Supplementary Material
Encapsulation of enzyme via one-step template-free formation of
stable organic-inorganic capsules: a simple and efficient method for
immobilizing enzyme with high activity and recyclability
Renliang Huang, Mengyun Wu, Mark J. Goldman, and Zhi Li*
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4
Engineering Drive 4, Singapore 117585, Singapore. E-mail: chelz@nus.edu.sg
List of content
1. Analytic methods
1.1 Scanning electron microscopy (SEM)
1.2 X-ray photoelectron spectroscopy (XPS)
1.3 Fourier transform infrared spectroscopy (FTIR)
1.4 Thermogravimetric analysis (TGA)
1.5 Zeta potential measurement
1.6 Specific enzyme loading and enzyme loading efficiency
2. Supplementary Figures
3. Supplementary Table
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1. Analytic methods
1.1 Scanning electron microscopy (SEM)
Before SEM measurement, all the samples were freeze-dried and sputter-coated with platinum. SEM images
were recorded by using a JSM-6700F field emission scanning electron microscope (FESEM, JEOL, Japan) at the
acceleration voltage of 5 kV.
1.2 X-ray photoelectron spectroscopy (XPS)
The elemental composition of the FP capsules and FPSi capsules was analyzed by XPS in a Kratos AXIS Ultra
spectrometer (Kratos Analytical Ltd, UK) with a monochromatic Al Ksource (h=1486.71 eV, 5 mA, 15 kV).
Base pressure was 1×10−9 Torr and operating pressure was 5×10−9 Torr. Survey spectra were recorded in the range
from 1100 to -5 eV with 1.0 eV step and 100 ms dwell time. High resolution C1s, N1s, O1s and Si2p spectra were
collected with a step size of 0.1 eV.
1.3 Fourier transform infrared spectroscopy (FTIR)
FTIR spectra of FP capsules and FPSi capsules were recorded on a FTIR-8400 spectrophotometer (Shimadzu,
Kyoto, Japan) with a KBr pellet method in the range 400-4000 cm−1. A total of 16 scans were accumulated with a
resolution of 4 cm-1 for each spectrum.
1.4 Thermogravimetric analysis (TGA)
Thermogravimetric analysis (TGA) of FP capsules and FPSi capsules was conducted on a DTG-60AH thermal
analyzer (Shimadzu, Kyoto, Japan) under the following conditions: measuring temperature range, 25–800 ˚C;
constant heating rate, 10 oC min-1; under Argon environment.
1.5 Zeta potential measurement
Zeta potential measurements of Fmoc-FF, PEI, and sodium silicate solutions were performed using the
Zetasizer Nano-ZS (Malvern Instruments, UK). The pH values of Fmoc-FF (2 mg mL-1) solution and PEI (1.0
wt%) solution were kept at 9.0 and 7.5, respectively. For sodium silicate solutions, the pH values (from 7.0 to 10.0)
were adjusted with concentrated HCl. A pH meter (SevenEasy, Mettler Toledo, Switzerland) was used to measure
the pH values of all the solutions.
1.6 Specific enzyme loading and enzyme loading efficiency
The amount of immobilized enzyme was deduced from the initial enzyme amount used for immobilization and
the measured enzyme amount in PEI solution after immobilization. The total dry weight (w) of enzyme-loaded
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FPSi capsules was measured after oven-drying for 24 h. The specific enzyme loading (EL) and immobilization
efficiency (Ei) were calculated according to the following equations, respectively:
m
m0 - c  v

w - m w - ( m0 - c  v )
(1)
m - c×v
m
× 100% = 0
× 100%
m0
m0
(2)
EL 
Ei =
where EL (mg g-1) represents the enzyme loading capacity of the capsules; m (mg) is the amount of enzyme
(SpEH) loaded into the capsules; w is the total dry weight of enzyme-loaded capsules; m0 (mg) is the initial
amount of enzyme before immobilization; c (mg mL-1) and v (mL) are the enzyme concentration and volume of
the PEI solution, respectively.
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2. Supplementary Figures
a)
b)
c)
d)
Fig. S1 SEM images or photograph of FPSi capsules with different size (a: ~200 m; b: ~400 m; c: ~1.2 mm; d;
~2 mm). The capsule synthesis conditions: 1.5 mL aqueous solution (pH 10.0) of Fmoc-FF (2 mg mL-1) and
sodium silicate (2 mg mL-1 SiO2), 5 mL aqueous solution (pH 7.5) of PEI (1wt%), 25 oC, 2 h.
a)
b)
Fig. S2 SEM images of the capsule membranes with a thickness of ~40 m (a) and 65 m (b). The capsule
synthesis conditions: 1.5 mL aqueous solution (pH 10.0) of Fmoc-FF (2 mg mL-1) and sodium silicate (2 mg mL-1
SiO2), 5 mL aqueous solution (pH 7.5) of PEI (1wt%), 25 oC, 2 h.
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a)
b)
c)
d)
Fig. S3 SEM images of freeze-dried and cleaved capsule (a) with a diameter of ~1.2 mm, capsule membrane (b)
and the thin layers (c-d). The capsule synthesis conditions: 1.5 mL aqueous solution (pH 10.0) of Fmoc-FF (2 mg
mL-1) and sodium silicate (2 mg mL-1 SiO2), 5 mL aqueous solution (pH 7.5) of PEI (1wt%), 25 oC, 2 h.
a)
b)
Fmoc-FF/PEI capusles
Silicate/PEI
Fig. S4 Photograph of Fmoc-FF/PEI (FP) capsules (a) and silicate/PEI mixture (b) formed in 1.0 wt% PEI
solutions at pH 7.5, respectively.
