A. Szelwicka et al.
Applied Catalysis A, General 574 (2019) 41–47
dicarboxylic acids in the literature.
thermobalance. Samples (approximately 10 mg) were heated from 25 °C
On the other hand, the application of enzymes generates potential
problems such as possible mechanical disintegration of the enzyme
structure, low stability of native lipases in organic environments and
sensitivity for pH and temperature changes [6]. As a consequence, the
stability of lipase may decrease and recycling of the biocatalyst could be
troublesome or economically impractical. Those difficulties are even
more striking for the development of continuous processes [7]. As we
have previously verified, Novozyme-435 was not promising for re-
cycling [8] and could be destroyed by mechanical damage. This resin
swells upon a contact with organic solvent and the enzyme loses its
activity. These problems could be overcome via immobilization of en-
zyme on a more mechanically stable solid support. Depending on the
support nature, the final features of the immobilized enzyme may be
different. Therefore, the selection of the support may be a critical step
in the design of a enzymatic biocatalyst [9].
Under favorable conditions, lipases can be immobilized via the hy-
drophobic surrounding of their active center, fixing them in an open
conformation. Interfacial activation on hydrophobic supports at low
ionic strength has been reported to be a simple and efficient method to
immobilize lipases on various supports [8–11]. The immobilization of
lipase on solid supports may additionally improve enzyme rigidity,
stability and integrity of the lipase-support hybrid [12,13].
to 800 °C at a rate of 10 °C/min in standard 70 μL Al
2 3
O crucibles under
a dynamic nitrogen flow of 60 mL/min.
Transmission Electron Microscopy (TEM) images were obtained
using a Tecnai F20 TWIN microscope (FEI Company, USA) equipped
with field emission gun, operating at an acceleration voltage of 200 kV.
Images were recorded on the Eagle 4k HS camera (FEI Company, USA)
and processed with TIA software (FEI Company, USA). The samples
were prepared on a copper grid with holey carbon film. Scanning
electron microscopy (SEM) images were obtained with a Phenom Pro
Desktop SEM instrument at accelerating voltage 15 kV and energy-
dispersive X-ray spectroscopy (EDX) used detector BSD full.
Nitrogen adsorption/desorption isotherms for carbon materials
were obtained using a Micrometrics ASAP 2420 M instrument at
−196 °C to calculate their specific surface areas (SBET) and pore vo-
lumes. The pore size was obtained using the Barrett–Joyner–Halenda
(BJH) method with the Kruk–Jaroniec–Sayari correction. Prior to the
experiments, samples of supports were outgassed at 200 °C and
−
3
1.33 × 10
Pa for 5 h. The sample of the (nano)biocatalyst was out-
−
3
gassed at 140 °C and 1.33 × 10
Pa for 5 h.
The presence of lipase in the filtrate after six reaction cycles was
determined using Lowry’s UV-VIS method of protein detection. UV-VIS
spectra (λ = 670 nm) were recorded on a Jasco V-650 spectro-
photometer at room temperature in aqueous solution.
Recently, nanoscale materials have opened opportunities in the field
of nanobiocatalysis [14–16]. Carbon nanomaterials have been de-
scribed as versatile supports for enzyme immobilization due to their
small size, large surface area, mechanical and thermal stability and
other unique properties. The simple combination of a nanoscale support
and an enzyme has led to a much higher enzyme loading and, more
importantly, increased enzyme stability [16]. The economy of processes
utilizing carbon nanotubes (CNTs) has become increasingly favorable as
the prices of industrial-grade MWCNTs (multi-walled carbon nano-
2
2
.2. Synthetic procedures
.2.1. Synthesis of pristine MWCNTs, N-MWCNTs, Fe-MWCNTs, ultra-
long curly MWCNTs (ulc-MWCNTs) and helical CNTs (h-CNTs)
Pristine MWCNTs, high Fe-content MWCNTs (Fe-MWCNTs) and ulc-
MWCNTs [17] were synthesized via catalytic chemical vapor deposition
(c-CVD) using a slightly modified protocol [18]. N-MWCNTs were
grown according a previously described protocol [19]. h-CNTs were
synthesized also via c-CVD using addition of copper microflakes
™
tubes) have achieved the level from 100$ per 1 kg (Nanocyl NC7000
™
MWCNTs) to 200$ per 1 kg (Cheap Tubes MWCNTs). In our previous
work, nanobiocatalysts from lipase non-covalently immobilized on
MWCNTs for the Baeyer-Villiger synthesis of lactones have been de-
monstrated [8]. Consequently, we deemed it important to test its po-
tential as a catalytic system in esterification – considered as an ex-
emplary enzyme-catalyzed reaction of a major importance.
