeric acid esterification evaluating the effect of different parameters
(biocatalyst load, presence of water, reaction temperature and stirring rate) in different alcoholic
media, and comparing two different methodologies: conventional heating and microwave irradiation.
Ethyl valerate yields were in the 80–85% range under optimum conditions (15 min, 12% m/v
biocatalyst, molar ratio 1:2 of valeric acid to alcohol). Comparatively, the biocatalysts were slightly
deactivated under microwave irradiation due to enzyme denaturalisation. Biocatalyst reuse was
attempted to prove that good reusability of these sol-gel immobilised enzymes could be achieved
under conventional heating.
Keywords: sol-gel biosilicification; esterification; conventional heating; microwaves
1
. Introduction
In recent years, the production of high added value products from biomass has become an important
subject of study in many scientific fields, as biomass encompasses a wide range of organic materials
characterized by its heterogeneous origin and composition. For instance, sewage sludge, urban solid
organic wastes and other residues generated from industrial processes [1] and other human activities
can be also considered as biomass sources. Moreover, biomass is considered the largest worldwide
renewable source of carbon, as it can be originated through biological pathways. Currently, biomass is
the fourth largest worldwide energy resource, becoming potentially a sustainable source of valuable
chemicals through various transformation pathways, among which enzymatic conversion should be
highlighted [2].
Carboxylic acids are common components of effluents and sub-streams from several industrial
procedures. While these compounds do not present interesting applications, their esters have numerous
uses in the food, cosmetic and pharmaceutical industries, representing a huge demand estimated to be
worth $23.5 billion in 2014 [
and their chemical synthesis via catalytic procedures requires severe reaction conditions with a quite
low selectivity of inorganic catalysts [ ]. In this sense, biological catalysts are a promising alternative
in the production of alkyl esters under moderate and mild conditions [ ]. They show a more
efficient and ecological alternative to the use of traditional inorganic catalysts for biomass valorisation
3,4]. The main issues of alkyl esters are their very limited natural supply,
5,6
3,6,7
Molecules 2018, 23, 2283; doi:10.3390/molecules23092283
www.mdpi.com/journal/molecules

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because of their good performance at environmentally friendly conditions, lower temperature and pH
values, and fewer by-products because of their regioselectivity and stereoselectivity [ ]. Therefore, it is
8
not surprising that biocatalysed processes have been steadily growing over recent years, with clear
outlooks indicating an increasing trend for enzyme commercialisation in the global market.
The use of these biocatalysts is not well established at industrial scale due to two main drawbacks:
their limited stability, being not compatible with continuous production processes [9], and the
complication of the substrates/products separation from the reaction medium, avoiding their reuse [10].
In order to overcome these important issues, research on enzyme immobilization is increasingly
relevant, allowing the development of several biocatalytic industrial processes towards economically
profitable applications. A simple immobilization protocol implemented in our group, denoted as
biosilicification, has been proved to improve enzyme stability in organic media, enhancing catalytic
efficiency at the same time [
synthetic procedure in which a silica precursor (e.g., tetraethylorthosilicate) is dropwise added to the
enzyme and hydrolysed, in order to partially encapsulate the enzyme by a silica layer [ ]. Although,
most of the research works have focused on the immobilization of hydrolytic enzymes, such as lipases,
more recent studies have employed the same procedure in laccases with similar excellent results [ 11].
9]. The enzyme biosilicification process is based on a simple sol-gel
9
9,
Furthermore, due to immobilization, the recovery and subsequent reuse of these biocatalysts is a
favoured characteristic, reducing the final production costs when compared with the use of free
enzymes [11].
Conventional heating (CH) methods have been reported as slow and inefficient energy transfer
production pathways due to the disadvantage that they present on the adequate convection currents
transference and the low thermal conductivity of the materials that they need to penetrate [12,13].
