Synthesis of papain-polyacrylamide hydrogel microspheres and their catalytic application

Article abstract of DOI:10.1039/d1nj02551a

Although immobilized enzyme technologies effectively improve the reusability and stability of an enzyme, the introduction of covalent bonds or/and charge interaction causes the change for the secondary structure of an enzyme, thereby reducing the catalytic activity. In this work, a mild and efficient strategy for enzyme immobilization is proposed. A size-controlled hydrogel microsphere was designed and synthesized as an immobilized enzyme carrier. Papain was immobilized on the surface and pores of hydrogel microspheres by hydrogen bonding interactions, which have no adverse effect on the secondary structure of an enzyme. The activity of the papain-polyacrylamide hydrogel microsphere was 106.41% of that of free papain. In addition, the immobilized papain maintained 59.60% of free papain activity after 10 cycles. The results of practical catalytic application proved that the obtained papain-polyacrylamide hydrogel microspheres were very suitable for catalytic hydrolysis of allergen protein in cow's milk without introducing any other hetero protein.

Full text of DOI:10.1039/d1nj02551a

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DOI: 10.1039/D1NJ02551A
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Synthesis of papain-polyacrylamide hydrogel
microspheres and their catalytic application
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Yuan Ma1, 2, Yao Li3, Xu Fei*1, Jing Tian*2, Longquan Xu 1, Yi Wang2
1. Instrumental Analysis Center, Dalian Polytechnic University, Dalian 116034, China
2. School of Biological Engineering, Dalian Polytechnic University, Dalian 116034,
China
3. School of Light Industry and Chemical Engineering, Dalian Polytechnic University,
Dalian 116034, China
* Correspondence to: Xu Fei, Instrumental Analysis Center, Dalian Polytechnic
University, 1# Qinggongyuan Road, Dalian 116034, P. R. China
E-mail: feixudlpu@163.com
Tel: +86-411-86323691-201
* Correspondence to: Jing Tian, School of Biological Engineering, Dalian Polytechnic
University, 1# Qinggongyuan Road, Dalian 116034, P. R. China
E-mail: tianjing@dlpu.edu.cn
Tel: +86-411-86324485
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DOI: 10.1039/D1NJ02551A
Abstract
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Although the immobilized enzyme technologies effectively improve the reusability
and stability of the enzyme, the introduction of covalent bonds or/and charge interaction
causes the change for the secondary structure of enzyme, thereby reducing the catalytic
activity. In this work, a mild and efficient strategy for enzyme immobilization is
proposed. A size-controlled hydrogel microsphere was designed and synthesized as an
immobilized enzyme carrier. Papain was immobilized on the surface and pores of
hydrogel microspheres by hydrogen bonding interactions, which have no adverse effect
on the secondary structure of enzyme. The activity of papain-polyacrylamide hydrogel
microsphere was 106.41% of that of free papain. In addition, the immobilized papain
maintained 59.60% of free papain activity after 10 cycles. The results of practical
catalytic application proved that the obtained papain-polyacrylamide hydrogel
microspheres were very suitable for catalytic hydrolysis of allergen protein in cow’s
milk without introducing any other hetero protein.
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Keywords: Hydrogel microspheres, Immobilized enzyme, Papain, Milk
desensitization
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1. Introduction
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Milk is a highly nutritious food that infants consume in great quantities during
lactation1. However, during the ingestion of milk, the immune system of some infants
will recognize certain proteins in milk as harmful substances, causing protein allergic
reaction (CMPA), which can lead to diarrhea, respiratory asthma, vomiting, and skin
irritation2, 3. In recent years, with the increase of milk feeding ratio, the prevalence of
CMPA has increased year by year. Many studies of milk protein allergic reaction have
shown that the major allergens in milk are α-lactoalbumin (α-LA) and β-lactoglobulin
(β-LG), especially for β-LG4. The main strategy to avoid CMPA is to eliminate the α-
LA and β-LG, and preserve the beneficial proteins to ensure that children receive
enough amino acids to promote their growth5. Enzyme biocatalysis is a very suitable
pathway for this goal because of its ability to carry out biochemical transformations
with amazing catalytic efficiency and substrate specificity under mild conditions.
Papain is a thiol protease and widely exists in fruits, stem and papaya roots6.
Papain belongs to cysteine protease family and is a macromolecule composed of 212–
218 amino acid residues7. Papain is widely used in food industry, biomedicine,
cosmetics, and textile industry, because of its plentiful raw materials, high thermal
stability and wide range of substrates7-9. However, there are some problems in the
utilization of free papain, such as recycling difficulty and product pollution. Enzyme
immobilization technology is an effective method to overcome the shortcomings of free
enzyme10. The immobilization of enzymes improves the stability of enzymes in the
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harsh environment, and enables the industrial reuse of enzymes to carry out more
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reaction cycles, which has been widely used in sustainable chemical processes11, 12
.
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Many methods have been reported for immobilization of papain on different supports13-
15. However, limited mass transfer, loss of activity and stability, and low reusability
are the main problems that limit wider application of immobilized papain16. In recent
decades, new enzyme immobilization techniques have developed, including adsorption,
embedding, cross-linking and covalent binding method14, 16-18. Among these methods,
adsorption is the simplest and most convenient method of immobilization.
