An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions

Article abstract of DOI:10.1016/j.bbrc.2016.03.049

A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of N_α-benzoyl-l-arginine ethyl ester to N_α-benzoyl-l-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas.

Full text of DOI:10.1016/j.bbrc.2016.03.049

Biochemical and Biophysical Research Communications 473 (2016) 54e60
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
An easily regenerable enzyme reactor prepared from polymerized
high internal phase emulsions
, b,
Guihua Ruan a *, Zhenwei Wu a, Yipeng Huang a, Meiping Wei a, Rihui Su a,
, b, **
Fuyou Du a
a Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional Materials, College of Chemistry and Bioengineering, Guilin University of
Technology, Guangxi 541004, China
b Guangxi Collaborative Innovation Center for Water Pollution Control and Water Safety in Karst Area, Guilin University of Technology, Guilin 541004, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith
Received 1 March 2016
Accepted 11 March 2016
Available online 17 March 2016
(polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal
polymerization of
a high internal phase emulsion containing styrene, divinylbenzene and poly-
glutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The
performance of the resultant enzyme reactor was assessed according to the conversion ability of Na-
benzoyl-L-arginine ethyl ester to Na-benzoyl-L-arginine, and the protein digestibility of bovine serum
albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited
high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C
could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and
residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with
4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good
permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor
was a good selector potentially applied in proteomics and catalysis areas.
Keywords:
Immobilized enzymatic reactor
Protein digestion
High internal phase emulsion
PolyHIPE monoliths
© 2016 Elsevier Inc. All rights reserved.
1. Introduction
membranes and nanofibers, [5,6], coreeshell magnetic nanoparticle,
[7,8], graphene oxide nanosheets [9] and organic-inorganic hybrid
Recently, protein digestion has been studied in many research
areas including proteomics, bioengineering and food industry, and
the high-throughput and high proteolytic enzyme reactor is ex-
pected for efficient protein digestion. Comparing with the traditional
protein digestion in solution suffered some drawbacks, such as
enzyme autodigestion, low efficiency, extended incubation time and
sample loss or contamination, [1], the immobilized enzymatic
reactors (IMERs) had great advantages in the aspect of protein
digestion [2e4]. Therefore, many IMERs have been studied by
immobilizing enzymes on different materials such as polymeric
monolith [10]. These rapid and low carry-over IMERs were success-
fully applied to protein digestion and proteomic analysis [11].
Among these IMERs, the monoliths, composed of porous solid
with small-sized skeletons and relatively large through-pores, could
offer fast mass transfer and high enzyme binding capacity [12,13].
Generally, monolithic supports were prepared as silica-based
monoliths [14,15] and organic polymers [16]. Silica-based mono-
liths had silica skeletons and through-pores via a solegel process or
hybrid organic-inorganic process [17,18]. which made them high
permeability, high mechanical strength, and good organic solvent
tolerance [19]. But, the preparation of these monoliths was compli-
cated, sometimes leading to poor reproducibility [20]. Another
problem was that the nonspecific adsorption of the silica-based
monolith was obvious due to the residual silanols, and the uncon-
trollable polymerization procedure lead it to an irregular pore size
and complicated absorption/desorption behavior at the surface of
the monolith. In comparison, with good porosity, mild biocompati-
bility and high-throughput conveying capacity, [21], [22] organic
polymer materials had been widely applied in many research areas
* Corresponding author. Guangxi Key Laboratory of Electrochemical and Mag-
netochemical Functional Materials, College of Chemistry and Bioengineering, Guilin
University of Technology, Guangxi 541004, China.
** Corresponding author. Guangxi Key Laboratory of Electrochemical and Mag-
netochemical Functional Materials, College of Chemistry and Bioengineering, Guilin
University of Technology, Guangxi 541004, China.
E-mail addresses: guihuaruan@hotmail.com (G. Ruan), dufu2005@126.com
(F. Du).
http://dx.doi.org/10.1016/j.bbrc.2016.03.049
0006-291X/© 2016 Elsevier Inc. All rights reserved.