These issues result in a higher temperature in the reaction tube than in the reaction medium, promoting
a reverse temperature gradient. This problem can be solved by using microwave (MW)-assisted heating
procedures (Figure 1), as the energy is directly transferred to reactive species by so-called “molecular
heating” processes, promoting more selective transformations that are not possible to achieve by
conventional heating [13–15]. MW radiation, as any electromagnetic radiation, is constituted by an
electric field and a magnetic field that propagates perpendicularly, although only the electric field
transfers the energy, through interaction of dipoles and ionic conduction, which leads to the heating of
the substances [13,16]. It is completely different from what happens in a conventional heating reactor,
where the heating takes place by conduction, irradiation and convection [13]. Conventional heating
also entails longer reaction times [17] in comparison with MW reactors but it leads to cost and energy
savings because of an easy and fast capacity to scale up in different industries.
Figure 1. Temperature profiles provided by (a) conventional heating or (b) microwave-assisted processes.
Esterification reactions can be defined as the transformation of carboxylic acids or their derivatives
into esters. The kinetic and process behaviour of this important type of reactions have been thoroughly

Molecules 2018, 23, 2283
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investigated since Berthelot and Gilles [18] began the first studies in 1862. The most widely employed
method comprises the direct esterification of carboxylic acid with alcohols in the presence of acid
catalysts. However, conventional acid catalysts during esterification reactions exhibit problems
associated with the generation of secondary reactions, corrosion of equipment, costly purification
procedures, and long reaction times [19]. Therefore, the need for an esterification reaction system
providing cleaner and shorter routes to synthesize a wide variety of industrial products, such as
valeric acid esters, results in being essential. Considering this, the aim of the present work was
to optimize the catalytic esterification of valeric acid to ethyl valerate (marketed as green apple
flavour) by using sol-gel biosilicified enzymes and comparing the performance of two different
methodologies: conventional heating and MW-assisted processes. Moreover, different experiments
with metal containing biocatalysts were carried out in order to improve their recovery and subsequent
reuse [20].
2
. Results and Discussion
The esterification reaction was carried out using valeric acid as substrate, as carboxylic acid, and
different short chain alcohols such as methanol (MeOH), ethanol, isopropanol (iPrOH) and 1-butanol
(
(
BuOH) as reactive media. The hydrolytic free and immobilized enzyme, Candida antarctica lipase B
CalB), was employed in order to study the efficiency of the encapsulation or biosilicification procedure,
conducted by CH and MW methodologies. The immobilized lipase was previously characterized by
our group using different techniques such as Brunauer-Emmett-Teller surface area analysis (BET),
thermogravimetric analysis (TGA) and X-Ray Photoelectron Spectroscopy (XPS) [20]. The reported
results indicated that the biocatalyst started being a non-porous material, becoming highly porous after
successive reuses due to the progressive loss of organic compounds. Despite these protein losses, the
biocatalyst still retained significant amounts of lipase, remaining highly active during the investigated
esterification reactions. These relevant results further supported the high stability of the immobilised
enzymes compared with the free enzyme, particularly under the investigated conditions [20].
2
.1. Protein Concentration and Enzymatic Activity
For each experiment 12% m/v of biocatalyst was introduced in the mixture of reaction, immobilized or
free form, presenting an enzymatic activity of 592 U/genzyme or 656 U/genzyme, respectively (Table 1).
As expected, the fresh free enzyme showed a higher value than synthetized biosilicified lipase. This was
due to the enzyme immobilization process that hid many active centres inside the silica crust. Leaching
was expected to take place after reuse, considering the methodology and reaction conditions at which
the immobilized enzyme was be exposed. On the other hand, the support does not show any enzymatic
activity which identifies the lipase as responsible for the esterification reaction, in this procedure.
Table 1. Enzymatic activity, protein loading and activity yield of the analysed systems.
System
Enzymatic Activity (U/genzyme)
Protein Loading Ratio (%)
Activity Yield (%)
Support SiO2
Free enzyme
Biosilicified lipase (R0)
-
-
-
656 ± 14
100
86
100
90
592 ± 13
2
.2. Determination of Optimal Conditions
The evaluation of the effect of different parameters (presence of water, biocatalyst load, stirring
rate and reaction temperature) on the valeric acid esterification behaviour was carried out by means a
set of experiments conducted using CH procedures for the subsequent evaluation of the conversion to
ethyl acetate by using both CH and MW methodologies (Figure 2).

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Figure 2. Conventional heating (CH) and microwave (MW)-assisted heating methodologies for the
esterification of valeric acid catalysed by biosilicified enzymes.
