The preparation and characterization of a laccase nanogel and its application in naphthoquinone synthesis

Article abstract of DOI:10.1002/cplu.201300066

Laccase from Trametes versicolor 51639 was encapsulated on the nanoscale. The laccase nanogel was prepared by polyacrylamide encapsulation as a result of N-acryloxysuccinimide modification and in situ polymerization. Size-exclusion chromatography, fluorescence resonance energy transfer, dynamic light scattering, and transmission electron microscopy were employed to characterize the laccase nanogel. Higher thermal, pH, and storage stability and organic-solvent tolerance of the laccase nanogel were indicated without activity loss. Moreover, the laccase nanogel was used to synthesize naphthoquinones from hydroquinones with a 10-15% greater overall yield than that of native laccase. Copyright

Full text of DOI:10.1002/cplu.201300066

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DOI: 10.1002/cplu.201300066
The Preparation and Characterization of a Laccase
Nanogel and Its Application in Naphthoquinone Synthesis
Honghua Jia,* Chao Zhong, Fan Huang, Chunming Wang, Lisha Jia, Hua Zhou, and
Ping Wei[a]
Laccase from Trametes versicolor 51639 was encapsulated on
the nanoscale. The laccase nanogel was prepared by polyacryl-
amide encapsulation as a result of N-acryloxysuccinimide modi-
fication and in situ polymerization. Size-exclusion chromatogra-
phy, fluorescence resonance energy transfer, dynamic light
scattering, and transmission electron microscopy were em-
ployed to characterize the laccase nanogel. Higher thermal,
pH, and storage stability and organic-solvent tolerance of the
laccase nanogel were indicated without activity loss. Moreover,
the laccase nanogel was used to synthesize naphthoquinones
from hydroquinones with a 10–15% greater overall yield than
that of native laccase.
Introduction
Laccases (EC 1.10.3.2) are multicopper blue oxidases widely ori-
ginated from plants, fungi, and bacteria.[1] They can oxidize
a broad range of substrates, such as phenols, anilines, benzo-
furans, and phenoxazines.[2] The processes catalyzed by laccas-
es are green and environmentally friendly with oxygen as the
electron acceptor to produce water as the only byproduct.
Nowadays, great interest has been focused on employing lac-
cases in many industrial fields. Over the past decade, many
publications have reported on the application of laccases in
the pulp and paper industry, food industry, textile industry, en-
vironmental protection, and so forth.[3] In particular, laccase
had been put into practice in biopulping based on the patent-
ed Lignozym process.[4]
Recently, nanostructured enzymes were thought to be
a promising method to enhance the stability of laccase with
series of studies published and parts of them reviewed.[10] g-
Chymotrypsin, trypsin, carbonic anhydrase, horseradish perox-
idase, lipase, and cytochrome c were used in these studies.[11]
Significant improvements in the thermal stability or solvent re-
sistance were observed and a preliminary mechanism was also
proposed by thermodynamic simulation.[12]
In this study, laccase from Trametes versicolor 51639 was en-
capsulated as a nanogel and used to synthesize naphthoqui-
none from hydroquinone. Firstly, laccase was modified by N-
acryloxysuccinimide followed by polymerization with acrylam-
ide in situ to produce a laccase nanogel encapsulated with
polyacrylamide. Secondly, the laccase nanogel was subse-
quently characterized by size-exclusion chromatography (SEC),
fluorescence resonance energy transfer (FRET), dynamic light
scattering (DLS), and TEM. The kinetics, thermal and pH stabili-
ty, and organic-solvent resistance of native laccase and laccase
nanogel were also assayed for comparison. Finally, the laccase
nanogel, prepared under optimal conditions, was applied effi-
ciently to synthesize naphthoquinones from hydroquinones.
The lower redox potential of laccase requires that its applica-
tion be accompanied with mediators such as the diammonium
salt of 2,2’-azinebis(3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS), 2,2,6,6-tetramethyl-1-piperidinyloxyl (TMPO), and hy-
droxybenzotriazole (HBT). The combination of laccase and
a mediator is called a laccase-mediator system (LMS).[5] Com-
mercialized LMSs, including DeniLite for fabric bleaching, Sub-
erase for cork modification, and Novozymes 51003 for pulp de-
lignification, have been developed by Novozymes (http://
www.novozymes.com).