Immobilization by adsorption can be achieved either by surface or pore adsorption
techniques. Adsorption immobilization is achieved by the interaction, such as hydrogen
bond, Van der Waals force, hydrophilic hydrophobic interaction, electrostatic
interaction, etc19. These interactions have little effect on the structure of the enzyme
protein and are conducive to maintaining the activity of the enzyme20. The suitable
support material plays an important role in the immobilization of enzyme. The selection
of support materials should consider the type of enzyme, reaction medium, reaction
conditions and application fields. The hydrophilic support materials should be selected
for the immobilized papain to hydrolyze the α-LAand β-LG in milk, which is beneficial
to the contact between substrate and enzyme.
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Hydrogel is a three-dimensional polymer network that absorbs large amounts of
water without dissolving and it has advantages of good biocompatibility, mechanical
and functionality properties21. Similar to biological tissue, hydrogels are widely used in
biomedical and related fields, such as tissue scaffolds22, wound dressings23 and
wearable flexible sensors24, etc. However, as support materials, hydrogels are not easily
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processed into micron or nanometer size particles due to their special foDrmOI:a1t0i.1o0n39/D1NJ02551A
mechanism includes physical crosslinking and chemical crosslinking. This
disadvantage hinders the further application of hydrogels in the field of enzyme
immobilization.
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In this work, polyacrylamide hydrogel microspheres (PAHMs) with different
particle sizes were synthesized by inverse emulsion polymerization, and papain was
immobilized on the PAHMs by hydrogen bonding interaction to form papain-PAHMs.
The papain-PAHMs showed higher catalytic activity than free papain, and were easy to
be separated from reaction mixture by precipitation. In particular, the stability of
papain-PAHM3 significantly improved, and it showed excellent reusability after
immobilization. The papain-PAHM3 was used to hydrolyzed the proteins in milk,
which successfully eliminated the α-LA and β-LG in milk. Based on this research, it
confirmed that this strategy improved the enzyme activity and the reusability, which
provided more opportunities for its application in the food industry.
2. Experimental
2.1 Materials
Papain was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (activity ≥
800U/mg). Acrylamide (AM, 99.5%) was produced by the Shanghai Guoyao Chemical
Co. Ltd. N, N-methylenebisacrylamide (MBA, 99.7%) was obtained from Tianjin
Aoran Fine Chemical Research Institute. Tween 80 (99.7%) purchased from Tianjin
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Guangfu Fine Chemical Research Institute. Ethyl N-benzoyl-L-arDgOinI:i1n0.a10te39/D1NJ02551A
hydrochloride (BAEE, 98%) was purchased from Shanghai Aladdin Biochemical
Technology Co., Ltd. DL-Dithiothreitol (DTT, 99.5%) was provided by Beijing
Solarbio Technology Co., Ltd. NaCl (99.5%) was provided by Tianjin Kermel Chemical
Reagent Co., Ltd.Cyclohexane, ammonium persulfate (APS), polyvinylpyrrolidone
(PVP), Na2HPO4 (99%), KH2PO4 (99.5%) and KCl (99.5%) were purchased from
Tianjin Damao Chemical Reagent Co., Ltd. The milk was produced by Nestle
Shuangcheng Co., Ltd. The water used in the experiment was deionized water.
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2.2 Characterizations
The activity of papain was determined by UV-Vis absorption spectra with a Perkin
Elmer LAMBDA35 from the United States. The FT-IR spectra were measured by
Spectrum Two Fourier transform infrared spectrometer (Perkin Elmer, USA). Scanning
electron microscope (SEM) of JEOL JSM7800F and energy dispersive X-ray
spectroscopy (EDS) of Oxford INCA were used to detect the surface morphology and
element distribution of the samples. Papain labeled with FITC fluorescein was
measured by IX73 inverted fluorescence microscope (Olympus Corporation, Japan).
Low-field nuclear magnetic resonance (LF-NMR) was used to measure the moisture
content of papain-polyacrylamide hydrogel microspheres. The mechanical properties
of the polyacrylamide hydrogel microspheres were tested by compressing the
microspheres at the speed of 3 mm/min.
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2.3 Preparation of Polyacrylamide Hydrogel Microspheres
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Polyacrylamide hydrogel microspheres (PAHMs) were prepared by inverse
emulsion polymerization (IEP)25, 26. First, the organic phase was prepared with
cyclohexane (45 mL) and Tween 80 (2 g, 4.67 mmol) and was put into a 250 mL three
port flask. The organic phase was stirred vigorously at room temperature until it was
completely dissolved. As the aqueous phase, the mixture of acrylamide (AM, 28.14
mmol), N, N-methylenebisacrylamide (MBA, 0.13 mmol), polyvinylpyrrolidone
(PVP,18 mmol) and water (4 g) were added slowly into the flask with nitrogen bubbling.
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The reaction system was heated to 65 C and stirred vigorously for 30 min to prepare
water-in-oil micelle droplets. And then, 1mL of 1% (V/V) ammonium persulfate (APS)
aqueous solution was added to the reaction system and the reaction was kept at 65 oC
for 2h. After the reaction, the precipitates were collected and washed with ethanol and
deionized water to remove the unreacted monomers. Different sizes of PAHMs were
prepared by adjusting the stirring speeds of 300, 500 and 700 rpm/min were named
PAHM1, PAHM2 and PAHM3, respectively.