2
.2.1. Presence of Water during Esterification Reaction
The presence of water (distilled water (DW)% m/v) in the reaction has been reported to favour
enzyme-substrate interaction in biocatalysed reactions. In the present study, this parameter did not
significantly affect the conversion of valeric acid (Table 2).
Table 2. Effect of water presence on the conversion of valeric acid to ethyl valerate and on the specific
activity of prepared biocatalysts.
Distilled Water (DW) % m/v
Conversion to Ethyl Valerate (mol %)
Specific Activity (U/gbiocatalyst)
5
87
85
84
269 ± 10
301 ± 9
364 ± 11
◦
1
2
0
0
Reaction conditions: 1:2 molar ratio valeric acid to EtOH, 12% m/v of biosilicified enzyme, 1000 rpm, 40 C, 2 h CH
in batch reactor.
None of the conversions achieved by the prepared biocatalyst or the specific activity demonstrated
presented remarkable differences between different percentages of DW studied. However, further
investigations are needed [15,21] for systems catalysed by sol-gel biosilicified enzymes.
2
.2.2. Influence of Biocatalyst Load
The optimal concentration of sol-gel biosilicified enzyme in the reaction was determined by a set
of experiments using the CH procedure (Figure 3).
Figure 3. Effect of amount of biosilicified enzyme on the conversion of valeric acid to ethyl valerate
◦
(
study conditions: molar ratio 1:2 of valeric acid to EtOH, 1000 rpm, 40 C, 2 h in CH batch reactor).
A 12% m/v of biocatalyst load provided a maximum conversion of 96% with 100% of reaction
selectivity, and no difference could be observed when higher amount of catalyst was added. Such an
enzyme concentration was consequently considered for subsequent experiments.

Molecules 2018, 23, 2283
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2
.2.3. Influence of Stirring Rate
Mass transfer limitations can affect the conversion efficiency of reactions, especially related to
heterogeneous catalysis [22]. Consequently, appropriate and efficient stirring is needed for uniform
mixing and contact between reactants in the reaction medium. In the present study, valeric acid
conversions between 73% and 95% were obtained for different acid-to-alcohol molar ratios, not being
significantly influenced by the stirring rate (Table 3). However, the higher conversion was achieved
at high stirring rates for medium and high acid concentrations (Figure 4a). Regarding enzymatic
activities, better values were obtained at higher stirring rate (501 ± 13 U/gbiocatalyst at 100 rpm).
Table 3. Influence of stirring rate on the conversion of valeric acid to ethyl valerate and on the specific
activity of prepared biocatalysts.
Conversion to Ethyl Valerate (mol %)
Specific Activity (U/gbiocatalyst
)
Acid-to-Alcohol
Ratio
2
50 rpm
500 rpm
750 rpm
1000 rpm
250 rpm
500 rpm
750 rpm
1000 rpm
1:3
1:2
1:1
73
89
92
76
81
87
75
87
87
81
95
90
478 ± 10
490 ± 11
497 ± 9
501 ± 13
◦
Reaction conditions: 12% m/v of biosilicified enzymes, 250–1000 rpm, 40 C, 2 h CH in batch reactor.
Figure 4. Effect of (a) stirring rate and (b) reaction temperature on the conversion of valeric acid to
ethyl valerate by CH procedures.
2
.2.4. Influence of Reaction Temperature
As reported in previous articles, temperature is key factor in the proper operation of biological
◦
◦
molecules such as enzymes [23]. Previous studies [24] reported 70 C as a critical temperature for the
stability of enzymes. Furthermore, temperatures higher than 50 C have been reported to promote
denaturation in the native state of proteins, with a complete loss of enzymatic activity. Comparatively,
lower temperatures may lead to aggregation in the reaction medium and a decrease in catalytic
efficiency [23]. Therefore, the study of the temperature in the optimization of CH esterification of
valeric acid by lipases is decisive. In the present work, immobilized and free enzymes were tested
◦
up to 50 C. Interestingly, the highest conversion rate (96%, 100% selectivity to ethyl valerate) was
◦
obtained at 40 C and with a 1:2 acid-to-alcohol molar ratio after 2 h (Table 4). Experiments conducted
at different acid-to-alcohol molar ratios and temperatures indicated that moderate to very good ethyl
valerate yields could be obtained (71–96% conversion at complete ethyl valerate selectivity) even at
high valeric acid concentration (Figure 4b).
