Activation of enzyme nanogel in organic solvents by PEG-substrate joint imprinting

Article abstract of DOI:10.1039/c4ra06660j

A substrate or polyethyleneglycol (PEG) imprinted lipase nanogel displayed increased apparent activity in organic solvents by 2.5-4.7 folds, compared to native lipase. It enabled a one-step synthesis of chloramphenicol palmitate with a yield of ～99% and purity of ～99%, indicating that the imprinted lipase nanogel is an appealing catalyst in organic media. This journal is

Full text of DOI:10.1039/c4ra06660j

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Activation of enzyme nanogel in organic solvents
by PEG–substrate joint imprinting†
Cite this: RSC Adv., 2014, 4, 40301
*
*
Rui Wang, Yifei Zhang, Jun Ge and Zheng Liu
Received 4th July 2014
Accepted 13th August 2014
DOI: 10.1039/c4ra06660j
www.rsc.org/advances
A substrate or polyethyleneglycol (PEG) imprinted lipase nanogel the mechanism underpinning the enhanced apparent activity,
displayed increased apparent activity in organic solvents by 2.5–4.7 using PEG and substrate.
folds, compared to native lipase. It enabled a one-step synthesis of
Fig. 1 shows the preparation of the lipase-encapsulated
chloramphenicol palmitate with a yield of ꢀ99% and purity of ꢀ99%, polyacrylamide nanogel and the imprinting procedure. The
indicating that the imprinted lipase nanogel is an appealing catalyst in transesterication reaction between chloramphenicol and vinyl
organic media.
palmitate to produce chloramphenicol palmitate was selected
to examine the catalytic performance of the lipase nanogel with
different imprinting treatments. Lipase from Thermomyces
lanuginosus (lipozyme TL 100 L) was selected as the model
enzyme and encapsulated into a nanogel using an aqueous two-
step in situ polymerization (Fig. 1).5 The acryloylation of lipase
with N-acryloxysuccinimide (NAS) was conducted at 30 ꢁC in
acetic buffer (pH 5.0, 50 mM) for 6 h, which generated vinyl
groups on the surface of lipase for subsequent polymerization
at room temperature using ammonium persulfate and
N,N,N0,N0-tetramethylethylenediamine as initiators, and acryl-
amide as the monomer. The encapsulation yield of lipase in the
nanogel was around 99%. The hydrolytic activity yield was 76%
with reference to its native counterpart.
The enzymatic synthesis of ne chemicals and therapeutic
molecules in organic solvents has been extensively pursued to
achieve a high chemo-, regio-, and stereo-selectivity for catalytic
reactions utilizing hydrophobic substrates and/or inhibiting the
adverse effects of water.1 However, most enzymes exist in an
aggregated and rigid form in organic solvents. This makes the
active site of the enzyme less accessible to its substrate and, also
hinders the conformational transition of the protein required
for catalysis.1 Thus, these reduce the apparent enzyme activity
by one to several orders of magnitude, as compared to that in an
aqueous solution.2 The recent advancement in nanostructured
enzyme catalysts3 resulted in the unlimited opportunities for
modifying the nature-made enzymes with tailor-made chemical
structures and functionalities to overcome the abovementioned
problems. We have demonstrated that nanostructured mate-
rials, such as polymer nanogels,4–6 inorganic nanoowers,7 and
polymer nanoconjugates8,9 are effective in enhancing the
performance of enzymes. Moreover, the apparent activity of the
enzyme-polymer nanogel, which is prepared by aqueous in situ
polymerization,5 in organic solvents can be elevated by
substrate imprinting, a method known to enhance the substrate
accessibility.10 Occasionally, we found that imprinting with
PEG, which will be discussed in the text, did not increase the
substrate accessibility, but led to a more signicant increase in
the apparent activity. Thus, we directed our efforts to explore
Transesterication between p-nitrophenyl palmitate and
n-butyl alcohol in n-heptane was selected as a standard assay to
evaluate the activity of the native lipase and lipase nanogels
with different treatments in organic media. The apparent
activity of the lipase nanogel and the mixture of lipase with
Department of Chemical Engineering, Tsinghua University, Beijing 100084, P. R.