G. Ruan et al. / Biochemical and Biophysical Research Communications 473 (2016) 54e60
55
like scaffolds for in vitro 3D cell culture, [23], protein separation and
phosphopeptides enrichment, [24], biomedical sensor support, [25],
biocatalysts [26] and biomacromolecules trapping [27]. But the
swelling of organic solvents might lead to the change of pore struc-
ture and the decrease of mechanical stability.
for 3 min to form a uniform emulsion. About 1.5 0.2 mL emulsion
was transferred into 2.5 mL sealed syringe and polymerized at
50 ꢁC for 24 h. The synthesized polyHIPEs was washed with
deionized water and ethanol-water solution (1:1 V/V). Finally, the
monoliths were washed and soaked with ammonium acetate buffer
(AAB, 0.1 mol/L, pH 8.0). It was measured that a dried polyHIPE
monolith was about 0.7 0.1 g.
The freshly prepared emulsions were placed for several hours
to observe the stability and variation then the two phases was
observed under the microscope in 400 and 1000 times. After
polymerization, deionized water was used to investigate the me-
chanical strength and flow velocity of the monoliths at a pressure
of 70 kPa. The morphology and cavity size distribution of mono-
liths were determined by SEM image analysis (S4800 field emis-
sion scanning electron microscope, Hitachi, Japan). Nitrogen
adsorption/desorption measurements were valuated on a Micro-
meritics® ASAP 2020 adsorption apparatus using a BET model for
surface area evaluation. To valuate the immobilization probability,
the experiments of FTIR spectrum (Nicolet iS10, Thermo Fisher,
USA) and elemental analysis (EA2400II, PerkinElmer, USA) were
carried out.
Notably, polymerized high internal phase emulsions (poly-
HIPEs) have been developed for extensive application in biocom-
patibility substrates [28,29]. After polymerization, polyHIPEs were
formed and the droplets of dispersed phase were removed yielding
a highly interconnected network pores with quite well defined
diameter. Depending on dispersed phase in emulsion, polyHIPEs
had adjustable meso- or macropore skeleton which avoided the
problems of nanoparticle agglomerating, low permeability and
surfactants demanding [30]. Moreover, the inherent meso- and
macropore of polyHIPEs were benefit for specific in-situ absorbents
of biological macromolecules when some functional group
reagents were added in the polymerization procedure [31].
Here, we developed an easy-preparing and regenerable poly-
HIPE based IMER for trypsin immobilization and protein digestion.
Our studies suggested that the developed IMERs not only have high
trypsin immobilization and bioactivity in protein digestion but also
have strong regeneration capacity and long-term durability. Ac-
cording to our knowledge, this is the first time report that using
polyHIPEs as immobilized enzyme reactor support for protein
digestion research.
2.3. Enzyme immobilization on polyHIPE monolith
For enzyme immobilization, the prepared monolith was con-
nected to a constant flow pump (HL-2D, Shanghai Huxi Analysis
Instrument Factory Co., Ltd) and kept at 37 ꢁC. 2.0 mL TPCK-trypsin
solution (0.2 mg/mL) was slowly injected and immobilized natu-
rally onto the monolith for 10 min. The residue trypsin solution was
pumped out and the reactor was washed with 2 mL AAB solution.
After the procedure of enzyme immobilization, the prepared IMER
was stored at 4 ꢁC. For long-term storage, the IMERs were soaked in
AAB and stored at ꢂ20 ꢁC in fridge.
2. Materials and methods
2.1. Materials and chemicals
Reagents including styrene (STY), divinylbenzene (DVB), sorbi-
tan monooleate (Span80), TPCK-trypsin (Type I, >10 000 units/mg
Na-benzoyl-
L-arginine ethyl ester hydrochloride, TPCK treated), Na-
benzoyl- -arginine ethyl ester (BAEE, 98%), Na-benzoyl-
L
L
-arginine
(BA, 98.5%), Tris (Hydroxymethyl)aminomethane (Tris), iodoace-
tamide (IAA), dithiothreitol (DTT), Cytochrome C (Cyt-c) and bovine
serum albumin (BSA) were purchased from Aladdin Reagent Co.
Ltd. (Shanghai, China). Potassium persulfate (K2S2O8, AR), Glutar-
aldehyde (GA, 25%, w/v aqueous solution) were obtained from
Xilong Chemical Reagent (Guangzhou, China). Methanol and
acetonitrile were of HPLC grade from Dikma Co., Ltd. (CA, America).