2.4 Preparation of papain-PAHMs
The papain was immobilized on PAHMs by physical process. The PAHMs and
papain were added into a beaker with the mass ratio of 1:4, and the beaker was placed
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in a shaker with 150 rpm at 37 C for 6-12 h (The conditions of immobilized papain
were detailed in the supporting information). The loading amount of papain on the
carrier was evaluated by Bradford protein assay27. UV/Vis was used to measure the UV
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absorption value of papain solution before and after adsorption of PAHM at 595 nm.
The protein standard curve in supporting information (Fig. S2) was used to calculate
the concentration of residual papain solution. The enzyme loading was determined by
the following equation:(1)
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Papain adsorption capacity (mg g-1) =퐶 푉 −퐶 푉
(1)
0
0
1 1
퐺
0
C0 (mg/mL) is the initial concentration of papain solution and C1 (mg/mL) is the
concentration of residual papain solution after adsorption with PAHM. V0 and V1 (mL)
are the volume of papain solution before and after adsorption, and G0 is the actual
weight of carrier (mg).
2.5 Measurement of enzyme activity
The enzyme activity of papain-PAHMs was determined by using the ethyl N-
benzoyl-L-argininate hydrochloride (BAEE) as substrate and dithiothreitol (DTT) as
the activator28. The BAEE was hydrolyzed by papain to generate N-α-Benzoyl-L-
Arginine (BA), and BA had absorption at 253 nm. Therefore, the absorption intensity
could be used to indicate the activity of papain. Firstly, 2 mL of PBS buffer (0.01M,
pH7.4) containing 0.04 mg of papain and 1 mL of DTT (30mM) were mixed to activate
for 10 min (37 oC, 180 rpm). Then, 3 mL of BAEE (2 mM) was added into the above
solution and the mixture was kept at 37 oC for 5 min. Finally, the reaction mixture was
centrifuged (4000 rpm, 5 min), and the UV absorption intensity of supernatant was
measured at 253 nm. The unit of enzyme activity was defined as the amount of enzyme
required for 0.001 increase of substrate absorbance per minute, which was calculated
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by the equation:
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∆퐴
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푈 =
(2)
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U is the activity of papain (U/mg). △A253 is the absorbance change of the reaction
solution at 253 nm. GE is the actual weight of papain (mg), and T is the reaction time
(min).
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2.6 The determination of reusability and stability
The reusability of papain-PAHM3 was evaluated by measuring the enzyme
activity after each catalysis. The reaction mixture was centrifuged (4000 rpm, 5 min) to
recover the precipitate after each catalytic hydrolysis reaction and the new reaction
substrate was added (see 2.5 for enzyme activity test method). The free papain and
papain-PAHMs were incubated in PBS buffer at pH 6-10 for 1 h at 25 oC to determine
their pH stability. The PBS buffer solution of free papain and papain-PAHM2 were
incubated at 30-70 oC for 1 h respectively to measure their thermal stability. BAEE was
used as the substrate to test the residual enzyme activity in the same way. The kinetic
parameters of free papain and papain-PAHM2 were measured by Lineweaver-Burk
double reciprocal graph method with BAEE as the substrate at the concentration of 1 ~
3 mM29.
2.7 Compressive and swelling properties of PAHMs
The cylindrical polyacrylamide (PA) hydrogel samples (diameter 15 mm,
thickness 20 mm) were prepared, and the compression resistance was verified by
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compression experiments at the speed of 3 mm/min. In order to analyze the swelling
behavior, PAHMs were evaluated in deionized water at 25 oC and were taken out every
5 min. After absorbing the water on the surface with filter paper, the PAHMs were
weighed immediately until the weight reached balance. The swelling degree (G) was
calculated by the following formula:
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푀 −푀
1
0
( )
퐺 % =
(3)
푀
0
M0 and M1 are respectively the weight of hydrogel before and after water absorption.
2.8 Application of catalytic hydrolysis
Papain-PAHM3 was used as a catalyst to hydrolyze milk protein. 10 mL of 1%
milk was mixed with 1g of papain-PAHM3, in a beaker and was kept in a shaker with
180 rpm at 37 oC for 45 min. The protein in milk was hydrolyzed by free papain under
the same condition as control. The milk hydrolysate was poured out and a new group
of milk continued to be added to the reaction system to realize the continuous catalysis
of papain-PAHM3. The degree of hydrolysis of milk was detected by SDS-PAGE gel
electrophoresis.
3 Results and discussion
3.1 Structural characterization of papain-PAHMs
PAHMs were prepared by inverse emulsion polymerization, which could generate
stable monodisperse aqueous droplets by using aqueous phase and surfactants in oil
phase with mechanical stirring30. In this study, cyclohexane was the oil phase, and the
solution containing the acrylamide, N, N-methylenebisacrylamide and
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polyvinylpyrrolidone was water phase. As shown in Fig. 1, the water-in-oil (W/O)
droplets with uniform size were formed by mechanical stirring. And these droplets were
successfully polymerized by initiation of ammonium persulfate (APS) to form PAHMs
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with porous structures at 65 C. A large number of amide groups on the surface of
PAHMs could form the hydrogen-bond interaction with the active functional groups of
the papain such as hydroxyl and amide groups. More specifically, the active sites of
papain were consisted of cysteine 25 (Cys), histidine 159 (His) and aspartic acid 158
(Asp). In neutral aqueous solution, the Cys25 protonated by His159 could nucleophilic
attack the C=O bond of the amide group on the surface of PAHMs to form the hydrogen-
bond31. This strong interaction not only makes the enzyme firmly fixed on the substrate,
but also maintains the maximum activity of the enzyme without destroying its spatial
conformation.