In the past, much attention has been devoted to the appli-
cation of laccases and LMS in organic synthesis.[6] The one-pot Results and Discussion
synthesis of 1,4-naphthoquinones, 1,4-naphthoquinone-2,3-bis-
Preparation of the laccase nanogel
(sulfides), pyridines, aminonaphthoquinones, and indole deriva-
tives catalyzed by laccase was reported recently.[7] Meanwhile,
laccases have also widely been employed to synthesize poly-
mers and antibiotics.[8] To improve activity and stability, laccas-
es have been immobilized by versatile methods.[9]
The procedure for the preparation of the laccase nanogel is
described briefly in Figure 1. First, the surface amino groups of
lysine were modified by an acryloylation reaction with N-acryl-
oxysuccinimide (NAS). The double bonds were grafted on the
laccases. Second, acrylamide was added to the solution of
modified laccase surrounding the single-modified laccase mol-
ecule with hydrogen bonds to form a modified-laccase mole-
cule–acrylamide assembly. Third, ammonium persulfate (APS)
and tetramethylethylenediamine (TEMED) were added to the
[a] Dr. H. Jia, C. Zhong, F. Huang, C. Wang, L. Jia, Dr. H. Zhou, Dr. P. Wei
College of Biotechnology and Pharmaceutical Engineering
Nanjing University of Technology
Nanjing 211316 (P.R. China)
E-mail: hhjia@njut.edu.cn
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Optimization of the concentration of acrylamide for laccase
nanogel preparation
As shown in Figure 3, the concentration of acrylamide can di-
rectly affect the activity of the laccase nanogel. The optimal
concentration of acrylamide was 20 mgmLÀ1, which corre-
Figure 1. Scheme of the preparation of the laccase nanogels.
mixture. The assembly was polymerized in situ to produce lac-
case nanogel encapsulated in polyacrylamide.
Optimization of pH for modification
Chemical modification has been widely used to enhance cata-
lytic efficiency and thermostability, and tolerance of enzymes
to organic solvents.[13] In this study, native laccase modified by
NAS was investigated at different pH levels. The effect of pH
on the relative activity and the degree of modification of modi-
fied laccase is shown in Figure 2. These results indicate that
a higher pH is actually beneficial to modification. The highest
Figure 3. Effect of the concentration of acrylamide in the nanogel. The rela-
tive activity is represented as a percentage with respect to native laccase.
sponds to a 26% increase in the relative activity after encapsu-
lation. This might be attributed to the formation of hydrogen
bonds between laccase and polylacrylamide that stabilize the
conformation of the enzyme. The activity of the laccase nano-
gel decreases for acrylamide concentrations greater than
20 mgmLÀ1. This is possibly explained by the limited mass
transfer that is caused by the formation of a compact gel layer
at higher concentrations of acrylamide.
SEC and FRET analysis
Figure 2. Effect of pH on modification. The relative activity is represented as
the percentage of the activity with respect to native laccase. The degree of
modification was calculated by using native laccase as the control.
SEC was used to determine the yield of encapsulation. As dis-
played in Figure 4, the eluting peak of the laccase nanogel ap-
degree of modification, 93%, was attained at pH 9.3. However,
at much higher pH the activity of modified laccase was found
to decline. The activity of the modified laccase was 1.68-fold
higher than that of the native at the optimal pH of 8.0. Similar
results from lipase modified by glycidyl methacrylate has been
reported.[14] It was deduced that a hydrophobic microenviron-
ment is formed after modification to facilitate substrate access
and simultaneously reinforce the ‘open-lid’ conformation favor-
ing the enzymatic activity based on molecular simulation.
Excess modification might occur at a much higher pH, which
could reduce the activity of the modified laccase.
Figure 4. SEC elution curves of native laccase and the laccase nanogel.