All other reagents were of analytical grade, such as sodium hy-
droxide, hydrochloric acid and ammonium acetate. The water was
deionized by an arium® 611 system (Sartorius, Germany) with
2.4. Investigation of monolithic polyHIPE based IMER
The TPCK-trypsin immobilization rate was calculated by con-
versation rate of BAEE to BA. Also, the conversion rate of BAEE to BA
was used to optimize the synthesis conditions of monolith and
assess the properties of IMER [33]. In brief, the prepared monolithic
polyHIPE based IMER was heated in a cabinet drier at constant
temperature of 37 ꢁC, and then 0.650 mL solution of TriseHCl and
BAEE (0.3 mL of 0.1 mol/L TriseHCl and 0.35 mL of 5.0 mg/mL BAEE)
was pumped through the IMER and reacted for 10 min at a constant
velocity. After that, 1.95 mL water was used to wash out the BAEE
and BA and deionized water was used to clean the IMER. All eluents
were respectively collected and the solution was filtered through
resistance ꢀ18.2 M
U
/cm. All solutions and deionized water were
m filter membrane before HPLC and LC-MS
filtered through 0.22
m
analysis.
Before using, STY and DVB were washed with 5% NaOH aqueous
solution and then with water to remove the polymerization in-
hibitor. After washed to pH 7.0 with water, the obtained solution
was dried with MgSO4.
0.22 mm filter membrane before HPLC analysis.
To confirm the limitation of reusability of IMER, it was used
many times. Once the enzyme activity of IMER was dramatically
decreased, 15 mL AAB was injected to monolith to clean up and
20 mL HCl (4 mol/L) was used to regenerate the monolith for
breaking the eC]Ne bond between the TPCK-trypsin and PGA
based on the Schiff base reaction. The remnant HCl was washed out
by 6 mL deionized water and followed by 15 mL AAB. After that,
TPCK-trypsin was immobilized on monolith once again and the
regenerated IMER was reused.
For protein digestion, 1 mL of 10 mg/mL BSA and Cyt-c were
respectively dissolved in 1 mL of 50 mM TriseHCl (pH 8.1) con-
taining 8 M urea and then the solution was reduced via 0.1 mL of
0.1 M DTT for 20 min at 50 ꢁC. After cooling to room temperature,
BSA and Cyt-c were alkylated in the dark with 0.1 mL 0.1 M IDA for
15 min at room temperature. The pretreated BSA and Cyt-C sample
(0.2 mg/mL) was pump through the IMER by using the peristaltic
pump at 37 ꢁC in cabinet drier for 10 min. After digestion, the
2.2. Preparation of polyHIPE monolith
Firstly, the dispersed phase of PGA and K2S2O8 solution was
prepared as following: polyglutaraldehyde (PGA) was obtained
with GA solution reacted with 1.0 M NaOH at pH 10.5 for 30 min
and then the solution was adjusted to pH 7.0 by 1 M HCl [32].
6.531 mL 0.02% (g/mL) K2S2O8 solution was mixed with 0.417 mL
PGA solution as dispersed phase for the preparation of HIPE.
0.772 mL continuous phase containing STY, DVB and Span 80
was added into centrifugation tube and rotated in a vortex mixer
(IKA MS3 basic, Germany) at 3000 rpm for 30 s, then 6.948 mL
dispersed phase was added drop-wise to the continuous phase
under condition of gentle stirring. The emulsion was finally stirred

56
G. Ruan et al. / Biochemical and Biophysical Research Communications 473 (2016) 54e60
enzymatic hydrolyzate was flushed out by 2 mL AAB solution and
collected for peptides analysis with LC-MS/MS.
compositions and proportions of high internal phase emulsion
greatly influence the morphology and porosity of polyHIPE, which
would finally affect the permeability and mechanical strength of
IMER. The monoliths were designed with different phase ratios of
STY/DVB (v/v) in oil phase. Five IMERs were first prepared and
different STY/DVB (v/v) ratio from 2:1 to 6.5:1 was investigated
(Fig. S1, Supplementary Material). The obtained results showed that
lower ratio of STY/DVB (2:1) in continuous phase developed larger
oil vacuoles and the excessive DVB made the monoliths compact-
ness with hardly impermeable and the 6.5:1 ratio of STY/DVB made
the monolith loose with the bubble-liking cavities obviously. The
monoliths had an ideal mechanical strength and permeability
when the STY/DVB (v/v) ratio was 3:1. Magnified electron micro-
scope dyed photograph of emulsion showed that the emulsion was
well formed and highly stable for more then 72 h when the STY/
DVB (v/v) ratio was 3:1 (Fig. 1).
