Rotating bed reactor packed with heterofunctional structured
silica-supported lipase or Novozym 435. Developing an effective system
for the organic solvent and aqueous phase reactions
Daria Kowalczykiewicz1,2, Katarzyna
Szymańska3*, Danuta Gillner1,2,
Andrzej B. Jarzębski3,4
1. Department of Organic Chemistry, Bioorganic
Chemistry and Biotechnology, Silesian University of Technology, B.
Krzywoustego 4, 44-100 Gliwice, Poland
2. Biotechnology Center, Silesian University of
Technology, B. Krzywoustego 8, 44-100 Gliwice, Poland
3. Department of Chemical Engineering and Process
Design, Silesian University of Technology, Ks. M. Strzody 7, 44-100
Gliwice, Poland
4. Institute of Chemical Engineering, Polish Academy
of Sciences, Bałtycka 5, 44-100 Gliwice, Poland
Funding: This work was supported by the by the National Centre for
Science – Poland, project UMO-2016/23/B/ST8/00627.
Abstract : Production of specialty chemicals increasingly makes
use of enzyme catalysts, and Novozym 435 (N435) is among most often
applied. However, its polymeric skeleton is unstable in many solvents.
In this context, we report results of a systematic study of the
biocatalysts, fabricated using highly porous siliceous pellets/enzyme
(MH), grafted with octyl (-O), amino (-A) and octyl and amino (-OA)
groups, deployed in a rotating bed reactor and tested in hydrolysis and
esterification reactions. N435 appeared the most active in both
reactions, when activity was related to the catalyst’s mass, mainly
owing to very large enzyme load. But its structure degraded in many
typical solvents, whereas no such effect was detected in MH-O- and
MH-OA-catalysts. MH-O showed the highest specific activity, however, a
significant enzyme leaching was observed in a hydrolytic reaction, in
contrast to MH-OA. In esterification reaction the MH-O-bound lipase was
not only most active but also quite stable.
Keywords: silica beads, lipase catalyst, Novozym 435, rotating
bed reactor, dedicated catalysts
INTRODUCTION
Supreme catalytic properties of enzymes offer an efficient and
environmentally friendly strategy for the production of value-added
chemicals, under mild conditions. Owing to good stability in diverse
media, promiscuity in recognition different substrates and
regioselectivity in both hydrolysis and inter-, trans/esterification
reactions in organic media, lipases emerge as the biocatalysts of prime
importance (Foukis et al., 2017; Thomas, Burich, Bandeira, Marques de
Oliveira & Piovan, 2017; Zare, Golmakani & Niakousari, 2019). For
practical reasons: easy separation, reusability and increased stability,
the industry favours the use of immobilized enzymes (Dwivedee, Bhaumik,
Rai, Laha & Banerjee, 2017; Mallin, Muschiol, Byström & Bornscheuer,
2013; Odrozek et al. 2017; Szymańska, Bryjak, Mrowiec-Białon &
Jarzębski, 2007; Szymańska, Odrozek, Zniszczoł, Pudło & Jarzębski,
2017). In industrial applications, the most common of lipase-based
biocatalysts is the Novozym 435 (N435), i.e. Candida antartica
lipase B (CALB) adsorbed on a hydrophobic macroporous acrylic resin -
the Lewatit VP OC 1600 (Foukis et al., 2017; Goundoju, Bokam,
Yalavarthi, Shaik & Ponnapalli, 2019; Huang et al., 2019; Ortiz et al.,
2019; Tang, Wang, Huang, Jin & Wang, 2015; Thomas, Burich, Bandeira,
Marques de Oliveira & Piovan, 2017; Wang, Gu, Cui, Wu & Wu, 2014; Wang
et al., 2016; Zare, Golmakani & Niakousari, 2019). Its particle size
below 1 mm, renders it a good candidate to be used in both stirred tank-
(STR) and packed bed (PBR) reactors (Foukis et al., 2017; Thomas,
Burich, Bandeira, Marques de Oliveira & Piovan, 2017; Wang, Gu, Cui, Wu
& Wu, 2014). However, despite a widespread application, N435 exhibits a
number of important negative features that are usually ignored, as
pointed out by others group (Ortiz et al. 2019; Jose, & Briand, 2010;
Jose et al., 2011; Zhao & Song, 2010). Owing to weak binding forces of
adsorption, the leakage of proteins thus attached to the carrier can
easily be triggered by a small change in temperature, pH and ionic
strength, or even the presence of substrates and solvents. Moreover,
fragility of the Lewatit particles, results in their comminution under
mechanical stirring, and this gives rise to serious separation problems
and also facilitates the enzyme leaching, that is often disregarded. And
not less importantly, the Lewatit matrix dissolves in many organic
solvents (Ortiz et al., 2019), and this leads to migration of monomers
or larger entities to the liquid phase (Jose et al., 2011) Zhao & Song
listed in this context 17 substances that migrated from N435 towards
organic solvents and ionic liquids, and some of them even turned out to
be active as acyl donors in the esterification and transesterification
reactions (Zhao & Song, 2010). All these observations clearly evidence
the kind of problems that may occur when N435 is used either in STR or
in organic media, or both.