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a)
b)
c)
d)
Fig. S5 SEM images of Fmoc-FF/PEI (FP) capsule (a-b) and its layers (c-d)
a)8000
b) 2800
C1s
7000
2400
NH-C*
4000
3000
2000
NH-C*=O
OH-C*=O
N1s
2200
Intensity
Intensity
6000
5000
O=C-N*H
C-C-N*H
C-C-N*H2
2600
C*H-CH
2000
1800
1600
1400
1000
1200
0
292
290
288
286
284
282
406
280
404
c)
6000
4000
3000
400
398
d)
396
Si 2p
1000
O1s
800
Si-O*-Si
O*=C-OC
Intensity
Intensity
5000
402
Binding energy (eV)
Binding energy (eV)
O*=C-NH
O=C-O*C
600
400
2000
200
1000
538
536
534
532
530
0
110
528
108
106
104
102
100
98
96
Binding energy (eV)
Binding energy (eV)
Fig. S6 XPS high-resolution spectra of C1s (a), N1s (b), O1s (c) and Si2p (d) for FPSi capsules. The capsule
synthesis conditions: 1.5 mL aqueous solution (pH 10.0) of Fmoc-FF (2 mg mL-1) and sodium silicate (2 mg mL-1
SiO2), 5 mL aqueous solution (pH 7.5) of PEI (1wt%), 25 oC, 2 h.
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a)
100
FP capsule
Transmittance (%)
90
80
70
60
50
40
30
20
4000
N-H
3500
Amide
3000
2500
2000
1500
1000
500
-1
Wavenumbers (cm )
b)
Transmittance (%)
100
FPSi capsule
90
80
70
60
50
Si-O-Si
40
30
4000
Amide
N-H
3500
3000
2500
2000
Si-O-Si
1500
1000
500
-1
Wavenumbers (cm )
Fig. S7 FTIR spectrum of FP capsule (a) and FPSi capsule (b). The synthesis conditions for FP capsule: 1.5 mL
aqueous solution (pH 10.0) of Fmoc-FF (2 mg mL-1), 5 mL aqueous solution (pH 7.5) of PEI (1wt%), 25 oC, 2
h.The synthesis conditions for FPSi capsule: 1.5 mL aqueous solution (pH 10.0) of Fmoc-FF (2 mg mL-1) and
sodium silicate (2 mg mL-1 SiO2), 5 mL aqueous solution (pH 7.5) of PEI (1wt%), 25 oC, 2 h.
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50
b) 20
25
Fmoc-FF solution
0
PEI solution
-25
Zeta potential (mV)
Zeta potential (mV)
a)
-50
10
0
Concentrations
(mg/mL SiO2)
0.5
1.0
2.0
Initial pH values
of silicate solution
7.0
8.0
9.0
10.0
-10
-20
-30
-40
Fig. S8 a) Zeta potential of 1.0 wt% PEI solution at pH 7.5 and the Fmoc-FF solution (2 mg mL-1) at pH 9.0. b)
Zeta potential of sodium silicate solution with different concentrations and pH values. The pH was kept at 9.0
when concentration was varied, while the concentration was kept at 2 mg mL-1 when pH was varied.
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a)
b)
c)
d)
e)
f)
g)
h)
Fig. S9 Photograph of BSA-containing Fmoc-FF/PEI/SiO2 (FPSi) or Fmoc-FF/PEI (FP) capsules. a-d) formed at
pH 9.0 and different sodium silicate concentrations (a: 0; b: 0.5 mg mL-1; c: 1.0 mg mL-1; d: 2.0 mg mL-1 SiO2).
e-h) formed at different initial pH values of silicate solution with 2 mg mL-1 SiO2 (e: pH 7.0; f: pH 8.0; g: pH 9.0;
h: pH 10.0).
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Specific activity (U/mg)
6
5
4
3
2
1
0
65
87
158
346
Specific protein loading (mg/g)
Fig. S10 Specific activities for the enantioselective hydrolysis of cyclohexene oxide (first 30 min) of FPSi-SpEH
(prepared by using pH of 9 for the initial enzyme solution) at different protein loadings.
Specific activity (U/mg)
10
Initial
After 8 days
8
6
4
2
0
Free SpEH Encapsulated SpEH
Fig. S11 Specific activities for the enantioselective hydrolysis of cyclohexene oxide (first 30 min) of free SpEH
and FPSi-SpEH before and after storage at 4 oC for 8 days.
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3. Supplementary Table
Table S1 The chemical compositions of FPSi capsules*
SiO2
Fmoc-FF
PEI
H2 O
Capsules
Synthesis
conditions
3 mg a
3 mg
50 mg
6.5 mL
10.2 mg
Theoretical
compositions
32.6%
32.6%
24.8% b
10% c
/
10%
/
Compositions
from TGA data
*
35%
55% d
The capsule synthesis conditions: 1.5 mL aqueous solution (pH 10.0) of Fmoc-FF (2 mg/mL) and sodium silicate (2
mg/mL SiO 2), 5 mL aqeous solution (pH 7.5) of PEI (1wt%)
a
The value respresents the corresponding amount to SiO2 for sodium silicate
b
The value is calculated by the following equation: CPEI = 100-CSiO -CFmoc-FF -CH O
2
2
c
The data is from the TGA analysis
d
The value represents the total content of Fmoc-FF and PEI
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