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peared at a retention time of around 11 min, and that of
native laccase at 21 min, which indicates that the laccase nano-
gel particles are bigger than native laccase. The yield of encap-
sulation was calculated to be about 80% based on the peak-
area ratio of encapsulated laccase to total laccase. The fluores-
cence emission spectrum of native laccase was similar to that
of the laccase nanogel, as seen from Figure 5. The maximal
fluorescence emission wavelength was 330 nm for both forms
of laccase, which indicates that no apparent change in the lac-
case structure occurred during the encapsulation process.
Figure 5. Fluorescence spectrum of native laccase and the laccase nanogel.
DLS and TEM analysis
The particle diameter was determined by DLS (Figure 6). The
results indicate that the diameter of native laccase and the lac-
case nanogel are in the range 3.16–10.0 nm with an average of
5.36 nm, and 5.62–100 nm with an average 28.33 nm, respec-
tively. In addition, the diameters of most of the nanogels were
within the range 10–56.23 nm. Figure 7a and b revealed the
high-resolution TEM images of laccase nanogel on the 500 and
100 nm scale. The diameter range for most of the laccase
Figure 7. TEM image of laccase nanogel: a) 500 and b) 100 nm.
nanogel molecules was within the range 10–40 nm, which was
to some extent identical to that of the DLS results. It was con-
sidered that the diameter of laccase from DLS is slightly larger
than that from TEM. The larger size was a result of the swelling
of the laccase nanogel in the buffer.
Effect of temperature and pH on the laccase activity
The optimal temperature and pH for native laccase and the
laccase nanogel are given in Figures 8 and 9, respectively. The
optimal temperature for native laccase is 408C, whereas it was
shifted to 558C for the laccase nanogel and high activity was
found between 30 and 608C. The optimal pH values were 3.5
and 3 for native laccase and the laccase nanogel, respectively.
No apparent shift was detected for the laccase nanogel.
Kinetic parameters
The kinetic parameters of native laccase and the laccase nano-
gel are shown in Table 1 based on the Lineweaver–Burk plot.
Figure 6. DLS analysis of native laccase and the laccase nanogel.
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may be attributed to the limited substrate diffusion to the
active site after encapsulation and a less-active conformation
of the laccase active site may also have been adopted.
Thermal and pH stability
The effects of temperature and pH on the stability of native
laccase and laccase nanogel are shown in Figures 10 and 11,
Figure 8. Effect of temperature on the activity of native laccase and the lac-
case nanogel. The activity was assayed at different temperatures by employ-
ing ABTS as the substrate.
Figure 10. The thermal stability of native laccase and the laccase nanogel.
Native laccase and the laccase nanogel were dissolved in phosphate buffer
(50 mm, pH 7.0) and incubated at 608C. Samples removed for activity tests
employed ABTS as the substrate.
Figure 9. Effect of pH on the activity of native laccase and the laccase nano-
gel. Activity was assayed over a pH range (2.0–7.5) by employing ABTS as
the substrate.
Table 1. Kinetic parameters of native laccase and the laccase nanogel.
Enzyme
Km
[mm]
Vmax
[mmminÀ1
Vmax/Km
[minÀ1] [ꢁ10À3
]
]
native laccase
laccase nanogel
54.1
30.3
21.2
16.8
391.9
554.5
Figure 11. The pH stability of native laccase and the laccase nanogel. Lac-
case and the laccase nanogel were each incubated in buffer (pH range 3.0–
8.0) at 308C for 2 h. The activity was assayed by employing ABTS as the sub-
strate.
The Michaelis constant (Km) and maximum rate (Vmax) of the
laccase nanogel were lower than those of native laccase. The
change of Km indicates that unlike the traditional encapsulation
of enzymes, the behavior here is similar to cross-linked enzyme
respectively. These results indicate that the thermal denatura-
tion curves of native laccase and the laccase nanogel are in
accord with the exponential model. The lifetime of the laccase
nanogel was 1.34 h, which was increased 15-fold compared
with 0.09 h of native laccase. Both native laccase and the lac-
case nanogel retained good stability within the pH range 6.0–
8.0. It was presumed that the effects of crowding and the con-
finement by the polyacrylamide gel were advantageous to sta-
aggregates (CLEAs) and polyethylene glycol (PEG)-modified en-
15]
zymes.[9b,
It was speculated that the structure of laccase
could be stabilized in favor of substrate binding after encapsu-
lation according to the difference in Km between the two lac-
case forms. As for Vmax, the reaction rate for the oxidation of
ABTS of the laccase nanogel decreased by about 25% with re-
spect to native laccase.[16] The decrease in the reaction rate
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bilizing the active conformation of laccase,[17] thereby resulting
in better thermal and pH stability of the laccase nanogel.