In the PBRs the catalysts load is much larger than in STRs, and that
leads to higher values of space-time yield (STY), and hence improved
process economy. Additionally, mass transfer to the catalysts particles
can also be more intensive, provided that fluid velocities are high. But
the latter implies the need for a high backpressure, and hence endangers
safety. A rational engineering solution may be the use of rotating bed
reactors (RBRs), that allow for a simultaneous mixing and percolation of
reactants through the bed of heterogeneous catalysts placed in a
cylindrical basket, mounted at the end of the shaft (Aurell, Karlsson,
Ponten, & Andersen, 2014; Mallin, Muschiol, Byström & Bornscheuer,
2013; Szymańska, Odrozek, Zniszczoł, Pudło & Jarzębski, 2017). The
problems of catalysts damage and separation are removed, and the
tortuous interconnected channels between the particles can induce
intensive lateral mixing and much enhanced mass transfer, which boost
the overall reaction rate (Xu et al., 2017). Moreover, similarly as in
PBRs the catalysts load can be high, and it translates into high values
of STY, without compromising process safety. For these reasons, the RBRs
attract increasing attention and were recently applied in a number of
practical situations: hydrogenation of D-xylose to D-xylitol, using
ruthenium decorated carbon foam (Pham et al., 2016), lipase catalyzed
regioselective lactamization (Aurell, Karlsson, Ponten, & Andersen,
2014), selective fractionation of hemicellulose and lignin (Eta,
Anugwom, Virtanen, Maki-Arvela & Mikkola, 2014) or hardwood (Eta &
Mikkola, 2016), and (S)-naproxen synthesis using immobilized
decarboxylase (Aβmann et al., 2017).
A limitation for the RBR application is posed by the size of catalyst
particles - it needs to be larger than ca. 0.4 mm (Aurell, Karlsson,
Ponten, & Andersen, 2014; Mallin, Muschiol, Byström & Bornscheuer,
2013; Szymańska, Odrozek, Zniszczoł, Pudło & Jarzębski, 2017), and this
eliminates the use of the most common mesoporous silica materials as
enzyme supports, but N435 meets this condition. A rational option for
the RBR application may be the use of silica beads/pellets which possess
an open, optimally hierarchical meso-macropore structure, that is stable
in various solvents, can easily be functionalized to attach enzymes and
allow for an unhindered transport of reactants to the catalytic sites.
These relatively new materials proved to be very effective as continuous
flow monolithic microreactors (Helm, 2019; Hou et al., 2019; Strub,
Szymańska, Hrydziuszko, Bryjak & Jarzębski, 2019; Szymańska et al.
2013; Szymańska et al., 2016a; Szymańska et al., 2016b), and also as 3
mm sized enzyme-activated packing of RBRs, applied in sucrose hydrolysis
(Szymańska, Odrozek, Zniszczoł, Pudło & Jarzębski, 2017) and oxidation
of benzoic acid (Odrozek et al., 2017). Not less importantly, unlike
polymeric enzyme supports, the siliceous beads could be regenerated and
reused several times, with no change in the pore structure.