On the other hand, the prepared biocatalysts present the optimum activity values temperature in
◦
a range between 35–45 C and at 1:2 acid-to-alcohol molar ratio. Immobilized catalysts were stirred in

Molecules 2018, 23, 2283
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aqueous solution for 2 h in a range of different temperatures. Table 4 shows a substantial decrease in
enzymatic activity with temperature under innocuous conditions, which results a determining factor
in case of biocatalytic processes.
Table 4. Effect of reaction temperature on the conversion of valeric acid to ethyl valerate and specific
activity of biocatalysts after esterification.
Conversion to Ethyl Valerate (mol %)
* Specific Activity (U/gbiocatalyst)
Acid-to-Alcohol
Ratio
50 ◦C
45 ◦C
40 ◦C
35 ◦C
30 ◦C
25 ◦C
20 ◦C
50 ◦C
40 ◦C
30 ◦C
20 ◦C
1:3
1:2
1:1
84
85
74
86
87
78
94
96
76
88
91
71
80
84
75
82
84
71
83
87
75
234 ± 11
485 ± 17
623 ± 14
630 ± 13
Conditions for esterification reaction: 12% m/v of biosilicified enzyme, 1000 rpm, 2 h CH in batch reactor. * To
determine the influence of temperature on the specific activity of the catalyst, 12% m/v of biosilified enzyme was
incubated in DW, 1000 rpm, 2 h CH in batch reactor at the different temperatures.
2
.3. Effect of Alcohol Type on Lipase Catalysed Esterification of Valeric Acid
2
.3.1. Production of Ethyl Valerate by Conventional Heating (CH) Procedures
Table 5 summarises the main results from a set of experiments conducted under optimised reaction
conditions with conventional heating. Similarly, experiments performed under CH in the absence of
biocatalyst (blank) or using SiO support (SiO crust without enzyme) did not lead to valeric acid
2
2
conversion for all tested alcohols. Comparably, the highest conversion rate (95%, 100% selectivity
to ethyl valerate) was obtained using a 12% m/v of biosilicified enzyme and a 1:2 acid-to-alcohol
molar ratio after 2 h (Table 5, entry 14). Product yields obtained using a 12% m/v free lipase and
different acid-to-alcohol ratios were about 80% (Table 5, entries 9–11). Moreover, reactions conducted
with MeOH as the alcohol provided negligible activities in the systems (Table 5, entry 12), in good
agreement with previous results from the group [9,11,25]. MeOH seems to be toxic for some enzymes,
especially CalB, and therefore they do not reach significant conversion in these alcoholic systems.
Previous studies reported that CalB, even if very stable in a wide range of organic solvents, is rapidly
deactivated in the presence of MeOH at molar ratios much lower than the theoretical optimum for
the conversion reaction [26]. However, the existence of specific activity after enzyme incubation in
MeOH medium indicated that the lack of valeric acid conversion was not only due to denaturation
mechanisms, but this alcohol specifically decreases the hydrolytic activity of CalB (Table 5, entry 12),
as reported in previous works [27]. The specific activity of the free enzyme after the esterification
reaction (600
±
12 U/gbiocatalyst) resulted comparably lower to that found for the fresh free enzyme
in Table 1) after enzyme incubation in the presence of EtOH. This indicated
(
656 14 U/g
±
biocatalyst
certain weathering of the biocatalyst, as several active centres could be deactivated by denaturing
processes. However, for the immobilized enzyme the specific activity increased from 592 13
see Table 1) to 640 17 U/gbiocatalyst after its use. During the immobilization process, many active
±
(
±
centres could be hidden inside the silica layer, being partially washed and released after the first use of
the biocatalyst, with the subsequent increase in specific activity.

Molecules 2018, 23, 2283
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Table 5. CH esterification of valeric acid using different alcohols (MeOH, EtOH, iPrOH, BuOH) and
different acid-to-alcohol molar ratios.