Organic-solvent tolerance experiments
Native laccase and the laccase nanogel were investigated by
dissolving each sample in various organic solvents. As seen
from Figure 12, native laccase was nearly inactive when incu-
Figure 13. Storage stability of native laccase, the laccase nanogel, and the
lyophilized laccase nanogel.
Synthesis of naphthoquinones from hydroquinones
2-Methoxyhydroquinone was selected as the substrate to in-
vestigate the effect of load of two forms of laccase on naph-
thoquinone synthesis (Figure 14). The results indicate that
Figure 12. Stability of native laccase and the laccase nanogel in organic sol-
vents. Native laccase and the laccase nanogel were incubated in different or-
ganic solvents at 508C for 3 h. The residual activity was detected as de-
scribed above and expressed as a percentage with respect to the initial ac-
tivity.
bated in DMSO, DMF, methanol, and ethanol at 508C for 3 h,
but the highest residual activity, 96% in DMF, and the lowest
of 74% in methanol were obtained for the laccase nanogel. Re-
sults showed that the laccase nanogel in organic solvents was
remarkably much more active than native laccase. The highest
relative activity of the laccase nanogel was 96-fold more than
that of native laccase in DMF, which could be ascribed to the
hydrophilic effect of the polyacrylamide gel network to expel
organic solvent and preserve the high water content necessary
for laccase activity. In addition, an intensified intramolecular
hydrogen bond within laccase can stabilize the conforma-
tion.[12, 18]
Figure 14. Effect of the load of laccase on the synthesis of 2-methoxy-1,4-
naphthoquinone catalyzed by native laccase and the laccase nanogel. The
solution consisted of 2-methoxyhydroquinone (0.1 g), 1-acetoxy-1,3-buta-
diene (0.16 g), a certain amount of native laccase and laccase nanogel,
DMSO (600 mL), and acetate buffer (20 mL, 100 mm, pH 4.5). The reaction
was carried out as described above.
Storage stability
The storage stability of native laccase, the laccase nanogel, and
the lyophilized laccase nanogel was observed at 48C. Figure 13
shows that the lyophilized laccase nanogel has good storage
stability with a slight decrease in activity for 6 weeks. An activi-
ty of about 68% can be maintained for the laccase nanogel in
a buffer, but only 31% for native laccase. The polyacrylamide
gel provided a suitable microenvironment for laccase to retain
its active conformation with similar results to those reported
on sol–gel-immobilized laccase in reference [19].
a higher load of enzyme affords a higher yield of product for
both forms of laccase and that 800 U per gram of substrate is
a suitable load of enzyme for both forms of laccase. The lac-
case nanogel showed a notably higher activity than native lac-
case. The reason for this is the thermal stability and higher or-
ganic-solvent tolerance of the laccase nanogel as reported
above.
The time course of the synthesis of 2-methoxy-1,4-naphtho-
quinone catalyzed by the laccase nanogel showed a clear in-
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crease in yield with an increase in time, as seen from
Figure 15. In particular, a rapid increase occurred within the
first 12 h of the reaction. The reaction was thought to be close
to equilibrium for 24 h with a yield of 2-methoxy-1,4-naphtho-
quinone of approximately 90%.
Table 2. Yield of naphthoquinones catalyzed by native laccase and the
laccase nanogel.[a]
Hydroquinones
Naphthoquinones
Yield [%]
Native
Nanogel
1
2
3
4
6
7
8
9
61.7Æ0.4
70.4Æ0.6
76.6Æ0.4
63.8Æ0.5
74.5Æ0.7
85.8Æ0.3
89.2Æ0.5
77.9Æ0.6
[a] The solution included hydroquinones (0.1 g), 1-acetoxy-1,3-butadiene
with a molar ratio of it to hydroquinones of 2:1, 800.0 U (per gram hydro-
quinones) of native laccase and the nanogel, DMSO (0.6 mL), and acetate
(20 mL,100 mm, pH 4.5) buffer.
fication and 26% increase in activity for the laccase nanogel at
optimal pH 8.0 and 20 mgmLÀ1 concentration of acrylamide.