In contrast to the extensive studies of mesoporous silica as enzyme
carriers (Hudson, Cooney & Magner, 2008), reports on the use of
macroporous siliceous carriers are scarce (Jiang et al., 2016; Zhou et
al., 2008), and we are familiar only with the study of matrices with
macropores ca. 0.5 µm dia. and small mesopores, ca. 5 nm (0.5
cm3g-1 mesopore volume). Meanwhile,
the silica beads developed recently in our group (Odrozek et al., 2017;
Szymańska, Odrozek, Zniszczoł, Pudło & Jarzębski, 2017), feature much
more open structure, an extensive (3
cm3g-1 pore volume) network of
macropores 20 - 40 µm in diameter, and mesopores of about 20 nm and 1
cm3g-1 pore volume. This makes them
easily penetrable, and hence potentially very attractive, especially
that the size of macropores in the packed bed enzyme carriers was found
to be critical for highly active catalytic beds (Szymańska, Odrozek,
Zniszczoł, Pudło & Jarzębski, 2017). Note, that owing to both thermal
and chemical stability of the silica, contamination of the reaction
mixture with degradation products is nonexistent. Moreover, silanol
groups allow for a facile grafting of its surface with various
functional groups to attach strongly the enzyme, and also other moieties
which stimulate expression of high enzymatic activity (Hou et al.,
2019;, Odrozek et al, 2017; Strub, Szymańska, Hrydziuszko, Bryjak &
Jarzębski, 2019; Szymańska et al. 2013; Szymańska et al., 2016a;
Szymańska et al., 2016b; Szymańska, Odrozek, Zniszczoł, Pudło &
Jarzębski, 2017).
To prevent enzyme leakage, either an ionic or a covalent bonding of the
enzyme by means of various organosilanes is recommended. More recently,
it evolved to apply hetero-, usually bi-functionalized carriers, the
surface of which was decorated with hydrophobic groups - responsible for
a proper conformation of lipase, and amino- or epoxy groups - that allow
for a more stable bonding of the enzyme (Bernal, Illanes, & Wilson,
2014; Hirata et al., 2016; Vescovi et al., 2016). The latter approach
was also recently shown to enable a preferential orientation of proteins
on the carrier, to make a full use of their catalytic potentials, and
hence emerges as being more effective (Wang, Zhang, Han, Wu & Wei,
2018).
Clearly, all these works pave the way to a new, integrated platform for
the intensive synthesis of fine chemicals. Proceeding along this path,
we considered it important to explore potentials of the RBRs filled with
biocatalysts, obtained using structured porous silica beads, the surface
of which was (hetero)functionalized to attach CALB, and to compare their
performance, in both an aqueous- and organic solvent system, with that
of the N435-packed same RBR. To obtain a deeper insight into the surface
effects, the beads were functionalized either separately with octyl- and
amino groups, or simultaneously with them both. The biocatalytic
properties were studied in esterification of levulinic acid and
hydrolysis of p- nitrophenyl acetate. The former reaction attracts
major interest since levulinic acid is a versatile renewable platform
molecule of high chemical reactivity and its esters have various
practical applications (Zhou et al., 2018). However, owing to a
precarious behavior of the N435 in various organic solvents on one hand,
and their widespread application on the other, prior to mainstream
studies we deemed it important to investigate solvents effect on the
N435 pore structure and catalytic activity after 24 h of soaking.
- MATERIALS AND METHODS
- Materials
Hexadecyltrimethylammonium bromide (CTAB), p- nitrophenyl acetate
(p- NPA, 97%) and Bengal Rose (85%) were purchased from Acros
Organics. Tetraethyl orthosilicate (TEOS, 98%), polyethylene glycol 35
000 (PEG), levulinic acid (98%), lipase B from Candida
antarctica , 3-aminopropyltrimetoxysilane and n-octyltrietoxysilane were
obtained from Sigma Aldrich. Glutaraldehyde (GLA, 25%) was from Avantor
Performance Materials (Poland). Novozym 435 was kindly provided by
Novozymes.
Fabrication of silica pellets
The siliceous pellets were prepared using methodology adapted from
Szymańska (Szymańska, Odrozek, Zniszczoł, Pudło & Jarzębski, 2017).
Briefly, polyethylene glycol 35 000 (PEG, 8.67 g) was dissolved in 1M
HNO3 (100 mL). After that, tetraethyl orthosilicate
(TEOS, 82.8 mL) was added dropwise in an ice bath and then
hexadecyltrimethylammonium bromide (CTAB, 3.8 g). The solution was mixed
and pipetted into small conical vessels (50 µL), left to gel at 40oC and aged for 7 days. After that time the
impregnation in ammonia solution (1M) was carried out for 9 h at 90oC, and then the obtained pellets were washed with
water, dried at room temperature and calcined at 550oC for 8 h.