Fig. 1 The schematic of the formation process of papain-PAHMs
The FTIR spectra of papain, PAHMs and papain-PAHMs were shown in Fig. 2.
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For papain, the broad peak between 3410-3310 cm-1 was attributed to the stretching
vibration of -OH and N-H and the peaks at 2940-2850 cm-1 were due to the stretching
vibration of -CH2. Meanwhile, the stretching vibrations of C=O group in amide I and
II were shown at the range of 1652-1538cm-1 32. As a sulfhydryl protease, papain had
the vibration absorption of the C-S bond at 1050-996 cm-1. FTIR spectrum of the
PAHMs showed the main characteristic absorption peaks at 3350-3200 cm-1 and 2920
cm-1 respectively, which represented the presence of N-H and -CH2 groups. Moreover,
the absorption peak of C=O group was found at 1660 -1620 cm-1. In the FTIR spectrum
of papain-PAHMs, all characteristic absorption peaks of papain and PAHMs were found,
indicating that papain was successfully loaded on the PAHMs. And the red shift of
stretching vibration peaks of -OH and N-H proved the formation of hydrogen-bonds.
N2 adsorption/desorption isotherm was performed on PAHMs to determine their
pore size and surface area. Fig. S3 showed the typical type IV curves for PAHMs, which
was due to the existence of mesopores33, 34. After the relative pressure of 0.01, the
uptake of the curve indicated the existence of mesopores. The hysteresis loop (H4) at
relative pressure at the range of 0.6-0.9, represented the slit pore type 33, 35. It indicated
that PAHMs particles had irregular shape with internal voids and broad size
distribution35. The Barrett−Joyner−Halenda (BJH) pore size was presented in table S1.
As the particle size of PAHMs decreased, its surface area increased from 2.22 m2/g to
8.12 m2/g. On the contrary, with the decrease of PAHMs particle size, the pore size of
PAHMs decreased gradually. The change in the mesoporous structure of PAHMs was
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provided possibility to adsorb more papain.
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Fig. 2 FTIR spectra of (a) free papain, (b-d) PAHMs and (e-g) papain-PAHMs
3.2 The SEM and EDS of PAHMs and papain-PAHMs
The morphologies and compositions of PAHMs and papain-PAHMs were
determined by SEM and EDS. In Fig. 3, the PAHMs showed porous spherical shapes,
and PAHM1, PAHM2 and PAHM3 had average diameters of about 600, 500 and 400
µm, respectively. The sizes of the hydrogel microspheres decreased with the increase
of stirring speed. It was because the shear force in the reaction system increased with
the increase of stirring speed, and then the water droplets in the reaction system
decreased with the increase of shear force36, 37. In Fig. 3c, f, and i, the surfaces of
PAHM1, PAHM2 and PAHM3 were porous network structures, which was conducive
to enzyme loading. In particular, the network of PAHM1 was loose with larger pores,
while the network of PAHM3 was the most compact. This result showed that PAHM3
with a large number of dense pores might offer a larger specific area for enzyme
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immobilization than PAHM1 and PAHM2. The SEM images of papain-PAHM1,
papain-PAHM2, and papain-PAHM3 were shown in Fig. 3j-l. It showed that the size of
papain-PAHMs was larger than that before adsorption of papain, and the surfaces were
covered with coarse particles. Moreover, compared with PAHM1 and PAHM2, the
surface of PAHM3 was more compact. It indicated that PAHM3 might adsorb more
papain.
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The immobilization of papain on polyacrylamide hydrogel microspheres was
mainly achieved by both surface and pore adsorption. Papain is a sulfhydryl protease,
which is rich in S element. In order to verify that the prepared hydrogel microspheres
and papain were combined by surface and pore adsorption, EDS analysis was
performed on papain-PAHMs. The result of Fig. S3 a-f showed that papain-PAHMs
contained S element not only on the surface, but also in the pores. With the decrease of
PAHM particle size, the content of S element gradually increased, indicating that more
papain was adsorbed on the carrier. In the analysis of EDS (Table S1), it was also found
that the surface concentration of element S in papain-PAHMs was lower than the total
content (Wt%). Therefore, it could be confirmed that papain and polyacrylamide
hydrogel microspheres were immobilized mainly by both surface and pore adsorption.
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Fig. 3 SEM images of PAHM1 (a-c), PAHM2 (d-f), PAHM3 (g-i) and papain-PAHM1 (j),
papain-PAHM2 (k), papain-PAHM3 (l), (m) EDS results of free papain, PAHMs and papain-
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3.3 The swelling rate, and mechanical properties of PAHM3
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Substances can be introduced into the internal structure of hydrogels through the
swelling process of hydrogels38. Hydrogels with large swelling capacity usually have
strong adsorption capacity39. The prepared polyacrylamide hydrogel microspheres were
subjected to water absorption and swelling experiments after freezing vacuum drying.