Volume fraction of dispersed phase (content of water phase in
emulsion, V/V) resulted polyHIPE monoliths differences at perme-
ability and mechanical strength (Fig. S2, Supplementary Material).
The lowest volume fraction of dispersed phase in HIPE resulted in
the hardest and the worst permeability for polyHIPE monolith. As
shown in Table 1, the IMER with different dispersed phase had
different permeability and enzyme immobilization efficiency. The
lower amount of dispersed phase resulted in a small through-pore
structure of monolith with bad permeability (columns 1 and 2) and
lower enzyme capacity as well as enzyme bioactivity. Only when
the dispersed phase volume fractions reach to 89% and 91%, the
TPCK-trypsin capacities both are 0.36 mg and the TPCK-trypsin
bioactivity reaches the highest (columns 4 and 5). In terms of the
permeability and mechanical strength of polyHIPE monolith, 90%
dispersed phase value fraction was selected. In summary, suitable
materials with well-organized framework, well permeable, large
enzyme capacity and high enzyme bioactivity are helpful for
enzyme immobilization and protein digestion. Full pretreatment
with the material, such as ordered, regular and porous structure at
controllability of synthesis process would benefit for protein
enzymatic hydrolysis [35,36].
2.5. HPLC analysis and LC-MS/MS analysis
HPLC (Shimadzu LC-20A, Kyoto, Japan) with photodiode array
detector (PAD) were employed to analyze BAEE and BA. Luna 5
m C18
(2) 100A (250 mm ꢃ 4.6 mm, 5
m
m, Phenomenex, USA) column was
used and the operation parameters were optimized. The main
determining wavelength was 253 nm. Gradient elution program
was set as follow: solution A: deionized water with 0.1vol% fomic
acid (FA), solution B: acetonitrile with 0.1 vol% FA. The flow rate was
0.80 mL/min gradient conditions: 10% B for 5 min, to 50% B in
15 min, to 10% B in 5 min and kept at 10% B for 5 min. 10
sample was injected.
mL of
Protein and peptides analysis were carried out with LC-ESI-MS/
MS (LTQ-Orbitrap, Thermo-Fisher, San Jose, CA, USA). Aeris PEPTIDE
XB-C18 column (250 mm ꢃ 4.6 mm, 3.6
mm, Phenomenex, USA)
was used with the flow rate of 300 L/min. The separation condi-
m
tion was optimized and water containing 0.1vol% formic acid (A)
and CH3CN (B) were used to generate a 60 min gradient, set as
follows: 5% B for 1 min, to 40% B in 33 min, to 95% B in 6 min, kept at
95% B for 6 min, to 5% B in 10 min and kept at 5% B for 4 min.10 mL of
sample was injected for LC-ESI MS3 analysis at positive ion mode.
The spray voltage was 3.0 kV, and the heated capillary temperature
was 300 ꢁC. The MS was operated in the data-dependent mode, in
which a survey full scan MS spectrum (from m/z 100 to 2000) was
acquired in the Orbitrap with a resolution of 30,000 at m/z 400.
This was then followed by MS2 scans of the most abundant ions and
the MS3 scans of first, second and third most abundant ions from
MS2. The resulting fragment ions were recorded in the linear ion
trap. The acquired mass spectrometric raw data was processed
using MassMatrix. The mechanism of searching could be explained
in details in the published papers of Xu et al. [34].
3. Results and discussion
At the point of most promising monolith, the IMER should be
easily prepared with the higher immobilization rate of trypsin on
the surface of monolithic porous polymers. It was reported that the
porous support covalent with PGA immobilized enzyme by both
absorption and covalent binding due to the presence of PGA func-
tional groups which could reacted with amino, thiol, imidazole and
phenol groups of proteins [37]. More PGA adding in emulsions
seems benefit for not only the permeability of polyHIPE monoliths,
but also for the TPCK-trypsin immobilization capacity and bioac-
tivity. Different concentrations of PGA in dispersed phase (V/V: 0%,
3%, 6%, 9%, 12%) were investigated. Table 2 shows the effects of PGA
concentration in dispersed phase. Except the 12% of PGA in
dispersed phase could not synthesis a homogeneous monolith after
3.1. Effect of contents in high internal phase emulsion
Variation in the chemistry of the polymerization mixture was
expected to change trypsin immobilization and mechanical
strength, and to some extent to influence the diameter of pore
around immobilization active sites. It was reported, [25], the
monolith composed of porous solid with facileness of functionality
and relatively large through-pores, could offer high rates of mass
transfer and high enzyme binding capacity. In our study, the pol-
yHIPE based IMER were prepared with styrene as monomer,
divinylbenzene as crosslinker and span80 as emulsifier. Meanwhile,
in the water phase, polyglutaraldehyde was used as functional
additive
for
TPCK-trypsin
immobilization.
