Molecular hydrogel-immobilized enzymes exhibit superactivity and high stability in organic solvents

Article abstract of DOI:10.1039/b615223f

This communication describes the use of a molecular hydrogel to immobilize enzymes for catalyzing reactions in an organic solvent to attain superactivity and exceptional stability. The Royal Society of Chemistry.

Full text of DOI:10.1039/b615223f

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COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Molecular hydrogel-immobilized enzymes exhibit superactivity and high
stability in organic solvents{
Qigang Wang, Zhimou Yang, Ling Wang, Manlung Ma and Bing Xu*
Received (in Cambridge, UK) 19th October 2006, Accepted 16th January 2007
First published as an Advance Article on the web 2nd February 2007
DOI: 10.1039/b615223f
This communication describes the use of a molecular hydrogel
to immobilize enzymes for catalyzing reactions in an organic
solvent to attain superactivity and exceptional stability.
Despite that enzymatic catalysis in organic solvents offers some
1
technological advantages (e.g., enhanced stability of enzymes,
easy recovery of products, and novel biosynthesis) for a variety of
applications and has already led to some successful commercial
1,2
processes, enzymes, however, often display drastically lower
3
activity in organic solvents than in water. It is believed that the
Fig. 1 Illustration of the molecular hydrogel-immobilized enzymes that
catalyze a reaction in organic media (E: enzymes; S: substrates; P:
products).
water layer on the molecular surface of enzymes determines their
1
activity in organic media, and there are three major causes of the
low activity—unfavorable substrate desolvation, suboptimal pH,
3
and reduced conformational mobility. Among several known
4
approaches to remedy these problems, it is quite effective to
nanofibers via the control of the structure of hydrogelators may
provide an optimal microenvironment for enzyme-catalyzed
10
biotransformations.
immobilize enzymes within an aqueous microenvironment in the
organic solvent. For example, enzymes bound inside polymer
It is simple and straightforward to make a molecular hydrogel
for confining an enzyme (Scheme 1). The mix of sodium carbonate
5
6
hydrogels or organic plastics show enhanced activity and stability
relative to native enzymes in organic media, and the enzymes
(
20 mg), Fmoc-L-lysine (1, 36 mg), and Fmoc-L-phenylalanine
2, 38 mg) into 0.9 mL water gives a suspension, which turns into
a clear solution upon heating to about 333 K. The addition of
.1 mL methemoglobin solution (Hb, 40 mg) into the solution
(
7
,8
within the water phase of reverse micelles exhibit near or even
higher activity in organic media than that in water. These
promising results led us to develop molecular hydrogels as new
materials to immobilize enzymes for catalysis in organic media.
Although molecular hydrogels, formed by self-assembly of
amphiphilic molecules, have served as scaffolds for tissue
engineering, medium for screening inhibitors of enzymes, and
0
at 308–313 K and subsequent cooling to room temperature
affords gel I. A similar procedure allows the immobilization of
other enzymes (e.g., horseradish peroxidase (HRP, 50 U), laccase
from trameters versicolor (Laccase, 3.6 U), or alpha-chymotrypsin
from bovine pancreas (Alpha-CT, 100 U)). Without the addition
of enzymes, the same process produces gel II as a control. A
9
biomaterials for wound healing, their application as matrices for
enzymatic reactions in organic media has yet to be explored. In this
work, we mixed enzymes into the molecular hydrogel formed by
the self-assembly of two simple derivatives of amino acids (1 and 2)
and carried out oxidation reactions in organic media (Fig. 1). We
found that the enzymes enclosed in the molecular hydrogel
exhibited higher activity (e.g., up to eight times) in the organic
media (e.g., toluene) than that of unconfined enzymes in water.
Moreover, the molecular hydrogel acts as a more effective carrier
of enzymes in organic solvents than a polymeric hydrogel does,
suggesting a unique role of the self-assembled nanofibers in
molecular hydrogels for the observed superactivity and stability
because covalently crosslinked random coils form the network in
polymeric hydrogels and self-assembled nanofibers of amphiphilic
molecules as the network in molecular hydrogels. This result is
particularly significant because it implies that tailoring the
5
crosslinked poly(acrylamide) hydrogel containing Hb serves as
another control (gel III).
The rheological test (Fig. 2(a)) confirms the elastic nature of gels
11
I and II. The dynamic storage modulus of gel I is ten times lower
than that of gel II, indicating that crosslinks in gel I exist at lower
density than that in gel II and suggesting that the interaction of
Hb with the nanofibers decreases the density of the crosslink.
TEM images (Fig. 3(a), (b)) reveal that Hb molecules aggregate
and shorten the nanofibers (y16 nm in diameter) made of the
11
hydrogelators, thus reducing the density of crosslink in gel I.
Both AFM and TEM images (Fig. 3) indicate that Hb molecules
mainly locate at the crosslink sites of the nanofibers, which agrees
with the rheological data. Little release of Hb from gel I into
Department of Chemistry, The Hong Kong University of Science &
Technology, Clear Water Bay, Hong Kong, China.
E-mail: chbingxu@ust.hk; Fax: 852 2358 1594; Tel: 852 2358 7351
{
Electronic supplementary information (ESI) available: Related proce-
dures, characterizations, reaction condition, activity test, and stability test.
See DOI: 10.1039/b615223f
Scheme 1 The preparation procedure of gels I, II and III.
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1
032 | Chem. Commun., 2007, 1032–1034