Fig. S4 showed the particle size distribution of dried PAHMs and PAHMs after swelling
equilibrium for 12 hours. As shown in Fig. S4 a, c, and e, the average particle diameters
of the prepared PAHM1, PAHM2 and PAHM3 are about 600, 500 and 400 μm,
respectively. Compared with dry PAHMs, the average particle size of PAHM1, PAHM2,
and PAHM3 (Fig. S4 b, d and f) after swelling increased by about one hundred microns,
which was about 700, 600 and 500 μm, respectively. Fig. 4a and 4b showed the
micrographs of PAHM3 before and after swelling equilibrium under an optical
microscope. Interestingly, the density of hydrogel microspheres decreased after
swelling, and light transmittance of PAHM3 increased significantly, making PAHM3
brighter. In Fig. 4c, the change of swelling ratio showed that the PAHM3 absorbed
water rapidly at first, then slowly. After 40 min, the water absorption equilibrium was
achieved, and the swelling ratio was 80%. The swelling phenomenon of PAHMs
showed that the polyacrylamide hydrogel microsphere was a three-dimensional
network structure, which was swollen and insoluble.
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In the process of catalytic application, immobilized enzyme will be constantly
affected by external forces, and good mechanical properties are crucial for the reuse of
catalyst. The mechanical properties of PAHM3 were determined by the compression
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experiment, which was performed at room temperature. As shown in Fig. 4d, with the
increase of compressive strain, the compressive stress reached 45 KPa. This result
indicated that PAHM3 had good compression resistance, which might be advantageous
for the further industrial application.
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Fig. 4 The morphology of PAHM3 (a) before and (b) after swelling equilibrium under optical
microscope, (c) the swelling radio of PAHM3, (d) compressive strain of PAHM3
3.4 The activity and stability of free and immobilized papain
After immobilization of papain, the adsorption capacities of PAHM1, PAHM2 and
PAHM3 were calculated to be 8.17, 12.23 and 14.67 (mg g-1), respectively, according
to formula (1). It showed that the adsorption capacity of PAHM3 was better than that
of PAHM1 and PAHM2. This might be due to the fact that the PAHM3 had more pores
and larger specific surface area than PAHM1 and PAHM2, which was conducive to
adsorb massive enzymes.
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The enzymatic activity is an important parameter to evaluate the effectiveness of
immobilization because the papain must maintain its proteolytic function after
immobilization. According to formula (2), the activities of free papain, papain-PAHM1,
papain-PAHM2, and papain-PAHM3 were calculated to be 203.44, 85.97, 92.97 and
216.48 U/mg, respectively. Compared with free papain, the relative activities of papain-
PAHM1, papain-PAHM2, and papain-PAHM3 were 42.26%, 45.70% and 106.41%,
respectively (Fig. 5). It indicated that papain-PAHM3 had higher activity than free
papain, papain-PAHM1 and papain-PAHM2. This result might be due to that more
papain was immobilized on the surface and pores of PAHM3, which was conducive to
contact with the substrate, thus improving the enzyme activity.
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The pH influence on the activities of free papain and papain-PAHM3 was
evaluated by testing the residual activity of solution after incubation at denaturing
conditions with pH 6.0 to 10.0 for 1 h. As shown in Fig. 5b, the maximum activity of
free papain maintained at pH 7.0. In addition, the residual activities of pH 6.0 and 10.0
were only 56.01% and 39.02% of the maximum activity. The maximum activity of
papain-PAHM3 was also at pH 7.0, and the activities of papain-PAHM3 at pH 6.0 and
10.0 were 61.73 and 62.49% maximum activity of free papain, which were higher than
that of free papain at pH 6.0 and 10.0. It indicated that immobilization did not change
the optimum pH (7.0) of papain and the working pH range of papain-PAHM3 was larger
than that of free papain. Furthermore, the papain-PAHM3 maintained exceptionally
high stability under harsh conditions since the porous PAHM3 support provided an
environment conducive to preserving protein folding, thus retaining the enzyme activity.
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Effects of temperature on enzyme activities of free papain and papain-PAHM3
were also assessed in the range of 30-70 °C. As shown in Fig. 5c, the relative activities
of papain were decreased with the increase of temperature. And the optimum
temperature of the free papain and papain-PAHM3 were both 30 oC. In particular, the
relative activity of papain-PAHM3 was 106.41% of maximum activity of free papain at
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30 C. And the residual activity of papain-PAHM3 could be maintained 76.71% of
maximum activity of free papain even at 70 oC. It showed that the immobilized papain
had a stable structure, which could reduce the sensitivity of papain to temperature and
expanded the application temperature range. This result might be due to the binding of
papain with surface of hydrogel microspheres, which limited the conformational
migration of papain at high temperature.