Particularly,
Fig. 1. Magnified electron microscope dyed photograph of high internal phase emulsions at STY/DVB ratio of 3:1 (v/v) and dispersed phase of 90% (v/v). a.1000 times; b. 400 times.

G. Ruan et al. / Biochemical and Biophysical Research Communications 473 (2016) 54e60
57
Table 1
Effects of different volume fraction of dispersed phase on preparation of polyHIPE monolithsa.
Column
1
2
3
4
5
STY-DVB(V:V ¼ 3:1)/mL
dispersed phase/mL (%b)
PGA/mL (9%c)
0.772
2.131 (73)
0.185
0.772
2.841 (79)
0.247
0.772
4.025 (84)
0.350
0.772
6.392 (89)
0.556
0.772
8.168 (91)
0.710
Morphology Hardnessd
Flow velocitye/
m
L$sꢂ1
e
0.7
0.28
58.86
34.8
0.32
68.31
45.5
0.36
71.95
71.4
0.36
70.03
Trypsin capacityf/mg
Trypsin bioactivityg/%
0.30
52.53
a
The continuous phase contained 0.445 mL of STY, 0.150 mL of DVB and 0.177 mL of Span 80 and the columns 1, 2, 3, 4, 5 were polymerized with 1.5 mL emulsion in 2.5 mL
plastic syringe at 50 ꢁC for 24 h.
b
Dispersed phase volume fraction (%) ¼ Vdispersed phase/(VSTYꢂDVB þ Vdispersed phase)*100.
c
Concentrations of PGA in the dispersed phase (%) ¼ PGA value/dispersed phase value.
d
The monoliths had several shape changes at pressure from tube handle. Very hard: no change; Hard: subtle compression; Hard-medium: little compression but full
resilience after withdrawing handle force; Medium: a little compression but full resilience; Soft: some compression but a little resilience.
e
Flushing with 1 mL deionized water at backpressure of 70 kPa; column: 15 mm ꢃ 5.4 mm I.D., 10 mm length. “-”: it was too hard to pump water.
f
TPCK-trypsin capacity was determined with the conversation rate of BAEE to BA with free TPCK-trypsin which was washed from the monolith. The total given amount of
TPCK-trypsin was 0.4 mg.
g
TPCK-Trypsin bioactivity was calculated with the conversation rate of BAEE to BA at TPCK-trypsin immobilized polyHIPE monolith comparing with 2 mL 0.2 mg/mL free
TPCK-trypsin solution.
24 h, the other monoliths were all synthesized successfully but the
performance of IMERs were little different in TPCK-trypsin immo-
bilization efficiency and bioactivity. With regard to enzyme
immobilization, the increase of the amount of porosity on the
support was desirable to enhance enzyme/substrate ratio and thus
the reaction rate [33,37]. Furthermore, the bioactivity of bound
trypsin was verified for higher digestion efficiency with optimized
PGA concentration. When PGA was not added in dispersed phase,
the synthesized monolith could absorb 0.37 mg trypsin (about 90%
given amount) but the bioactivity of loaded enzyme was only
38.85%. The substantial decrease of trypsin bioactivity suggested
that the trypsin should be randomly adsorbed and strongly
agglomerated on the surface of HIPE microsphere and the affinity
sites of the agglomerated enzyme were not fully activated. The
homogeneous distribution of the functional groups on the surface
of the support would largely reduce the stereochemical hindrance,
thus increasing the amount and bioactivity of immobilized trypsin.
In our study, when the PGA concentration was increased to 6%, the
permeability, pore volume and specific surface area of HIPE
monolith was improved and the bioactivity of immobilized trypsin
was 90.75% with 0.36 mg immobilized trypsin. The higher trypsin
bioactivity was thought to be attributed to the lower enzyme/
substrate ratio to avoid the reactant blocking. However, excessive
PGA would result a soft and nonelastic monolith and it was not
suitable for trypsin immobilization.