Acid-to-Alcohol Conversion to Alkyl
* Specific Activity
(U/gbiocatalyst)
Entry
System
Alcohol
Ratio
Valerate (mol %)
1
2
3
4
MeOH
EtOH
iPrOH
BuOH
Blank
All
No conversion
-
5
6
7
8
MeOH
EtOH
iPrOH
BuOH
Support SiO2
Free enzyme
All
No conversion
-
9
1:1
1:2
1:3
82
82
87
1
1
0
1
EtOH
MeOH
EtOH
600 ± 12
-
Biosilicified
enzyme
1
2
All
No conversion
13
14
15
1:1
1:2
1:3
81
95
90
Biosilicified
enzyme
640 ± 17
1
1
1
6
7
8
1:1
1:2
1:3
57
92
75
Biosilicified
enzyme
iPrOH
BuOH
194 ± 15
291 ± 10
1
2
2
9
0
1
1:1
1:2
1:3
60
89
77
Biosilicified
enzyme
Conditions for esterification reaction: 12% m/v biosilicified enzyme (equal quantity for free enzyme using buffer
◦
solution) 1000 rpm, 40 C, 2 h CH in batch reactor. * To determine the influence of different alcohols on the specific
activity of the catalyst, 12% m/v of biosilificated enzyme was incubated in MeOH, EtOH, iPrOH, BuOH, respectively,
◦
1
000 rpm, 40 C, 2 h CH in batch reactor.
Finally, the use of other alcohols (such as iPrOH and BuOH) mediated conversions to alkyl valerate
of about 90% (Table 5, entries 17, 20) in a 1:2 acid:alcohol molar ratio, but in general with significantly
lower yields than when EtOH was used. Moreover, considering specific activity values, CalB appeared
to be more stable in ethanol than in iPrOH and BuOH because after the biocatalyst incubation in the
presence of iPrOH or BuOH, the remaining specific activity of the immobilized enzyme was much
lower compared to the catalyst incubated in the presence of EtOH. During esterification experiments
using these two alcohols, the biocatalyst behaviour was different, and certain deposition/precipitation
of particles was observed. Thus, partial steric impedance could occur due lower solubility and explain
the decrease of the catalyst’s specific activities in these alcoholic media. On the other hand, the support
does not show any enzymatic activity which identifies the lipase as responsible for the esterification
reaction, in this procedure. The effect of the alcohol used on the catalyst is studied in Table 5. As it could
be expected, the lipase incubated with ethanol maintains the highest enzymatic activity 640 U/genzyme
The stability and reusability of the enzyme under the investigated CH conditions was subsequently
tested (Table 6). Results pointed out that the biosilicified lipase keeps its conversion capacity in the
ethanol stable. This is not so for iPrOH and BuOH due to their toxicity [28]. Selectivity to alkyl valerate
was fully preserved after reuses in each case. Comparably, the free enzyme provided 82% conversion
at complete selectivity in the first run, followed by a significant reduction of conversion of the enzyme
.
(<50% after 1 reuses, R1), most probably due to denaturalisation under temperature.

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Table 6. Reusability studies of biosilicified enzymes for esterification reactions with different alcohols
under CH.
Conversion to Ethyl Valerate (mol%)
Specific Activity (U/gbiocatalyst)
Reuse
MeOH
EtOH
IPrOH BuOH
Free Enzyme
MeOH
EtOH
iPrOH
BuOH
Free Enzyme
R0
R1
R2
R3
R4
-
-
-
-
-
95 ± 8
88 ± 7
94 ± 6
92 ± 8
93 ± 6
92 ± 7
89 ± 8
80 ± 9
70 ± 7
69 ± 9
60 ± 7
82 ± 6
75 ± 8
68 ± 5
60 ± 8
61 ± 6
418 ± 14
506 ± 16
505 ± 12
415 ± 18
400 ± 14
403 ± 11
508 ± 14
427 ± 12
307 ± 14
270 ± 12
254 ± 17
207 ± 16
352 ± 12
307 ± 16
301 ± 15
272 ± 9
42 ± 7
207 ± 12
400 ± 19
-
-
-
-
-
-
367 ± 15
-
-
260 ± 13
Reaction conditions: 12% m/v biosilicified enzyme (equal quantity for free enzyme using buffer solution), 1000 rpm,
◦
4
0 C, 2 h CH in batch reactor.
2
.3.2. Production of Ethyl Valerate Using Microwave (MW) Irradiation
Compared to the CH procedure, blank runs (in the absence of biocatalyst) including those with
the silica support (SiO crust without enzyme) did not provide any valeric acid conversion (Table 7).