Herein, we present the design of an alternative, highly stable and
active nanobiocatalyst based on Candida antarctica lipase B anchored
onto carbon nanomaterials as a catalytic system for the synthesis of
plasticizers based on aliphatic diesters (dicarboxylates) with the po-
tential to become industrially relevant processes. Our work seeks to
realize the challenge of environmentally and economically sustainable
processes yielding safer and environmentally friendly industrial-scale
polymer additives.
(
1–10 wt.%) to a ferrocene/toluene feedstock as the catalyst growth
under otherwise unchanged conditions.
2
.2.2. Immobilization of the Candida antarctica lipase B on nanocarbon
solid supports
The immobilization step was carried out according to a previously
published procedure [8]. A Candida antarctica lipase B (0.25 g – 1 g),
nanocarbon solid support (0.1 g) and demineralized water (3 mL) were
introduced into a 100 mL round bottom flask. The immobilization step
was carried out for 3 h at 20 °C in a thermostatic shaker (180 rpm).
After that, the mixture was filtered under vacuum and washed with
20 mL of demineralized water. The catalyst was then dried for 3 d in a
desiccator at 5 °C.
2. Experimental
2.1. Materials and methods
2.2.3. General esterification procedure
A (nano)biocatalyst (10–200 mg/l mmol of dicarboxylic acid) was
introduced into a 10 mL round-bottom flask. Next, decane (20 wt. % per
acid, internal standard), solvent (0–2 mL/L mmol of acid), dicarboxylic
acid (1.0 mmol) and alcohol (2.0–32.8 mmol) were successively added.
The reaction mixture was then inserted into the thermostatic shaker
(250 rpm) at 25–45 °C and the reaction was carried out for 2–24 h.
During the reaction, 10 μl of the samples (diluted with acetonitrile)
were periodically collected to monitor the reaction progress by GC-FID.
After the completion of the reaction, the (nano)biocatalyst was filtered
and washed with 20 mL of cyclohexane. The filtrate was concentrated
using a rotary evaporator (7 mbar, 110 °C, 6 h for di-n-butyl esters and
5 mbar, 135 °C, 8 h for 2-ethylhexanol esters) to remove cyclohexane
and alcohols. The esters were purified by column chromatography
Solvents, alcohols, aqueous solutions of Candida antarctica Lipase B
and decane were purchased from Sigma-Aldrich; all acids were pur-
chased from Chemat, Poland. Industrial grade MWCNTs were pur-
™
chased from Cheap Tubes Inc. (United States), Nanocyl NC7000
MWCNTs were purchased from Nanocyl (Belgium); active carbon and
graphite were commercial materials obtained from Avantor
Performance Materials (Poland) and the United Quantum Factory
Poland), respectively.
GC analyses were performed using a SHIMADZU GC-2010 Plus
(
equipped with a Zebron ZB-5MSi column (30 m × 0.32 mm × 0.25 μm
1
13
film). H NMR spectra were recorded at 600 MHz and C NMR at
1
50 MHz (Varian system).
Lipase loadings on the surface of the carbon materials were de-
using Al
2 3 2 2
O as the stationary phase and CH Cl as the eluent. NMR
termined by thermogravimetry (TGA) using a Mettler Toledo TGA851e
spectra are available in Supplementary Information (Figs S9-S24).
42