Table 2
Effects of PGA concentration in dispersed phase on preparation of polyHIPE
monolithsa.
Column
A
B
C
D
STY-DVB(V:V ¼ 3:1)/mL
dispersed phase/mL
0.772
6.948
0.000 (0)
12.56
0.095
22.13
40.0
0.772
6.740
0.208 (3)
10.87
0.101
30.26
55.6
0.772
6.531
0.417 (6)
18.31
0.126
45.11
71.4
0.772
6.323
0.625 (9)
14.00
0.019
5.48
100
0.17
55.90
PGA/mL (%b)
Average pore widthc (nm)
Pore volumec (cm3/g)
Specific surface areac (m2/g)
3.2. Characteristic performance of polyHIPE monolith IMER
The detailed morphological and structural features of the as-
prepared monolith were characterized using scanning electron
microscopy (SEM), fourier transform infrared spectrometer (FT-IR)
and material elemental analysis. The SEM images of the HIPE
microsphere in Fig. 2a and b indicated that they are well-dispersed
Flow velocityd/
m
L sꢂ1
Trypsin capacitye/mg
Trypsin bioactivityf/%
0.37
38.85
0.37
72.70
0.36
90.75
a
The continuous phase contained 0.445 mL of STY, 0.150 mL of DVB and 0.177 mL
of Span 80 and the columns F, G, H, I were polymerized with 1.5 mL emulsion in
2.5 mL plastic syringe at 50 ꢁC for 24 h.
porous microsphere with the average size of 10
mm which are
b
Percentage of PGA(%) ¼ VPGA/Vdispersed phase*100.
composed of about 1.5 m many holes side by side. After the IMER
m
c
Average pore width, pore volume and specific surface area were obtained from
regeneration via HCl flushing, the obtained microspheres showed
original porous surface and non-destructive 3D structure (Fig 2c),
indicating monolith stabilization of long-term protein digestion. By
comparison, the TPCK-trypsin re-immobilized microspheres with
the high porosity still retain almost the same 3D structure and
specific surface area as their precursor microspheres. FITR analysis
provided information regarding the changes in TPCK-trypsin
immobilization of the monolith. FITR images (Fig. 3) revealed the
Micromeritics ASAP (accelerated surface area and porosimetry) for BET test.
d
Flushing with 1 mL deionized water at backpressure of 70 kPa; column:
15 mm ꢃ 5.4 mm I.D., 10 mm length. “-”: it was too hard to pump water.
e
Trypsin capacity was determined with the conversation rate of BAEE to BA with
free trypsin which was washed from the monolith. The total given amount of trypsin
was 0.4 mg.
f
Trypsin bioactivity was calculated with the conversation rate of BAEE to BA at
trypsin immobilized polyHIPE monolith comparing with 2 mL 0.2 mg/mL free
trypsin solution.

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G. Ruan et al. / Biochemical and Biophysical Research Communications 473 (2016) 54e60
changes of synthesized IMER (STY-DVB-PGA) before and after
conjugation with TPCK-trypsin. It can be concluded that the spec-
troscopy of STY-DVB-PGA monolith before conjugation with TPCK-
trypsin had the main characteristic band of the C]O at 1735 cmꢂ1
with weak peak. After conjunction with TPCK-trypsin, the main
characteristic band of the C]O at 1735 cmꢂ1 was stronger. The
nitrogen compositions of two kinds of IMERs revealed that TPCK-
trypsin immobilized polyHIPEs contained a higher quantity of
-NH2 than the monolith without TPCK-trypsin by the determina-
tion of element analysis. These results proved that the majority of
TPCK-trypsin was successfully immobilized on the monolith. The
SEM images shows that polyHIPE monoliths exhibited a highly
crosslinked structure which has substantial three-dimensional
microsphere with thin wall and interconnected small chambers
(Fig. 2a and b). Also, the stable skeleton of polyHIPEs monoliths
provides the proteolytic support for enzymes. The monolithic ma-
terial could offer large specific surface area while the macropores
and high porosity afforded better permeability, leading to increased
trypsin immobilization capacity. In our study, the meso-/macro-
porous shell structure of the polyHIPEs monoliths not only poss
high specific surface and amounts of affinity sites for the high
loading capacity of trypsin but also provide large pores for protein
ingress and small pores to allow diffusion of reactants and resulting
digests (Fig. S3, Supplementary Material).