Functionalization of silica pellets
Briefly, 1 g of dry siliceous pellets was modified with amino groups
using 0.27 mL 3-aminopropyltrimethoxysilane, dissolved in 32.5 mL of dry
toluene, and gently stirred and refluxed at 85 °C, for 48 h. Then the
obtained pellets (MH-A) were dried on the air. To attach octyl groups,
dry silica pellets (1 g) were gently stirred and refluxed with 0.49 mL
of n-octyltriethoxysilane, dissolved in 32.5 mL dry toluene, at 85 °C,
for 48 h. After that the pellets (MH-O) were dried similarly to MH-A. To
attach both amino and octyl groups, 1 g of dry monolith carriers was
gently stirred and refluxed with 0.24 mL n-octyltriethoxysilane and 0.13
mL 3-aminopropylotrimethoxysilane dissolved in 32.5 mL of dry toluene,
for 48 h, at 85 °C, and then the pellets (MH-OA) were dried on the air.
Lipase immobilization on silica pellets
Prior to attachment of the lipase, pellets were washed with ethanol (5
h, 50 °C), then with distilled water (3 x 30 min x 30 mL), and after
that with 0.1 M phosphate buffer (pH 7.0; 3 x 30 min). Then the MH-O
pellets were immersed in 30 mL of the lipase solution in 0.1 M phosphate
buffer (pH 7.0), for 2.5 h, at 25 °C, and then left at 6 °C overnight.
After that they were washed with 30 mL of 0.1 M phosphate buffer (pH
7.0) for 30 min, to remove excess proteins, and finally filtered and
vacuum dried at 4 °C. The amount of immobilized enzyme was determined
with Lowry method, using bovine serum albumin as a standard. To attach
the lipase on to MH-A and MH-OA, the pellets were washed as described
before, and then activated with glutaraldehyde (2.5% glutaraldehyde
solution in 0.1 M phosphate buffer, pH 7.0). Excess of glutaraldehyde
was removed by washing with distilled water, then with 0.1 M phosphate
buffer pH 7 (3 x 30 min x 30 mL). Afterward, the pellets were immersed
in the lipase solution as described for MH-O, and then treated in the
same way.
Characterization of supports
The supports were characterized by elemental analysis (CHNS) using LECO
model TruSpec Micro elemental analyzer and by FTIR spectroscopy,
applying Nicolet iS50 instrument from Thermo Fisher Scientific. The
pellets were grated with KBR (5% mass of sample). The mesopore
structure of the carriers was determined by the nitrogen adsorption at
-196 °C, using a Micromeritics ASAP 2020 instrument, and in
meso- and macropore size range, by mercury porosimetry (Quantachrome
Pore Master 60). The hydrophobicity of the modified pellets was
determined by adsorption of Bengal Rose dye, using the method described
by Vescovi [36] and Lima [38] (details are given in
Supplementary Information).
Activity and stability of the biocatalysts in aqueous
system and in organic solvent
All experiments were carried out using a commercial RBR (SpinChem™) with
100 mL working volume. The hydrolysis activity of immobilized lipase was
determined using the procedure described by Gustafsson et al.
(Gustafsson, Thorn & Holmberg, 2011). The enzyme-activated pellets
(0.14 g) or 0.05 g N435 were placed in the reactor’s basket. The
substrate solution (15 mL of p- nitrophenyl acetate in
acetonitrile, 5.5 mM) dissolved in 50 mM phosphate buffer pH 7 (85 mL)
was placed in RBR. The reaction was carried out at 37 °C, at rotation
speed of 500 rpm. The amount of released p- nitrophenol was
measured spectrophotometrically at 400 nm. The calibration curve was
prepared using p- nitrophenol as standard. The operational
stability of the biocatalysts in aqueous media was checked in 4 runs.
After each run the biocatalysts were washed with 50 mM phosphate buffer
(pH 7.0).