China. E-mail: junge@mail.tsinghua.edu.cn; liuzheng@mail.tsinghua.edu.cn
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra06660j
Fig. 1 Synthesis of imprinted lipase nanogels and application in the
enzymatic synthesis of chloramphenicol palmitate in organic solvents.
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polyacrylamide was 219% and 171% compared to that of the lipase nanogel and the palmitic acid imprinted one was 189%
native lipase, respectively. The increased apparent activity and 293%, respectively, compared to the native lipase imprinted
induced by polyacrylamide, as shown by both the mixture and with palmitic acid showed activity similar to the native one. To
the nanogel, is attributed to the improved dispersion of the investigate the mechanism of the increased activity of the pal-
lipase in organic solvents and consequently, the accessibility to mitic acid imprinted lipase nanogel, we compared the adsorp-
substrates. This is more signicant in the case of lipase nano- tion of palmitic acid by these imprinted lipase nanogels to that
gel, in which the enzyme molecules are more evenly encapsu- by non-imprinted lipase nanogel. FITC-labeled palmitic acid
lated and are covalently bound with the polyacrylamide porous with a concentration of 0.05 mg mLꢂ1 in acetonitrile was added
network.
to 0.1 mg of imprinted (or non-imprinted) lipase nanogels at
Palmitic acid, and PEG with molecular weights of 200, 1000, 25 ꢁC, followed by an immediate analysis of the uorescence
4000, and 12 000 were selected to prepare the imprinted lipase intensity within the lipase nanogels under a laser scanning
nanogel. During the experiment, palmitic acid or PEG was confocal microscope (LSCM). The increase in the uorescence
added to an acetic buffer (50 mM, pH 5.0) solution containing intensity within the lipase nanogel in a selected region of
the pre-synthesized lipase nanogel (2 mg mLꢂ1 of protein) with interest reects the adsorption of the substrate. Fig. 3a–c
the molar ratio of the imprinting molecule to lipase being 5 : 1, present the uorescence images of FITC-labeled palmitic acid
with vigorous stirring. Then, the mixture was lyophilized and adsorbed by the palmitic acid imprinted lipase nanogel,
subjected to extraction with toluene to remove palmitic acid or showing a remarkable increase in the uorescence intensity
PEG, resulting in the imprinted lipase nanogel in powder form. aer 0 s, 250 s, and 500 s incubation. As shown in Fig. 3d, the
The transmission electron microscopy (TEM) images of the palmitic acid imprinted lipase nanogel showed a higher
negatively stained non-imprinted lipase nanogel, lipase nanogel adsorption rate and capacity than the non-imprinted lipase
imprinted with palmitic acid and PEG are shown in Fig. 2a–c, all nanogel. From the abovementioned facts it is concluded that
of which appear spherical in shape with diameters ranging from the substrate imprinting favors the substrate uptake and thus
20 to 30 nm. The scanning electron microscopy (SEM) images of results in a higher apparent activity.10
the lyophilized non-imprinted, palmitic acid and PEG imprin-
For the PEG imprinted lipase nanogel having a non-porous
ted lipase nanogels in powder form are shown in Fig. 2d–f. Both structure (Fig. 2f), the mass transfer hindrance is expected to be
the non-imprinted and the palmitic acid imprinted lipase signicant and thus is disadvantageous for the catalysis.