2
Table 7. MW-assisted esterification of valeric acid using different alcohols (MeOH, EtOH, iPrOH,
BuOH) and different acid-to-alcohol molar ratios.
Acid-to-Alcohol
Ratio
Conversion to Alkyl
Valerate (mol %)
Specific Activity
Entry
System
Alcohol
(U/gbiocatalyst
)
1
2
3
4
MeOH
EtOH
iPrOH
BuOH
Blank
All
All
No conversion
No conversion
-
5
6
7
8
MeOH
EtOH
iPrOH
BuOH
Support SiO2
Free enzyme
-
9
1:1
1:2
1:3
70
75
71
1
1
0
1
EtOH
MeOH
EtOH
270 ± 13
-
Biosilicified
enzyme
1
2
All
No conversion
13
14
15
1:1
1:2
1:3
58
82
73
Biosilicified
enzyme
376 ± 7
1
1
1
6
7
8
1:1
1:2
1:3
39
40
37
Biosilicified
enzyme
IPrOH
BuOH
300 ± 13
337 ± 5
1
2
2
9
0
1
1:1
1:2
1:3
79
81
80
Biosilicified
enzyme
Reaction conditions: 12% m/v biosilicified enzyme (equal quantity for free enzyme using buffer solution), 100 W,
◦
4
0 C, 1 h MW radiation.
The highest conversion rate (82%, with 100% selectivity to ethyl valerate) was obtained using a
12% m/v of biosilicified lipase and a 1:2 acid:alcohol molar ratio after 1 h (Table 7, entry 14). Product
yields obtained using a 12% m/v of free lipase and different acid-to-alcohol ratios were found to be
higher than 70% (Table 7, entries 9–11). Moreover, reactions conducted with MeOH as alcohol provided
negligible activities in the systems (Table 7, entry 12). Finally, the use of other alcohols (such as iPrOH
and BuOH) mediated conversions to alkyl valerate of 40% in iPrOH and 80% in BuOH (Table 7, entries
1
7, 20) in a 1:2 acid-to-alcohol molar ratio. This technique aimed to simplify the esterification procedure
as the reaction energy is transferred directly to reactive species [13 16]. In the case of enzymatic activity,
,
it could be said that CH is an efficient procedure where biocatalyst remains stable without loss of their
catalytic efficiency. It was also found that CH methodology increased the catalytic efficiency under the
pressure- and temperature-mild conditions required for lipases, whereas a reaction conducted using

Molecules 2018, 23, 2283
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MW as the heating source resulted quite focused on the reactive species, interfering with the thermal
stability of the proteins.
In Table 8, the results of the reaction investigated under optimised conditions throughout the
present work are shown. As observed, after 15 min of reaction the conversion to ethyl valerate was
higher than 80% with a 100% of selectivity in both cases. These values kept almost equal for longer
MW-assisted reactions, whereas higher yields were obtained for CH conducted procedures (90% at 30
and 60 min). On the other hand, a clear increase of the enzymatic activity with the reaction time was
observed for both CH and MW methodologies.
Table 8. Effect of reaction time on MW and CH conducted esterification of valeric acid under the main
optimised reaction conditions.
Conversion to Ethyl Valerate (mol %)
Specific Activity (U/gbiocatalyst)
Methodology
1
5 min
30 min
60 min
15 min
30 min
60 min
MW
CH
80
86
80
90
82
90
394 ± 18
384 ± 16
357 ± 10
460 ± 11
◦
545 ± 14
495 ± 11
Reaction conditions: molar ratio 1:2 of valeric acid to EtOH, 12% m/v biosilicified enzyme, 100 W (MW), 40 C (CH).
Although these two methodologies offered good results for the valeric acid esterification reaction,
and even if MW could result in a more efficient heating procedure, CH still provides a more
environmentally sustainable, economically profitable and easily scaled-up procedure, due to the lower
costs related with the energy requirements that it provides. Conditions reached during MW-assisted
reaction were too severe for lipases, despite their high stability reached under sol-gel biosilicification,
and significantly lower specific activities were observed in MW reactions in comparison to those
observed for CH.