The maximum enzyme immobilization ability and the remained
bioactivity of immobilized enzyme on the prepared polyHIPE
monoliths were investigated according to the immobilization of
TPCK-trypsin. Table 3 reveals that the TPCK-trypsin uploading ca-
pacity almost remains about 90% but the bioactivities of TPCK-
trypsin are gradually decreased when the trypsin amount over-
upload over 0.4 mg. The great TPCK-trypsin uploading capacity
was attributed to the substantial three-dimensional microsphere
and crosslinking structure which gave sufficient space to absorb
TPCK-trypsin but the IMER modified with 6% PGA (0.417 mL) could
not immobilize all the TPCK-trypsin. Indeed, the suitable amount of
loading trypsin should be not more than 0.4 mg.
For the regeneration of polyHIPEs monoliths, the IMER was pro-
posed to treat with hydrochloric acid solution (HCl) to break the
covalent bond between -NH2 and -CHO. The IMER was successfully
regenerated with HCl solution without any structure change in long-
term or large scale protein digestion (Fig. 2c). The same three
dimension structures and sizes as their precursor microspheres
indicated that the monolith could be further reused and regenerated
when the bioactivity of uploaded enzyme dramatically decreased.
Actually, in our experiments, the polyHIPE monoliths still remained
57.9% TPCK-trypsin bioactivity after ten times protein digestion. The
Fig. 2. SEM of PolyHIPE monoliths (a) STY-DVB-PGA-trypsin monoliths at 1000 times;
(b) STY-DVB-PGA-trypsin monoliths at 5000 times; (c) regenerated STY-DVB-PGA-
trypsin monoliths at 1000 times.
Fig. 3. FTIR and Elementary Analysis of polyHIPEs. (a) STY-DVB-PGA monolith. (b). STY-DVB-PGA-TPCKtrypsin reactor.

G. Ruan et al. / Biochemical and Biophysical Research Communications 473 (2016) 54e60
59
Table 3
Trypsin immobilization and trypsin bioactivity of polyHIPE IMER.
Table 4
Peptides analysis by LC-ESI-MS/MS from BSA and Cyt-C.
Column
J
K(H)
0.772
6.948
0.4
0.36
90.20
L
M
N
Protein
No
Sequence
numbers
MW
Peptide sequence
STY-DVB(V:V ¼ 3:1)/mL
dispersed phase/mL
Uploaded trypsin/mg
Trypsin capacity/mg
Trypsin bioactivity/%
0.772
6.948
0.2
0.772
6.948
1.0
0.772
6.948
1.6
1.56
77.20
0.772
6.948
2.0
1.96
70.80
BSA
1
2
3
4
5
6
7
8
9
25e34
35e44
66e75
66e88
1193.6026
1249.6219
1163.6309
2608.2026
1463.5927
1419.6980
1946.0191
545.3414
2542.1654
649.3352
906.4722
706.3555
1195.5911
508.2523
1001.5889
689.3734
847.5036
922.4891
789.4741
1684.8227
1532.7834
1291.6044
1567.7446
1439.8135
2355.1430
1068.4417
2529.2223
1479.7980
1639.9393
817.4903
432.2600
1166.4926
1724.8395
898.4819
1539.8215
1869.0254
1138.4999
1881.9327
2471.1888
609.2886
2498.1908
2867.4293
1907.9223
2277.1604
1142.7143
1014.6207
818.4266
1399.6932
1003.5800
762.4872
634.3924
1433.7760
1168.6244
1296.7172
1584.7674
1826.9027
1456.6705
1698.8078
2009.9607
2138.0503
806.4775
779.4492
907.5436
1306.692
1434.7954
1092.6327
434.1887
DTHKSEIAHR
FKDLGEEHFK
LVNELTEFAK
0.17
0.96
107.63
81.10
LVNELTEFAKTCVADESHAGCEK
TCVADESHAGCEK
SLHTLFGDELCK
SLHTLFGDELCKVASLR
VASLR
QEPERNECFLSHKDDSPDLPK
IETMR
IETMREK
CASIQK
CASIQKFGER
FGER
ALKAWSVAR
76e88
89e100
89e105
101e105
118e138
205e209
205e211
223e228
223e232
229e232
233e241
236e241
242e248
249e256
257e263
286e299
298e309
300e309
347e359
360e371
402e420
413e420
413e433
421e433
437e451
452e459
456e459
460e468
469e482
483e489
483e495
483e498
499e507
508e523
508e528
524e528
524e544
524e547
529e544
529e547
548e557
549e557
562e568
569e580
598e607
9e14
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
AWSVAR
LSQKFPK
AEFVEVTK
LVTDLTK
YICDNQDTISSKLK
LKECCDKPLLEK
ECCDKPLLEK
DAFLGSFLYEYSR
RHPEYAVSVLLR
HLVDEPQNLIKQNCDQFEK
QNCDQFEK
QNCDQFEKLGEYGFQNALIVR
LGEYGFQNALIVR
KVPQVSTPTLVEVSR
SLGKVGTR
Fig. 4. Stability and regeneration ability of polyHIPE IMER.