In the case of esterification of levulinic acid, 0.355 M of levulinic
acid and 0.710 M of n-butanol were mixed with diisopropyl ether (total
volume of 100 mL), and incubated for 15 min. at 50 °C. After that 1 g of
the enzyme-activated pellets, or 0.24 g of N435, were placed in RBR
basket and mixed at rotation speed of 500 rpm. The amount of n-butyl
levulinate was determined using gas chromatography. The calibration
curve was prepared using n- butyl levulinate as a standard. To evaluate
the operational stability of the biocatalysts in organic solvent, the
reaction was carried out four times. After each run the biocatalysts
were washed with diisopropyl ether at room temperature.
To evaluate the stability of immobilized lipases in different organic
solvents, 0.1g of the catalyst was incubated with 1 mL of chosen organic
solvent (acetonitrile, ethyl acetate, hexane, butanol, acetone and
diisopropyl ether), at 37 °C for 24 h. Then the samples were dried under
vacuum and enzymatic activity was measured using hydrolysis ofp- NPA as a standard reaction. Additionally, the N435 pore
structure was examined by low temperature nitrogen adsorption and
mercury porosimetry.
RESULTS and DISCUSSION
Our earlier studies (Szymańska, Odrozek, Zniszczoł, Pudło & Jarzębski,
2017) showed that activity of the enzyme immobilized on silica pellets
strongly depends on pore structure in the macropore size range. Thus,
the catalysts with 20-40 µm (dia.) macropores proved to be much more
active than those with 10 µm pores or less, for the same (macro)pore
volume. For this reason, in the reported studies, the applied lipase
carriers (of conical shape) also had the very open pore structure, and
external sizes of 5.1 mm x 3.3 mm (H/d). Hg porosimetry showed an ample
presence of macropores 20-50 µm in size (pore volume ca. 3
cm3g-1) and mesopores 20-30 nm
(volume ca. 1 cm3g-1) (Fig.1). But
SEM images also revealed the presence of even larger macropores
interconnected in a tortuous network of flow-through channels (Fig.1).
These images also very clearly show openness of the structure and
accessibility of the internal surface. Large macropores and textural
mesopores of the silica skeleton enabled unhindered penetration by
various moieties. Modification of the surface appeared to affect
porosity in the meso- but not macropore-size scales. The specific
surface area (SBET, Table 1) of 310
m2g-1 before modification decreased
almost by half after amino groups implementation (MH-A), but only about
10% in the octyl group modified pellets (MH-O). The decrease in the
pore volume and its size after functionalization was insignificant, but
even then the modified carriers still offered large surface area easily
accessible for both the enzyme molecules and reactants. Moreover, the
applied RBR filled in one-third with the structured siliceous carriers
offers the surface-to-volume ratio of over 106m2 m-3. That is at least an order of
magnitude more than feature much acclaimed microreactors made of 100 µm
capillaries, the capacity of which is only a tiny fraction of the
reactor used in these studies (Strub et al., 2019).
Chemical character of the surface is a critical property for enzyme
immobilization. FTIR spectroscopy (Supporting Information, Fig. S1) was
used to confirm the presence of functional moieties on the support’s
surface. The unmodified silica featured a broad band between 3200
cm-1 and 3600 cm-1 that could be
ascribed to the Si–OH stretching vibrations, additionally a sharp and
intense band at 3740 cm-1, could be unambiguously
assigned to the OH symmetric stretching vibration of isolated silanol
groups (Karbowiak et al., 2010). Adsorption bands around 1000-1250
cm-1 and 800 cm-1 are attributed to
asymmetric and symmetric stretching vibrations of the Si-O-Si framework
(Ciemięga, Maresz & Mrowiec-Białoń, 2018). Appreciable changes in all
the spectra were observed after modification of the monolithic support
with organic moieties. First, the intensity of the band at 3740
cm-1 was notably reduced due to incorporation of
organic groups. The amino groups presence were identified by the
methylene stretching bands of the propyl chain in the region of
2850–2950 cm-1, seen in FTIR spectra of both MH-A and
MH-OA, and their deformation bands at 1410–1455 cm-1.
The N–H absorption bands overlapped with O–H bands at 3200–3600
cm-1 (Szymańska, Bryjak, Mrowiec-Białon & Jarzębski,
2007). Methylene stretching bands of the octyl chain (MH-O) in the
region of 2850–2950 cm-1 is poorly visible mostly
because of small amount of the incorporated octyl groups.