Enzyme reusability is an important feature in industrial application40. Therefore,
the reusability of papain-PAHMs was investigated through repeated enzymolysis
experiments of the substrate (BAEE). The relative activity of free papain was set to
100%. As shown in Fig. 5d, the enzymatic activities of papain-PAHM1, papain-PAHM2,
and papain-PAHM3 were maintained 9.68%, 20.23% and 59.60% activities of free
papain after 10 consecutive cycles. This result was attributed to the strong binding of
amide group on PAHMs with papain, which provided the excellent condition for the
reuse of immobilized papain. Particularly, the high relative activity of papain-PAHM3
demonstrated that the large specific surface area was conducive to the binding with
papain. Compared with papain-PAHM1 and papain-PAHM2, papain-PAHM3 had a
more compact porous structure, which could reduce the leaching of the enzyme and
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further improve the possibility of effective interaction between the enzyme and
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In addition, the relative activity of papain-PAHM3 was slighter decrease to 41.04%
activity of free papain after 12 consecutive cycles. The decrease in activity was usually
attributed to factors such as enzyme desorption, product inhibition, and enzyme
deformation41. The loss of papain-PAHM3 activity might be the product inhibition and
enzyme deformation, because the amount of enzyme loaded in the hydrogel
microspheres did not significantly decrease after 12 cycles under fluorescence
microscope (Fig. 5e-f). Fig. 5e clearly showed that papain-PAHM3 was full of papain
labeled by FITC before recycling. After 12 cycles, the FITC-labeled papain could still
be observed in papain-PAHM3 (Fig. 5f). However, the fluorescence image of papain-
PAHM3 was a little weaker than before recycling because that the hydrogel
microsphere swelled with water absorption and increased in volume during the catalytic
process.
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To measure the effect of immobilization on the catalytic activity of papain, the
kinetic parameters (Km and Vmax) of the papain-PAHM3 and free papain were compared
by Michaelis-Menten model42. The Km value can reflect the affinity of the enzyme to
the substrate. The smaller the Km value is, the greater the affinity between enzyme and
substrate is. Vmax represents the maximum rate of the reaction between the enzyme and
the substrate to form a product. A larger Vmax means a faster reaction rate between the
enzyme and substrate. In this work, Km and Vmax of the free and immobilized papain
were calculated by their plots of inverse reaction rate (1/V) against concentration of
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substrate (1/[S]) (Fig. 5g). As shown in Table1, the Km values of free papain andDpOaI:p1a0.i1n03-9/D1NJ02551A
PAHM3 were calculated to be 1.77 and 0.86 mM, respectively. The Km value of papain-
PAHM3 was lower than that of the free papain, indicating that the immobilized papain
had a better affinity to the substrate. The favourable affinity of papain-PAHM3 to
substrates could be attributed to the porous structure, which could capture more
substrates43. The Vmax values of free papain and papain-PAHM3 were 7.51 and 4.38
mM min-1, respectively (Table1). The decrease of Vmax value of papain-PAHM3
indicated that the reaction rate of immobilized papain with substrate was slower than
that of free papain. This could be ascribed to the additional diffusion resistance between
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the supports and enzyme molecules44, 45
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In addition, the kcat/Km ratio of catalytic efficiency was examined. The kcat/Km
ratios of free papain and papain-PAHM3 were 0.14 and 1.15 M-1s-1, respectively. The
higher kcat/Km ratio of papain-PAHM3 demonstrated that the catalytic efficiency of
papain-PAHM3 significantly improved. This result might be due to the pore of the
support could capture more substrates and promote the diffusion of substrate and
product molecules, which was conducive to catalytic reaction.
Table 1 The kinetic parameters of free papain and papain-PAHM3
Km
(mM)
Vmax
(mM min-1)
kcat
(s-1)
kcat/Km
(M-1s-1)
Types of enzymes
Free papain
1.77
0.86
7.51
4.38
0.25
0.99
0.14
1.15
Papain-PAHM3
F-NMR and the T2 inversion method was used to analyze the mobility of water
molecules on PAHM3, free papain and papain-PAHM346. The relaxation time was
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divided into three parts. T21 was defined as bound water formed in a mDoOleI:c1u0.l1a03r9/D1NJ02551A
structure with a relaxation time shorter than 10 ms. T22 was defined as water that was
not easy to move, and its relaxation time range was 10 ~ 1000 ms. T23 was attributed
to free water with relaxation time longer than 1000 ms. In Fig. 5h, the T21 peak of
water in the free papain indicated the presence of bound water in the free papain. And
the relaxation time (T23) of water in PAHM3 showed that PAHM3 was full of free
water. The papain-PAHM3 showed the relaxation time of T21, indicating that the
papain in papain-PAHM3 still had the bound water and the conformation did not change.
In addition, the peak of T23 disappeared might be due to the free water on PAHM3 was
substituted by the papain molecules.
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Fig. 5 (a) The activity of free papain, papain-PAHM1, papain-PAHM2 and papain-PAHM3,
(b) the pH stability of the free papain and papain-PAHM3, (c) the temperature stability of the
free papain and papain-PAHM3, (d) recycle of papain-PAHM1, papain-PAHM2 and papain-
PAHM3 (with the activity of free papain as 100%), the fluorescence microscope images
(e) before and (f) after 12 cycles, (g) Lineweaver–Burk plot of free papain and papain-
PAHM3, (h) the LF-NMR of free papain, PAHM3 and papain-PAHM3
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3.5 Catalytic application
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β-LG and α-LA are the main allergenic proteins in milk. In addition, 60% ~ 80%
of patients have abnormal β-LG immune response46, 47. Therefore, it is significant to
develop a kind of milk that is friendly to allergic patients. To accomplish this objective,
we used papain-PAHM3 to hydrolyze the β-LG and α-LA in milk powder solution.