nanogels show porous structure, which is advantageous for the Surprisingly, the PEG imprinted lipase nanogel gave a relative
substrate to access the enzyme encapsulated in nanogels. The activity of 223–313% compared to native lipase (Table 1), while
mercury intrusion porosimetry method was used to analyze the the PEG imprinting increased the apparent activity of the native
specic surface area of different forms of lipase nanogels, and lipase by 169–252%. To elucidate the mechanism underpinning
showed that the palmitic acid imprinted lipase nanogel has the the enhanced activity, we examined the adsorption of the
largest total pore area (181.917 m2 gꢂ1), whereas the non- substrate by the PEG imprinted lipase nanogel using the LCSM
imprinted lipase nanogel has a smaller one (88.127 m2 gꢂ1). In method under the abovementioned conditions. As shown in
contrast, from the SEM image, the PEG imprinted lipase Fig. 3d, the PEG imprinted lipase nanogel showed considerably
nanogel did not appear a porous structure, with a total pore area slow adsorption of palmitic acid compared to that for both the
of 63.327 m2 gꢂ1
.
non-imprinted and the palmitic acid imprinted lipase nanogel.
The abovementioned lipase nanogels were subjected to the This indicates the transport of the substrate in the PEG-
transesterication reaction between p-nitrophenyl palmitate imprinted lipase nanogel is hindered, as expected from the non-
and ethanol in n-heptane, using native lipase as the reference. It porous structure illustrated by SEM (Fig. 2f). Considering the
is shown in Table 1 that the relative activity of non-imprinted improved apparent activity achieved at a hindered mass trans-
port of the substrate, it is concluded that the increased apparent
activity of PEG-imprinted lipase nanogel is attributed to the
activation effect of PEG. Otero, et al.11 has reported that PEG
Table 1 Relative activities of different preparations of the lipase
catalysts compared with that of native lipase
Apparent activity (%)
Imprinting molecule
Native lipase
Lipase nanogel
Non-imprinted
Palmitic acid
PEG (Mw: 200)
PEG (Mw: 1000)
PEG (Mw: 4000)
PEG (Mw: 12 000)
PEG (Mw: 1000) + palmitic acid
100.00
89.00
189.05
292.66
281.69
313.15
255.31
223.12
476.30
220.58
252.22
247.78
169.54
240.01
Fig. 2 TEM images of (a) non-imprinted, (b) palmitic acid imprinted, (c)
PEG imprinted lipase nanogel. SEM images of the powder of (d) non-
imprinted, (e) palmitic acid imprinted, (f) PEG imprinted lipase nanogel.
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between the two hydroxyl groups,4,15,16 may enable a one-step
synthesis of the target molecule. Using native lipase as the
control, the lipase catalysts prepared with the above mentioned
imprinting procedures were subjected to the synthesis of
chloramphenicol palmitate in acetonitrile at 20 ꢁC. Enzyme
samples applied in the transesterication reaction including
free lipase, lipase nanogel and imprinted lipase nanogel were
all obtained in powder form by lyophilisation, with the same
water content of ꢀ5% w/w as measured by the Karl-Fischer
titration. As shown in Fig. 4, the native lipase gave a yield below
40% in 24 h, which was mainly because of the lipase deactiva-
tion caused by the extraction of the ‘essential water’ by aceto-
nitrile. The non-imprinted lipase nanogel, which inhibited the
extraction of essential water, elevated the yield to ꢀ99% in
24 h.4 The palmitic acid imprinted lipase nanogel took only 12 h
to reach a ꢀ99% yield because of the facilitated uptake of the
substrate and the preservation of the essential water of the
enzyme by the polyacrylamide network. This conclusion was
supported by measuring the water content in reaction media of
the catalysis by using different enzyme samples. For native
Fig. 3 LSCM images of palmitic acid-imprinted lipase nanogel incu-
bated with FITC-labeled palmitic acid in acetonitrile for (a) 0 s, (b) 250
s, (c) 500 s. (Red circles indicate the selected region for analysis). (d)
Increase in relative fluorescence intensity within different lipase
nanogels (all data were obtained from three parallel experiments).
induces a conformational change, which uncovers the ‘lid’ lipase, the water content in the acetonitrile solution was
covering the active site of lipase. It is possible that in our study measured as 0.01% w/w, while for lipase nanogel (non-
the activated conformation of lipase attributed to PEG imprinted and imprinted) it was 0.002% w/w which demon-
imprinting was retained in organic solvents, giving an elevated strates the capability of preservation of water by polyacrylamide
catalytic activity.