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Fig. 2 (a) Frequency dependence of the dynamic storage modulus (G9)
and the loss modulus (G0) of hydrogel with and without Hb at a strain of
Fig. 4 (a) Comparison of activities of Hb(I) and Hb(U) in various media.
(b) The reaction course in the first minute of pyrogallol (10 mM) and
21
2%. (b) and (c) UV-Vis spectra of the Hb(U), Hb(I) and Hb(III) at various
2 2
H O (30 mM) catalyzed by various Hb (0.1 g L ) displays zero-order
wavelengths. (d) CD spectra of Hb(U), Hb(I), and Hb(III)
.
kinetics (all r .0.99), and is therefore used to calculate the initial rate. (c)
The ratios of activities of E(I) (I: immobilized by the molecular hydrogel) in
1
0
toluene and E(U) (U: unconfined) in water. The observed activities of
(U) in water are indicated. (d) The extended 15 min reaction course of (b).
All the concentrations were calibrated according to the molar extinction
E
11
coefficient of the product in different solvents.
solvents at room temperature. Using the oxidation of pyrogallol by
as the model reaction, we obtained the activity of Hb by
2 2
H O
11
monitoring the concentration of purpurogallin. The control, gel
11
II, exhibits no activity. As shown in Fig. 4(a), Hb(I) exhibits
almost the same activities as Hb(U) in water, suggesting that the
structures of Hb(I) and Hb(U) differ little, which agrees with the
UV-Vis and CD analysis. Hb(I) exhibits higher activities than
Hb(U) in the same organic media tested, confirming the protective
effect of the aqueous microenvironment provided by gel I. The
activities of both Hb(I) and Hb(U) increase with a decrease of the
polarity of the organic solvent (from acetonitrile to toluene), which
agrees with the established trend of the activity of an enzyme in an
Fig. 3 TEM images of (a) gel I and (b) gel II. AFM images of (c) gel I
13
and (d) gel II.
organic solvent. The Lineweaver–Burk plots constructed by the
initial reaction rates at different pyrogallol concentrations are used
to estimate their kinetic constant values. As shown in Table 1, the
1
1
solvents also confirms that the non-covalent interaction between
the enzyme and the nanofibers is strong enough to ensure the
immobilization.
21
21
activity of Hb(I) in toluene (7.98 mmol min mg ) is eight times
21
21
that of Hb(U) in bulk water (0.92 mmol min mg ). According
to Fig. 4(b), the initial rate of Hb(I) at 10 mM pyrogallol
concentration is much larger than that of Hb(U) and Hb(III). These
results represent the first observation of the superactivity of an
We used UV-Vis and circular dichroism (CD) spectra to probe
the state of Hb in different matrices. As shown in Fig. 2(b), the
almost same absorption peaks at 408 nm of the unconfined Hb
8
(
Hb(U)), gel I bound Hb (Hb(I)), and gel III bound Hb (Hb(III)
)
enzyme immobilized in a medium other than reverse micelles.
indicate the undisturbed spectral activity of the active heme
Other enzymes immobilized in the molecular hydrogel also display
superactivity in organic media (Fig. 4(c) and Table 1), indicating
that the superactivity is generally conferred by the molecular
hydrogel.
12
group. Hb(U), Hb(I) and Hb(III) all have a series of weak absor-
bances at about 500 and 630 nm (Fig. 2(c)), which indicate the
III
same HbFe form of the three Hbs. The CD spectra of the three
Hbs (Fig. 2(d)) show the double absorbance at 210 and 222 nm,