The amount of functional group present on silica surface was calculated
from elemental analysis data (Table 1) assuming complete hydrolysis and
subsequent condensation of ethoxy or methoxy groups from organosilica
precursor during functionalization. As can be seen (Table 1), the amount
of octyl groups on silica surface was at least two times lower than that
of amino groups, and this could be explained by much higher reactivity
of methoxysilanes than ethoxysilanes.
For lipases, hydrophobicity of the support is of particular importance
(Bernal, Illanes, & Wilson, 2014; Garmroodi et al., 2016; Ortiz et al.,
2019; Vescovi et al., 2016). In these studies, it was evaluated from the
amount of a hydrophobic compound (Bengal Rose dye) adsorbed on the
carrier. The chemical modification of the silica (Table 1) increased its
capacity to adsorb the hydrophobic dye from 0.0149 mg
g-1 (for pure silica MH) to 0.133, 0.233 and 0.311 mg
g-1, respectively for MH-O, MH-A and MH-OA. For MH-A
and MH-OA, the higher adsorption of Bengal Rose could be explained by an
ionic interaction between ionized groups present in both the dye and the
support (Vescovi et al, 2016). It is noteworthy that the amount of dye
adsorbed on MH-OA approximately equals to an aggregate of values found
for MH-A and MH-O. Thus, surface ability to adsorb Bengal Rose dye quite
well correlates with the amount of the attached lipase (Table 1).
Owing to specific character of the applied functional groups, for each
of the carriers the first step of lipase immobilization is adsorption on
the support, and in the case of MH-A and MH-OA it is followed by a
covalent stabilization of the enzyme by reactive moieties (amino groups)
(Vescovi et al., 2016). Since CALB is the same enzyme as the one applied
in N435, we could directly compare its content in all the catalysts. It
is noteworthy that amino functionalized silica appeared to bound more
enzyme than the silica grafted with octyl groups, owing to ionic forces
which hugely impact the lipase immobilization yield. Thus not
surprisingly, the largest amount of protein was attached to
heterofunctionalized silica (MH-OA, 27 mg/g). But even then, it was at
least two times less than determined for N435 (Table 1).
Once the MH family of catalysts was prepared we investigated the effect
of organic solvents treatment – 24 h soaking - on catalytic activity,
also of N435, and confronted it with the changes in its pore structure.
As can be seen from Table 2 (and also Fig. S2 and Fig. S3, SI) even the
most typical organic solvents appeared to exert a significant effect on
pore structure of N435, and surprisingly enough, it appeared to be
different in micrometer and nanometer scales. Only the diisopropyl ether
slightly reduced porosity fairly symmetrically in both domains, in a
stark contrast to butanol, acetonitrile and ethyl acetate, which caused
a nearly total collapse of mesopores (Fig. S3, SI). First two of the
latter solvents had little effect on the volume of very large pores
(macropores), unlike ethyl acetate in which that volume nearly doubled,
a clear sign of a significant restructuring and swelling of Lewatit
(Fig.S3, SI). These structural changes of the polymeric support are
likely to be accompanied with, and stimulate the release of various
chemical entities to the solvent/reaction system, as was first reported
by Zhao & Song (2010) and most recently also by other authors (Jose &
Briand, 2010; Jose et al. 2011). In the reported study we did not tackle
this issue in more detail.
Investigations of the catalysts activity after the solvent treatment
showed (Table 2) that a hydrolytic activity of the N435 remained high,
except for ethyl acetate and somewhat less acetonitrile, which proved to
be detrimental. It can be observed that a notable decrease of N435
activity quite well correlates with major changes in pore structure of
the polymeric support (Table 2, Fig. S2 and Fig. S3). However, the
effect of organic solvents on catalytic activity of the supported
enzymes is likely far more complex; we did not go much into these
details. More importantly, in a stark contrast to N435 all of the
functionalized silica-supported biocatalysts proved not susceptible to
organic solvents, and they were consistently highly active (Table 2).
Owing to some sort of a symmetrical effect on both enzyme carriers, the
diisopropyl ether was selected to be used in a more systematic study of
both N435 and the MH-series of biocatalysts.