The particle diameters of the laccase nanogel ranged from 10
to 40 nm in an 80% yield of encapsulation without conforma-
tional change as determined by SEC, FRET, DLS, and TEM char-
acterization. The kinetic parameters and enzymatic properties
were characterized and compared for native laccase and the
laccase nanogel. The laccase nanogel showed greater thermal,
pH, and storage stability, and a higher organic-solvent toler-
ance than native laccase. The half-life of the laccase nanogel
increased 15-fold relative to that of native laccase and the rela-
tive activity of the laccase nanogel was observed to be 96-fold
higher than that of native laccase in DMF.
Figure 15. Time course of the synthesis of 2-methoxy-1,4-naphthoquinone
catalyzed by the laccase nanogel. The solution contained 2-methoxyhydro-
quinone (1.0 g), 1-acetoxy-1,3-butadiene (1.6 g), laccase nanogel (800.0 U),
DMSO (6.0 mL), and acetate (200 mL, 100 mm, pH 4.5) buffer. The reaction
was carried out as described above and aliquots of samples were removed
periodically.
In addition, the naphthoquinones could be efficiently syn-
thesized from hydroquinones by employing laccase and the
laccase nanogel. The load of enzyme and the time course were
studied for 2-methoxy-1,4-naphthoquinone as an example.
About 800 U per gram of hydroquinones was a suitable load
of enzyme and 24 h was a suitable reaction time for both
forms of laccases. The products were characterized by GC–MS
and NMR spectroscopy. The results indicate that the yields of
naphthoquinones catalyzed by the laccase nanogel are clearly
higher than those catalyzed by native laccase.
Four kinds of naphthoquinones (1,4-naphthoquinone (6), 2-
methyl-1,4-naphthoquinone (7), 2-methoxy-1,4-naphthoqui-
none (8), 2-chloro-1,4-naphthoquinone (9)) were synthesized
from hydroquinones catalyzed by native laccase and the lac-
case nanogel (Scheme 1 and Table 2). The yields of 8 (77 and
89%, Table 2) are similar to those reported by Witayakran and
Ragauskas (79%).[7a] The results indicate that the yields of reac-
tions catalyzed by native laccase are consistent with literature
reports. The laccase nanogel was more efficient in the synthe-
sis of the four naphthoquinones with 10–15% more overall
yield obtained compared with native laccase.
Experimental Section
Laccase from Trametes versicolor 51639, NAS, ABTS, and 2,4,6-trini-
trobenzene sulfonic acid (TNBS) were all obtained from Sigma–Al-
drich (USA). DMSO, TEMED, acrylamide, and trehalose were pur-
chased from Sinophar Chemical Reagent Co. 1-Acetoxy-1,3-buta-
diene and 2-methoxyhydroquinone were purchased from Alfa
Aesar. Hydroquinone and 2-methylhydroquinone were supplied by
Aladdin. 2-Chlorohydroquinone, APS, and BCA Protein Assay Kit
were provided by Adamas Reagent, Shanghai Lingfeng Chemical
Reagent Co., and Beyotime Institute of Biotechnology. All other
chemicals were of analytical grade. All UV/Vis spectrophotometric
analyses were performed on a Thermo Scientific Genesys 10S UV/
Vis spectrophotometer.
Scheme 1. Synthesis of naphthoquinones.
Conclusion
Preparation of the laccase nanogel
The laccase nanogel was prepared by polyacrylamide encapsu-
lation. The pH and concentration of the acrylamide modifica-
tion were optimized and resulted in an 86.8% degree of modi-
The laccase nanogel was prepared according to published proce-
dures.[20] The process was divided into two steps: modification and
encapsulation.
