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ChemComm
Page 3 of 4
DOI: 10.1039/C7CC00607A
ChemComm
COMMUNICATION
affect the configuration of the protein nor impose obvious mass-
transfer limitations, we observed nearly the same levels of enzyme
activity as that of the free enzyme in solution. The high degree of
retained enzyme activity confirmed our hypothesis that by using
metal ion-surfactant complexes, the encapsulated enzyme can
exhibit higher levels of activity as compared with other enzymes
encapsulated within inorganic nanocrystals and metal-organic
frameworks.3,5 And PDA@CRL-MSNC has similar pH stability with
native CRL (Fig. S4).
Free enzymes usually undergo denaturation in highly polar
organic solvents, resulting in their exhibiting almost no activity
following exposure to these solvents, such as ethanol, methanol, or
dimethyl sulfoxide (DMSO).23–25 The poor stability of enzymes in
polar organic solvents limits their application in reaction mediums
often necessary for simultaneously dissolving both hydrophobic and
hydrophilic substrates. In this study, we observed that the CRL-
MSNC and PDA@CRL-MSNC retained >68% and >88% of their
original activities, respectively, following incubation in pure ethanol,
methanol, and DMSO for 5 min at room temperature (Fig. 3b). By
contrast, free lipase exhibited no activity under the same
conditions. The greatly enhanced enzyme stability displayed by CRL-
MSNC and PDA@CRL-MSNC in polar organic solvents was
comparable to that of enzymes incorporated in metal-organic
frameworks.5,6 One possible explanation of the high degrees of
enzyme stability in polar organic solvents is that the encapsulation
of enzymes in hydrophilic nanoconfined spaces enhances
protection of the protein structure against the denaturing effect of
polar organic solvents, thereby allowing the retention of essential
water molecules the protein surface.26,27 Moreover, after PDA
coating, the PDA@CRL-MSNC retained >93% of the original activity
after 10 cycles of reuse in aqueous solution, suggesting a higher
degree of reusability for PDA@CRL-MSNC as compared with CRL-
MSNC (Fig. 3c and d).
Fig. 4 (a) CRL-catalyzed synthesis of vitamin E succinate. (b)
Synthesis of vitamin E succinate by different types of lipase. (c)
Reusability of CRL-MSNC and PDA@ CRL-MSNC in DMSO. (d) Effect
of reaction time on the synthesis of vitamin E succinate catalyzed by
PDA@CRL-MSNC and free CRL.
this reaction system. By contrast, CRL-MSNC lost most of its activity
after only one use, primarily due to the disassociation of CRL-MSNC
in DMSO after >30 min (Fig. S6). The PDA coating on the CRL-MSNC
created more covalent and noncovalent interactions between the
PDA shell and the CRL-MSNC, which produced insoluble and stable
black nanoparticles (Fig. S6) and strengthened the structural
stability of the biocatalyst in DMSO. Comparison of time-course
experiments measuring the synthesis of vitamin
E succinate
catalyzed by PDA@CRL-MSNC and free CRL (Fig. 4d) revealed that
use of free CRL as the catalyst resulted in <20% conversion after 12
h due to the poor stability of CRL in DMSO. A previous study18 also
reported a slow reaction rate for the synthesis of vitamin E
succinate using CRL as the catalyst (a yield of 46.95% after 18 h),
supporting the poor stability of CRL in DMSO. Using PDA@CRL-
MSNC as the catalyst increased the initial reaction rate 4-fold as
compared with that observed using free CRL, with a conversion
rate of ~80% after a 6-h reaction.
The high degree of stability of CRL-MSNC and PDA@CRL-MSNC
allowed us to investigate their application for the lipase-catalyzed
synthesis of vitamin E succinate in organic solvents (Fig. 4a). This
reaction requires the use of highly polar organic solvents to dissolve
the substrates. Here, we first investigated the influence of polar
organic solvents [including ethanol, acetonitrile, methanol,
dimethylformamide (DMF), and DMSO] on the reaction catalyzed by
free CRL (see the ESI† for experimental details). As shown in Fig. S5,
the yield of vitamin E succinate was higher in DMSO as compared
with that observed in other solvents, including ethanol, acetonitrile,
methanol, and DMF (~12% conversion of vitamin E at a substrate
molar ratio of 1:5 at 55°C for 4 h). Under the same conditions and
using CRL-MSNC and PDA@CRL-MSNC as the catalysts (Fig. 4b),
conversion of vitamin E reached ~55% after 4 h at 55oC, which was
>4-fold higher than that observed for free CRL. Additionally,
compared with commercially available lipase catalysts, such as
Lipozyme Thermomyces lanuginosus lipase (TLIM) and Novozyme
435 (immobilized lipase), CRL-MSNC and PDA@CRL-MSNC exhibited
enhanced levels of substrate conversion.
In conclusion, we described
a co-precipitation method to
encapsulate enzymes in metal ion-surfactant nanocomplexes. These
complexes can be further cross-linked with PDA to form a highly
effective nanobiocatalyst capable of exhibiting both high levels of
activity and stability, especially in protein-denaturing polar organic
solvents. This new type of lipase-encapsulated metal ion-surfactant
nanocomposite exhibited much higher catalytic capability, stability,
and reusability in the presence of polar organic solvents as
compared with free lipase and commercially available immobilized
lipases, thereby demonstrating its potential for applications
requiring enzymatic catalysis under harsh conditions.
This work was supported by the National Key Research and
Development Program (2016YFA0204300, 2014AA021703), and the
Synergetic Innovation Center for Advanced Materials.
We then investigated the recyclability and long-term use of CRL-
MSNC and PDA@CRL-MSNC for the aforementioned reaction. As
shown in Fig. 4c, the relative activity of PDA@CRL-MSNC decreased
by 55% after 10 rounds of use, suggesting adequate reusability for
Notes and references
1
J. Ge, J. Lei and R. N. Zare, Nat. Nanotechnol., 2012, 7, 428.
This journal is © The Royal Society of Chemistry 20xx
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