The stability and reusability of the enzymes used under the above investigated MW conditions
were subsequently tested (Table 9). Results pointed out that the biosilicified lipases gradually lost
their conversion capacity when reuses were performed in EtOH medium (from 82 to 35% after
3
uses), but importantly the enzymes were not fully denaturalised after several cycles under the
investigated conditions. Lipases require quite moderate reaction conditions that cannot be established
in microwaves due to the rapid heating to which the molecules in reaction are subjected [16].
Table 9. Reusability studies of biosilicified enzymes in the esterification reactions using MW radiation.
Reuses
Conversion to Ethyl Valerate (mol %)
Specific Activity (U/gbiocatalyst
)
R0
R1
R2
R3
82 ± 9
66 ± 4
51 ± 6
35 ± 8
357 ± 14
310 ± 9
285 ± 11
210 ± 12
◦
Reaction conditions: molar ratio 1:2 of valeric acid to EtOH, 12% m/v biosilicified enzyme, 100 W, 40 C, 1 h
MW radiation.
3
. Materials and Methods
3
.1. Materials
The reagents Tetraethoxysilane 98% (TEOS), n-dodecylamine 98%, buffer solution of enzyme
Candida antarctica lipase B 5000 LU/g (CalB), titanium dioxide 99.5%, p-nitrophenyl butirate 98%
(
(
pNPB), valeric acid 99% and Bradford reagent were purchased from Sigma Aldrich. Ethanol 98%
EtOH), methanol 99.5% (MeOH), isopropanol 99.5% (iPrOH), 1-butanol 99.5% (BuOH), acetonitrile
99.9% and Cobalt (II) chloride hexahydrates 98% were purchased from Panreac and used without
further purification.

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3
.2. Enzyme Biosilicification
According to the protocol previously described [29], TEOS (20.80 g, 0.10 mol) was added to a
continuously stirred solution containing n-dodecylamine (5.10 g, 0.03 mol), 50 g of acetonitrile and
0 g CalB buffer solution at room temperature. For the synthesis of immobilized enzymes with two
5
different metal precursors, after addition of the acetonitrile and n-dodecylamine, titanium dioxide and
cobalt (II) chloride hexahydrates (1% wt.) were added under continuous stirring, before the CalB buffer
solution and TEOS addition. The resulting solutions presented a visible solid precipitate after a few
minutes, so they were further stirred for another 3 h. Finally, the synthetized biocatalysts were filtered,
twice washed with ethanol, and dried at room temperature for 24 h. For the preparation of the SiO2
reference support, the same protocol was conducted but without the addition of CalB buffer solution.
3
.3. Protein Concentration and Enzymatic Activity
For enzyme characterization, protein concentration and enzymatic activity were evaluated for
SiO reference support, free CalB, and biosilicified samples.
2
Protein concentration was determined according to the Bradford colorimetric method [29], using
protein bovine serum albumin as standard. All solutions were prepared by employing ultrapure water.
◦
Enzymatic activity was determined by hydrolysis of pNPB in acetonitrile, at pH 7 and 25 C
according to the protocol described by García-Galán et al. [30]. p-nitrophenol, released as the hydrolysis
product of pNPB to the reaction medium was spectrophotometrically measured at 400 nm. In the
present work, an activity unit (U) was defined as the amount of enzyme capable to hydrolyse 1
of substrate per minute at pH 7 and 25 C (Equation (1)).
µmol
◦
umol of substrate hydrolysed
Enzymatic activity (U) =
(1)
minute
The specific activity, as an important measure of enzyme purity, was expressed as
substrate hydrolysed per g of enzyme per min of incubation, being corrected for blank values
Equation (2)).
µmol of
(
ꢀ
ꢁ
U
Specific activity U/g
=
(2)
biocatalyst
g of enzyme
3
.4. Immobilization Efficiency
Immobilization efficiency was evaluated with two parameters: protein loading ratio (Equation (3))
and activity yield (Equation (4)).
protein loaded
protein introduced
Protein loading ratio (%) =
× 100
(3)
(4)
specific activity (immobilized lipase)
specific activity (free lipase)
Activity yield (%) =
× 100
Protein loading ratio (%) was used to compare the amount of enzyme that finally remains in the
biocatalyst with the free enzyme. Activity yield (%) equally determined the difference between specific
activity in case of the biosilicified enzyme and the free form.