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Firstly, the laccase was modified by NAS as follows. Laccase (20mg)
was dissolved in different buffers (4mL; 50mm, pH 4.0 acetate
buffer; 50mm, pH 6.0 phosphate buffer; 50mm, pH 8.0 borate
buffer; and 50mm, pH 9.3 borate buffer, respectively). The solu-
tions were then dialyzed against the same buffer for 24h. Soon af-
terwards, NAS (10 mg) was dissolved in DMSO (600 mL) and then
slowly added to the laccase solution. The mixtures were collected
after incubation at 308C for 4h and dialyzed against phosphate
buffer (50mm, pH 7.0) for 24h to remove unreacted reagents.
Secondly, the modified laccase was encapsulated. The different
amounts of acrylamide (25–150 mg) were added to the modified
laccase solutions (5 mL) with agitation. Then APS (15 mg) and
TEMED (15 mL) were added directly and polymerized at 308C for
12h with agitation. The mixtures were collected and dialyzed
against phosphate buffer (50 mm, pH 7.0) for 24h. After that, the
products were dialyzed in distilled water to remove unreacted re-
agents and stored at 48C for the following experiments. The
lyophilized nanogel was prepared with addition of 2% trehalose
(m/m) for the storage stability experiment. Where 2% trehalose
(m/m) is defined as the mass ratio of trehalose to laccase nanogel.
DLS analysis
The DLS analysis of native laccase and the laccase nanogel was car-
ried out at 258C and a 908 scattering angle on a Brookhaven BI-
100SM laser light scattering system.
FRET analysis
The FRET measurements excited at 280 nm of native laccase and
the laccase nanogel were monitored from 300 to 500 nm with a Hi-
tachi F-700 spectrofluorometer.
TEM analysis
The samples for TEM were prepared by dropping a sample of the
laccase nanogel (5 mL, 0.01 gLÀ1) dispersion onto a carbon-coated
copper grid, followed by air drying. The TEM observation of the
laccase nanogel was conducted on a Hitachi H-7650 transmission
electron microscope operated at 99 kV.
Assay of the kinetic parameters
Determination of protein concentration
The kinetic parameters, Km and Vmax, of native laccase and the
nanogel were calculated by using the Lineweaver–Burk plot. Reac-
tions were conducted based on the determination of the activity
using 0.05–0.6 mm ABTS. All the data are the average values of
triplicate tests with a standard deviation lower than 1%.
Protein concentration was determined spectrophotometrically at
562 nm according to the BCA Protein Assay Kit using BSA as the
standard. Where BSA (Bull Serum Albumin) with concentration of
1 mgmLÀ1 was used as the standard for detecting the protein con-
centration, obtaining the standard curve of “y=0.0665xÀ0.0027”
with R2 =0.9975.
Organic-solvent tolerance test
A certain amount of native laccase and the laccase nanogel were
dissolved in DMSO, DMF, methanol, and ethanol, respectively. All
the mixtures were incubated at 508C for 3 h. Then the activity was
determined as described above.
Determination of laccase activity
The laccase activity was measured according to a published proce-
dure.[21] ABTS was selected as a substrate for native laccase and the
laccase nanogel, the activity of each was determined by using
a spectrophotometric method. First, an equal volume of ABTS
(1 mm) and acetate buffer (0.1 m, pH 4.5) was mixed and incubated
under stirring at 308C for 10 min. Then an appropriate amount of
native laccase and laccase nanogel was added to the mixture
(3 mL). The absorbance (OD) was recorded at 420 nm for 3 min.
One unit of activity was defined as the amount of laccase that oxi-
dized 1 mmol ABTS per minute.
Storage stability
All laccase preparations (native laccase and the laccase nanogel in
acetate buffer (100 mm, pH 4.5) and the lyophilized laccase nano-
gel) were kept at 48C. Samples were removed for the laccase activ-
ity assay periodically over 6 weeks. The relative activity of laccase
was expressed as a percentage of the initial value for each sample.