VGTR
CCTKPESER
tenth TPCK-trypsin re-immobilized IMER could hold about 50.4%
bioactivity for protein digestion. Fig. 4 shows that with 4.0 M or
higher concentration HCl (20 mL) treating for 10 times, the remnant
of TPCK-trypsin in monolith was very little (the residual TPCK-
trypsin was about 0.01 mg at Fig. S4, Supplementary Material). The
high bioactivity of this regenerated IMER after facile operations for
fresh enzyme uploading significantly opens up a new potential
employment of polyHIPE-IMER for different enzyme immobilization
and an easy renewable way to digest various proteins.
MPCTEDYLSLILNR
LCVLHEK
LCVLHEKTPVSEK
LCVLHEKTPVSEKVTK
CCTESLVNR
RPCFSALTPDETYVPK
RPCFSALTPDETYVPKAFDEK
AFDEK
AFDEKLFTFHADICTLPDTEK
AFDEKLFTFHADICTLPDTEKQIK
LFTFHADICTLPDTEK
LFTFHADICTLPDTEKQIK
KQTALVELLK
3.3. Application property of polyHIPE-IMER
QTALVELLK
ATEEQLK
TVMENFVAFVDK
LVVSTQTALA
KIFVQK
The reusability of the IMER is important for routine proteolysis
procedure in proteomic analysis. In this study, the STY-DVB-PGA
polyHIPE-IMER monolith was successfully tested by repeated
digestion of BAEE solution. Fig. 4 showed that the uploaded trypsin
of IMER decreased gradually as the regeneration number increased.
However, the IMER still showed a higher retainable of retention
activity, where 58.3% retention activity was found after ten re-
generations. This indicates that the IMER had a very operational
stability for a large quantity of uploaded enzyme with high bioac-
tivity and high recovery activity of trypsin re-immobilization.
Table 4 lists the peptide generated from BSA and Cyt-c digestion.
About 59% sequence coverage with 49 peptides in BSA digestion
and 78% sequence coverage with 18 peptides in Cyt-c digestion
were obtained. In addition, the successful regeneration of
polyHIPE-IMER for BSA and Cyt-c digestion verified that the IMER
could be used as a fundamental support for recycle digestion
though a long-term protein digestion (Fig. S5, Supplementary
Material). The molecular weights (MW) of peptides digested from
BSA and Cyt-c mainly ranged from 1000 to 1750 Da and from 750 to
1500 Da, respectively (Fig. S6, Supplementary Material). Comparing
with former studies of IMER, [36], although the sequence coverages
of BSA and Cyt-C were not quite high, the polyHIPE-IMER was more
easily manufactured with two-step strategies (HIPE polymerization
Cyt-c
10e14
27e39
29e39
29e40
40e54
40e56
41e54
41e56
57e73
57e74
74e80
81e87
IFVQK
HKTGPNLHGLFGR
TGPNLHGLFGR
TGPNLHGLFGRK
KTGQAPGFSYTDANK
KTGQAPGFSYTDANKNK
TGQAPGFSYTDANK
TGQAPGFSYTDANKNK
GITWGEETLMEYLENPK
GITWGEETLMEYLENPKKKYIPGTK
PGTK
MIFAGIK
81e88
MIFAGIKK
90e100
90e101
93e101
102e105
GEREDLIAYLK
GEREDLIAYLKK
EDLIAYLKK
ATNE
and enzyme immobilization) and had the advantages of large-scale
digestion and easy regeneration. Furthermore, this monolith IMER
can be tentatively applied in online fast and high-throughput
protein digestion when it is coupled with other accelerated

60
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The authors declare no conflict of interest.
Acknowledgments
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