3
.5. Biocatalytic Esterification Activity
The conversion of valeric acid to ethyl valerate was investigated in order to evaluate the activity
of the synthetized biocatalysts. Valeric acid and different alcohols (EtOH, MeOH, iPrOH, BuOH) were
used as reaction substrates.
For optimization purposes, different experimental sets were conducted in order to evaluate the
◦
biocatalyst load (molar ratio 1:2 of valeric acid to EtOH, 1000 rpm, 40 C, 2 h in CH batch reactor),
the presence of water (1:2 molar ratio valeric acid to EtOH, 12% m/v of biosilicified enzyme, 1000 rpm,

Molecules 2018, 23, 2283
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◦
4
0 C, 2 h CH in batch reactor), the stirring rate (different molar ratios of valeric acid to EtOH, 12% m/v
◦
of biosilicified enzymes, 250–1000 rpm, 40 C, 2 h CH in batch reactor) and the reaction temperature
different molar ratios of valeric acid to EtOH, 12% m/v of biosilicified enzyme, 1000 rpm, 2 h CH in
batch reactor).
In a typical esterification reaction, valeric acid was added to each alcoholic solution at 1:1, 1:2 and
:3 molar ratios. In all the experiments 12% m/v of biosilicified lipase was used. Mixtures were placed in
a microwave reactor (CEM-Discover, Matthews, NC, USA) or in a Carousel Reaction Station (Radley
(
1
™
Discovery Technologies, Saffron Walden, UK) for MW or CH experiments, respectively. Esterification
was conducted for 2 h in all cases, with continuous temperature measurement and periodic sampling.
Blank reactions containing the free CalB were run under identical conditions using comparable enzyme
quantities (12% m/v of not-immobilized CalB).
3
.6. Evaluation of Biocatalytic Esterification Behaviour
From each conducted esterification reaction, the resulting products were quantified by gas
chromatography (GC) analysis in an Agilent 7890 GC model fitted with a capillary Petrocol column
−
1
(
100 m
Collected samples were 1:6 diluted in the corresponding alcoholic reaction media and filtrated with a
m syringe filter before injection. The GC program was as follows: oven from room temperature
×
0.25 nm
×
0.5
µ
m) and a flame ionization detector (FID) and a N carrier flow of 30 mL min
.
2
45 µ
(
◦ ◦ ◦
RT) to 220 C (40 min), inlet at 250 C (split mode), and detector at 300 C.
4
. Conclusions
Optimised conditions were studied in the esterification of valeric acid by biosilicified enzymes via
conventional and microwave-assisted heating, resulting in about 80% of valeric acid conversion to ethyl
valerate after 15 min using both methodologies under mild reaction conditions. Comparably, the reuse
of the enzyme remained stable after different cycles of esterification via conventional heating, while
protein folding (leading to enzyme denaturalisation) seemed to take place under microwave-assisted
conditions due to the rapid increase of temperature. It can be established that, in spite of exhibiting
similar performances, conventional heating assisted esterification required less energy, resulting in
being a simpler procedure for implementation in many industrial fields but also allowing easier reuse
of this novel biocatalysts.
Author Contributions: S.C.-G. performed all experiments in the lab and contributed to the first draft; A.M.B. and
A.G. revised and completed the draft before submission; R.L. planned all experiments, curated/discussed all
data, supervised the work and eventually revised and completed the manuscript (including the revised version)
for submission.
Funding: Funding from MINECO under project CTQ2016-78289-P, co-financed with FEDER funds.
Acknowledgments: Soledad Cebrián-García is grateful to Junta de Andalucía for the contract European Social
Fund-Program Youth Employment-PTA and Araceli García would like to thank the Spanish Ministry of Economy,
Industry and Competitiveness for the postdoctoral contract Juan de la Cierva Incorporación IJCI-2015-23168.
The publication was prepared with support from RUDN University Program 5-100.
Conflicts of Interest: The authors declare no conflict of interest.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).