which are characteristic peaks of the a-helix. No change in the
peaks of the CD spectra indicates little or no structural difference
among Hb(U), Hb(I) and Hb(III). The employed spectroscopic
methods have confirmed that the Hb remains at the same state
as the unconfined form in water when it was immobilized by
the hydrogels.
We suggest that it is likely that several factors contribute to the
superactivity of Hb . (i) Hydrophilicity promotes the substrate
(I)
(i.e., pyrogallol) across the microinterface to enter the hydrogel,
7,8
similar to the case of reversed micelles.
(ii) Amphiphilic
14
character and/or the molecular superstructure of the self-
assembled nanofibers in gel I may assist the substrates to approach
Hb and the products to leave Hb. This assumption agrees with the
much lower activity of Hb(III) (i.e., Hb immobilized by a randomly-
crosslinked poly(acrylamide) hydrogel) than that of Hb(I). (iii) The
III
The HbFe form of the three Hbs allow them to serve as a
1
2
substitute for peroxidase for catalyzing oxidation in different
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Table 1 The catalytic data of the hydrogel-immobilized enzymes in toluene and the unconfined enzymes in water
Activity (half-life/min) Activity (half-life/min)
Ratio of
activity
a
Enzyme
Substrate
Product
of E(U) in H
2
O
of E(I) in toluene
b
0.92 (1.9)
b
7.98 (7.0)
Hb
HRP
Laccase
Alpha-CT
Pyrogallol
Pyrogallol
o-Phenyldiamine
N-Benzoyl-L-tyrosine-
p-nitroanilide
Purpurgallin
Purpurgallin
Phenazine-2,3-diamine
p-Nitroanilide
8.7
2.7
c
802 (2.8)
c
2165 (5.3)
b
c
54.6 (4.2)
c
229.3 (13.6)
b
4.2
5.5
c
1.06 (5.9)
c
5.83 (28.4)
b
a
b
21
Ratio of activity: activity of E(I) in toluene/activity of E(U) in water. mmol min mg
21
c
21
21
U .
.
nmol min
large pore sizes of the nanofibrous networks in gel I (TEM and
will benefit industrial biotransformations. Moreover, the principle
illustrated in this work may allow the immobilization of catalysts
11
AFM confirm 0.2–2 mm and 5–6 nm pores in gel I and III,
respectively) facilitate the mass transport in gel I.
15
16
in organogels to carry out reactions in water. Our future work
will expand this general strategy to a variety of catalysts and gels.
B. X. acknowledges the partial financial supports from RGC
(Hong Kong), EHIA (HKUST), and Central allocation grant
(HKU2/05C).
We also found that molecular hydrogels significantly improve
the stability of the enzymes. As shown in Fig. 4(d), Hb(I) has
improved stability in toluene compared with that of Hb(U) in
water. The quantitative analysis of their reaction course shows the
highest stability of Hb(I) in toluene, as indicated by their half lives
(t1/2) of Hb. To evaluate the potential industrial application of the
Notes and references
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catalyzed oxidization in toluene because the oxidative product of 3
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mycin A). The initial rate of Hb(I) in toluene is slightly lower than
that of Hb(U) in water, indicating that the superactivity is also
substrate dependent, a characteristic feature of enzymes. The
molecular hydrogel significantly improves the stability of Hb(I) in
toluene (t1/2 = 27.8 min) and leads to the additional production of
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1
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