After being hydrolyzed at 37 oC, the hydrolysates of 1% (w/w) milk powder at different
time were determined by SDS-PAGE method.
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To determine the reasonable hydrolysis reaction time, the hydrolysates of milk
powder aqueous solution catalyzed by papain-PAHM3 were determined by
electrophoresis every 10 min and the result was shown in Fig. 6a. The molecular
weights of protein marker were 10-140 kDa. Compared with the marker, the bands with
the molecular weight between 25 and 35 kDa belonged to casein, and the molecular
weights of β-LG and α-LA were 14 and 18 kDa, respectively. The electrophoresis
diagram of milk hydrolysates showed that bands of β-LG and α-LA in the hydrolysates
decreased significantly after 10 min. At 30 min, almost all the bands of β-LG and α-LA
in the milk powder disappeared, indicating that the hydrolysis was complete. Therefore,
30 min was chosen as the best enzymolysis time. To determine the reusability, the
papain-PAHM3 was filtered and poured into a new milk powder aqueous solution after
each catalytic reaction. The results of papain-PAHM3 cyclic catalysis were shown in
Fig. 6b. It could be seen from the results of three cycles of catalysis that papain-PAHM3
could effectively enzymolyze the β-LG and α-LA in milk powder for many times. It
was reported that the papain-Cu3(PO4)2·3H2O-magnetic nanoflowers were prepared to
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hydrolyze milk powder. However the preparation process of this nanomaterial were
complex. Compared with papain-Cu3(PO4)2·3H2O-magnetic nanoflowers, the
preparation of papain-PAHM3 was simpler and showed good biocompatibility. The
papain-PAHM3 could be separated from the milk quickly without introducing new
hetero protein. The results showed that the immobilized papain on polyacrylamide
hydrogel microspheres had great potential for industrial application.
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Fig. 6 (a) SDS-PAGE profile of M: protein marker (10-140 kDa), 0: 1% (w/w) milk powder
aqueous solution, 1-4: hydrolysate after 40 min, 30 min, 20 min, 10 min; (b) SDS-PAGE
profile for the hydrolysate of the reuse experiment, M: protein marker (10-140 kDa), 0:1%
(w/w) milk powder aqueous solution, 1-3: hydrolysate of different reuse times (one, two,
three times)
4 Conclusions
The novel papain-PAHMs with high enzyme activity were prepared by combining
papain with the porous PAHMs. The papain was immobilized on PAHMs through
strong hydrogen bonds. This physical action does not change the structure of the
enzyme, which is conducive to maintaining the activity of the enzyme. Compared with
the free papain, the papain-PAHM3 exhibited high catalytic activity, good stability, and
great reusability. Further, papain-PAHM3 was successfully used to hydrolysis the β-LG
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and α-LA. In conclusion, the combination of enzyme and polyacrylamide hydrogel
microsphere could provide a new opportunity for the application of green catalyst in
food industry.
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Conflicts of interest
The authors report no conflicts of interest in this work.
Acknowledgements
This work was supported by National Natural Science Foundation of China (31771914,
31800498), Research on science and technology of education department of Liaoning
province project (J2020058).
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References
1.
M. Dutra Rosolen, A. Gennari, G. Volpato and C. F. Volken de Souza, Enzyme
Res, 2015, 2015, 806240.
M. D. L. Aasma Shaukat, Brent C. Taylor, et al. , Annals of Internal Medicine,
2010, 152, 797-803.
2.
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
50
51
52
53
54
55
56
57
58
59
60
3.
4.
L. Dong and Q. Zhong, J Agric Food Chem, 2019, 67, 6559-6568.
S. Abbring, G. Hols, J. Garssen and B. van Esch, Eur J Pharmacol, 2019, 843,
55-65.
D. Munblit, M. R. Perkin, D. J. Palmer, K. J. Allen and R. J. Boyle, JAMA
Pediatrics, 2020, 174.
S. Hussain, A. Khan, S. Gul, M. Resmini, C. S. Verma, E. W. Thomas and K.
Brocklehurst, Biochemistry, 2011, 50, 10732-10742.
C. Xu, L. Cao, G. Su, W. Liu, H. Liu, Y. Yu and X. Qu, Journal of Hazardous
Materials, 2010, 176, 807-813.
C. Xu, P. Shin, L. Cao and D. Gao, The Journal of Physical Chemistry C, 2010,
114, 125-129.
I. Ocsoy, E. Dogru and S. Usta, Enzyme Microb Technol, 2015, 75-76, 25-29.
S. Cantone, V. Ferrario, L. Corici, C. Ebert, D. Fattor, P. Spizzo and L. Gardossi,
Chem Soc Rev, 2013, 42, 6262-6276.
A. Rodriguez-Abetxuko, D. Sanchez-deAlcazar, P. Munumer and A. Beloqui,
Front Bioeng Biotechnol, 2020, 8, 830.
Z. Ashkan, R. Hemmati, A. Homaei, A. Dinari, M. Jamlidoost and A. Tashakor,
International Journal of Biological Macromolecules, 2021, 168, 708-721.
J. Yu, X. Chen, M. Jiang, A. Wang, L. Yang, X. Pei, P. Zhang and S. G. Wu, RSC
Advances, 2018, 8, 2357-2364.