First, the biocatalysts were tested in the hydrolysis ofp- nitrophenyl acetate in a phosphate buffer (pH 7.0) using RBR
operating at 500 rpm (centrifugal acceleration ca. 11.5g ). In
this reaction the highest activity showed N435, whereas activity of MH-O
and MH-OA was lower, but much similar in value (Fig. 2A). Least active
appeared to be MH-A, even though its enzyme load was comparable with
MH-O. If however, the catalytic activity was related to the amount of
the immobilized lipase, the most active appeared to be MH-O, followed by
MH-OA (Fig. 2B). This can be partly explained by much better
accessibility of the lipase, but mainly by a hydrophobic character of
the silica surface and its huge effect on conformation and hence
activity of the attached lipase. The latter is usually termed as an
‘interfacial activation’ and it has been extensively studied before
(Ortiz et al., 2019).
From a practical point of view, more important than catalyst’s activity
is its stability, and it was checked in four successive cycles. N435,
MH-A and MH-O showed (Fig. 3) very good stability, whereas the MH-O
attached lipase was strongly leaching from the carrier, and it is in
accord with previous observations (Ortiz et al., 2019). Thus, a
hydrophobic interaction between the lipase and the carrier, while
essential for expressing lipase activity, was not strong enough to
counter a water-protein affinity and also the effect of forces exerted
by the flowing fluids. To conclude, taking into account the catalytic
activity and stability, and also the lack of susceptibility to organic
solvents, the heterofunctional MH-OA emerges as a rational option for
the lipase-catalyzed hydrolytic reaction. Note that in RBRs the
potentially lower catalytic activity can easily be compensated by much
larger catalyst load (3.5 g of MH-type of catalysts in the applied
reactor), so to give in effect very high values of space-time yield. For
that packed with MH-OA catalyst the STY value was about 2 g
h-1 L-1, and it is respectable.
Activity of the biocatalysts was also checked in the esterification of
levulinic acid dissolved in diisopropyl ether (Fig. 4). Similarly as in
the hydrolysis reaction the highest activity (per gram of catalyst)
showed Novozym 435 (Fig. 4A); the one revealed by MH-O was notably lower
but still more than twice the value of heterofunctional MH-OA, even
though the latter bound twice as much proteins (Table 1). Guajardo et
al. (Guajardo, Bernal, Wilson & Cabrera, 2015) also reported lower
activity of CALB immobilized on heterofunctional supports:
octyl-silica-epoxy and undecanol-Sepharose-glyoxyl, than on
monofunctional carriers: octyl-silica and octyl-Sepharose, and the same
trend was also reported when CALB-on-octyl-agarose-glyoxyl system was
compared with that of CALB-on-octyl-agarose (Hirata et al., 2016). Most
likely, additional covalent links of the hydrophobic support with the
lipase attached in its open/active form, stabilized the system thus
preventing enzyme leaching, but they also contributed to an excessive
stiffness of the attached protein, which hampered activity (Guajardo,
Bernal, Wilson & Cabrera, 2015; Stauch, Fisher & Cianci, 2015; Vescovi
et al., 2016). Similarly as in the case of hydrolytic reaction, the
highest specific activity, i.e. refereed to the amount of attached
proteins, featured MH-O-based biocatalyst (Fig. 4B), and that of N435
was only slightly lower, whereas the lipase immobilized on MH‑A showed
no activity at all. All of the tested catalysts demonstrated very good
stability in the esterification of levulinic acid (Fig. 5).
To summarize, N435 emerges as the most effective catalyst for the
esterification reaction under study, with a reservation that the
reported results apply to the diisopropyl ether as a solvent. If another
organic solvent is applied in this or related reaction, we may also
expect to observe a Levatit-solvent interaction (cf. Table 2) the final
outcome of which, while hard to predict, is likely to be negative from a
catalytic perspective. In this respect, the use of MH-O-supported lipase
which entails no such problems may be worth considering.
CONCLUSIONS
The biocatalysts made of the enzyme – lipase, supported on structured
siliceous beads functionalized with octyl and amino groups are highly
active and stable in organic solvents, in contrast to Novozym 435 which
degrades in similar situations. Grafting with both octyl- and amino
entities appeared most effective in hydrolytic application – the
catalysts were stable and highly active. For reactions in organic
solvents structured beads grafted with octyl groups are recommended. The
proposed enzyme supports can easily be modified with various functional
groups and enzymes, according to the needs, and applied in rotating bed
reactors to give highly efficient synthetic systems.