Synthesis of naphthoquinone by native laccase and laccase
nanogel
Determination of the modified amino group
The modified amino group in native laccase was measured spec-
trophotometrically by using the TNBS method at 420 nm as report-
ed.[22]
The naphthoquinones were synthesized from the reaction of 1-ace-
toxy-1,3-butadiene and hydroquinones and 1-acetoxy-1,3-buta-
diene using native laccase and the laccase nanogel as catalysts. A
certain amount of hydroquinone (1,2-methylhydroquinone (2), 2-
methoxyhydroquinone (3), and 2-chlorohydroquinone (4)) was dis-
solved in DMSO, and then mixed with acetate buffer (100 mm,
pH 6.0). The amount of 1-acetoxy-1,3-butadiene (5) with a molar
ratio of it to hydroquinones of 2:1 and native laccase or laccase
nanogel were added successively to the mixtures after introducing
oxygen for 30 min. Subsequently, the solutions were incubated at
558C for 24 h. After that, the solutions were extracted using ethyl
acetate, and the products were purified by silica column chroma-
tography employing ethyl acetate and petroleum ether (1:5, v/v)
as the mobile phase. The yields were calculated as the percentage
of the amount of products with respect to the calculated value.
SEC analysis
SEC measurements were executed on a TSK-GEL G4000SWXL size-
exclusion column equipped with a Shimadzu RF-10AXL fluores-
cence detector at 340 nm. The phosphate buffer (100 mm, pH 6.7)
containing sodium sulfate (100 mm) was used as the mobile phase
at a flow rate of 0.5 mLminÀ1. The encapsulation ratio of the lac-
case was expressed as the percentage of the elution peak area of
the nanogel with respect to the total elution areas of the nanogel
and residual laccase.
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Identification of naphthoquinones
All the naphthoquinones were identified by GC–MS and 1H and
13C NMR spectroscopy. A Thermo Scientific TRACE GC-DSQ system
equipped with an Agilent DB-5 MS (30 mꢁ0.25 mmꢁ0.25 m)
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1
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1,4-Naphthoquinone (6): H NMR (500 MHz, CDCl3): dH =8.03 (s, 2H;
PhÀH), 7.72 (s, 2H; PhÀH), 6.94 ppm (s, 2H; CH); 13C NMR
(126 MHz, CDCl3): dC =184.78 (2C=O), 138.49 (2CH), 133.74 (2CH),
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2-Methyl-1,4-naphthoquinone (7): 1H NMR (500 MHz, CD3Cl): dH =
8.10 (ddd, 2H; PhÀH), 7.75 (dd, 2H; PhÀH), 6.87 (s, 1H; CH),
2.22 ppm (s, 3H; CH3); 13C NMR (126 MHz, DMSO): dC =185.54 (C=
O), 184.97 (C=O), 148.17 (C), 135.67 (CH), 133.61 (2CH), 126.51
(2C), 126.08 (2CH), 16.46 ppm (CH3); MS: m/z (%): 172.
1
2-Methoxy-1,4-naphthoquinone (8): H NMR (500 MHz, CD3Cl): dH =
8.14 (dd, 2H; PhÀH), 7.76 (dt, 2H; PhÀH), 6.20 (s, 1H; CH),
3.94 ppm (s, 3H; CH3); 13C NMR (126 MHz, DMSO): dC =184.73 (C=
O), 180.02 (C=O), 160.39 (CÀO), 134.29 (2CH), 133.30 (2C), 126.64
(2CH), 109.87 (CH), 56.40 ppm (CH3); MS: m/z (%): 188.
2-Chloro-1,4-naphthoquinone (9): 1H NMR (500 MHz, CDCl3): dH =
8.11 (s, 2H; PhÀH), 7.77 (s, 2H; PhÀH), 7.20 ppm (s, 1H; CH);
13C NMR (126 MHz, CDCl3): dC =182.97 (C=O), 182.52 (C=O), 135.87
(C), 134.37 (CH), 134.00 (2CH), 127.43 (CH), 127.15 (CH), 126.69
(2CH); MS: m/z (%): 192.
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Acknowledgements
We thank Professor Zheng Liu and Associate Professor Diannan Lu
from the Department of Chemical Engineering, Tsinghua University,
for their enthusiastic assistance. The research was financially sup-
ported by NSFC (20906048), the State Key Basic Research and De-
velopment Plan of China (2009CB724700), Jiangsu Province Natural
Science Foundation Innovation Project (SBK200910195), PAPD, and
PCSIRT (IRT1066).
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Keywords: enzymes · gels · nanostructures · polymers ·
quinones
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Received: February 25, 2013
Published online on April 4, 2013
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