W. Sheng, Y. Xi, L. Zhang, T. Ye and X. Zhao, Int J Biol Macromol, 2018, 114,
143-148.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
R. N. F. Moreira Filho, N. F. Vasconcelos, F. K. Andrade, M. F. Rosa and R. S.
Vieira, Colloids Surf B Biointerfaces, 2020, 194, 111222.
16. C. Hou, Y. Wang, H. Zhu and H. Wei, Chemical Engineering Journal, 2016, 283,
397-403.
17.
18.
H. Dai, S. Ou, Z. Liu and H. Huang, Carbohydr Polym, 2017, 169, 504-514.
A. Soares, L. M. O. Goncalves, R. D. S. Ferreira, J. M. de Souza, R. Fangueiro,
M. M. M. Alves, F. A. A. Carvalho, A. N. Mendes and W. Cantanhede,
Carbohydr Polym, 2020, 243, 116498.
S. Mei, J. Shi, S. Zhang, Y. Wang, Y. Wu, Z. Jiang and H. Wu, ACS Applied Bio
Materials, 2019, 2, 777-786.
Y. Fan, X. Cao, T. Hu, X. Lin, H. Dong and X. Zou, The Journal of Physical
Chemistry C, 2016, 120, 3955-3963.
Z. Wang, Y. Cong and J. Fu, J Mater Chem B, 2020, 8, 3437-3459.
X. Zhang, W. Liu, J. Cai, J. Huang and X. Qiu, Journal of Materials Chemistry
19.
20.
21.
22.
A, 2019, 7, 26917-26926.
K. Wang, J. Wang, L. Li, L. Xu, N. Feng, Y. Wang, X. Fei, J. Tian and Y. Li,
23.
27

New Journal of Chemistry
Page 28 of 28
1
2
3
4
5
6
7
8
9
View Article Online
DOI: 10.1039/D1NJ02551A
Chemical Engineering Journal, 2019, 372, 216-225.
24.
25.
26.
S. Xia, S. Song, F. Jia and G. Gao, J Mater Chem B, 2019, 7, 4638-4648.
P. A. Lovell and F. J. Schork, Biomacromolecules, 2020, 21, 4396-4441.
T. Zhang, R. A. Sanguramath, S. Israel and M. S. Silverstein, Macromolecules,
2019, 52, 5445-5479.
27.
28.
29.
30.
31.
32.
W. Ren, Y. Li, J. Wang, L. Li, L. Xu, Y. Wu, Y. Wang, X. Fei and J. Tian, New
Journal of Chemistry, 2019, 43, 11082-11090.
H. Zhang, X. Fei, J. Tian, Y. Li, H. Zhi, K. Wang, L. Xu and Y. Wang, Catalysis
Communications, 2018, 116, 5-9.
N. Feng, H. Zhang, Y. Li, Y. Liu, L. Xu, Y. Wang, X. Fei and J. Tian, Food Chem,
2020, 311, 125911.
K. Raghupathi, S. J. Eron, F. Anson, J. A. Hardy and S. Thayumanavan, Mol
Pharm, 2017, 14, 4515-4524.
L. Boon, E. Ugarte-Berzal, J. Vandooren and G. Opdenakker, Crit Rev Biochem
Mol Biol, 2020, 55, 111-165.
K. A. Mahmoud, E. Lam, S. Hrapovic and J. H. T. Luong, ACS Applied
Materials & Interfaces, 2013, 5, 4978-4985.
K. S. W. Sing, Pure and Applied Chemistry, 1985, 57, 603-619.
L. Chen, T. Ji, L. Brisbin and J. Zhu, ACS Appl Mater Interfaces, 2015, 7,
12230-12237.
M. Kruk and M. Jaroniec, Chemistry of Materials, 2001, 13, 3169-3183.
M. Barari, M. ABD ELAHI and M. Hemati, 2011.
A. C. Daly, L. Riley, T. Segura and J. A. Burdick, Nat Rev Mater, 2020, 5, 20-
43.
H. Jia, Q. Ren, W.-F. Pu and J. Zhao, Energy & Fuels, 2014, 28, 6735-6744.
I. Gajic, S. Ilic-Stojanovic, A. Dinic, A. Zdravkovic, L. Stanojevic, V. Nikolic
and L. Nikolic, Polymers (Basel), 2021, 13.
E.-J. Woo, H.-S. Kwon and C.-H. Lee, Chemical Engineering Journal, 2015,
274, 1-8.
B. Saha, J. Saikia and G. Das, Analyst, 2015, 140, 532-542.
M. Simões and F. X. Malcata, Journal of Chemical Education, 2020, 97, 2308-
2313.
Y. Wei, Z. Cai, M. Wu, Y. Guo, P. Wang, R. Li, A. Ma and H. Zhang, Carbohydr
Polym, 2020, 240, 116273.
K. Cheng, F. Svec, Y. Lv and T. Tan, Small, 2019, 15, e1902927.
Y. Dai, J. Yao, Y. Song, X. Liu, S. Wang and Y. Yuan, J Hazard Mater, 2016,
317, 485-493.
J. Wu and S. Chen, Langmuir, 2012, 28, 2137-2144.
J. Liu, X.-y. Feng and D.-s. Wang, Petroleum Science and Technology, 2019, 37,
1123-1135.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
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