shells. The PEG (Mw: 1000) imprinted lipase nanogel displayed
The above results encouraged us to probe a new imprinting similar performance compared to that of the substrate
strategy, which is using PEG and substrate simultaneously, to imprinted one, which again conrmed the activation effect of
achieve a facilitated substrate uptake and an activated enzyme. PEG. The lipase nanogel jointly imprinted with PEG and pal-
This idea was validated by an improved apparent activity, up to mitic acid reached a ꢀ99% yield within only 6 h, because of
476% compared to that of native lipase (Table 1), in the trans- both the activation effect by PEG imprinting and the facilitated
esterication reaction between p-nitrophenyl palmitate and uptake of substrate by palmitic acid imprinting. Interpreted
ethanol. The substrate adsorption behavior of the palmitic from the initial reaction rate, the catalytic activity of the non-
acid–PEG imprinted lipase nanogel was also examined under imprinted, palmitic acid imprinted, PEG imprinted and pal-
the same conditions. The increase in the uorescence intensity mitic acid–PEG imprinted lipase nanogel was increased by 4.0,
within the palmitic acid–PEG imprinted lipase nanogel in a 6.8, 8.2, 15.5 fold, respectively, compared to the native lipase.
selected region of interest is shown in Fig. 3d, which indicates Moreover the two by-products, (1R,2R)-2-[(dichloroacetyl)
that the adsorption rate by the PEG and palmitic acid jointly amino]-1-(4-nitrophenyl)-3-(palmitoyloxy)propylpalmitate
imprinted lipase nanogel was lower than that of the palmitic [(20R,30R)-chloramphenicol 10,30-dipalmitate and (1R,2R)-2-
acid imprinted one, but higher than the PEG imprinted one. [(dichloroacetyl)amino]-3-hydroxy-1-(4-nitrophenyl)propyl dec-
The specic area analysis showed that the PEG and palmitic anoate (20R,30R)-chloramphenicol 10-palmitate, which are the
acid jointly imprinted lipase nanogel has a total pore area of 10,30-di-substituted product and the 10-mono-substituted
109.381 m2 gꢂ1. The complementary input of activity and product were not detected by high performance liquid
structure assay led us to the conclusion that the increased
activity shown by the palmitic acid–PEG jointly imprinted lipase
nanogel can be attributed to the activation effect by PEG
imprinting and increased accessibility of the substrate by pal-
mitic acid imprinting. Although the molecular imprinting12,13
and PEG treatment11 of native enzymes have been well studied
to increase enzymatic activity in organic media, in this study, we
for the rst time demonstrated that the substrate and PEG joint
imprinting can signicantly increase the apparent activity of
lipase nanogel, which is a robust nanostructured enzyme cata-
lyst with promising applications in industrial biocatalysis.
The chemical synthesis of chloramphenicol palmitate, a
broad-spectrum bacteriostatic antimicrobial already used in
clinics14 requires selective modication with the protection and
Fig. 4 Yields of chloramphenicol palmitate using different lipase
deprotection steps. Lipase, which exhibits regioselectivity catalysts.
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chromatography (HPLC),17 suggesting that the imprinted lipase
nanogel gave the expected regioselectivity.
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In summary, we described a PEG and substrate joint
imprinting method, which activates the encapsulated lipase
and facilitates the uptake of the substrate, leading to an
enhanced apparent catalytic activity in organic media. The
enzyme-catalyzed transesterication reaction between chlor-
amphenicol and vinyl palmitate demonstrated the high
performance of the PEG and substrate jointly imprinted lipase
nanogel and its advantages in terms of enhanced stability,
improved apparent activity, in addition to the high selectivity,
making the imprinted enzyme nanogels appealing for indus-
trial biocatalysis.
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This work was supported by the National High Technology
Research and Development Program (“863” Program) of China
under the grant number of 2014AA020507, the National Natural
Science Foundation of China under the grant number of
21036003 and 21206082, Tsinghua University Initiative Scien-
tic Research Program under the grant number of
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