PEI-crosslinked lipase on the surface of magnetic microspheres and its characteristics

Journal Pre-proof PEI-crosslinked lipase on the surface of magnetic microspheres and its characteristics Yi-Ping Cao, Yu-Pei Xia, Xiao-Fei Gu, Li Han, Queting Chen, Gao-Ying Zhi, Dong-Hao Zhang

PII:

S0927-7765(20)30104-1

DOI:

https://doi.org/10.1016/j.colsurfb.2020.110874

Reference:

COLSUB 110874

To appear in:

Colloids and Surfaces B: Biointerfaces

Received Date:

26 September 2019

Revised Date:

19 January 2020

Accepted Date:

12 February 2020

Please cite this article as: Cao Y-Ping, Xia Y-Pei, Gu X-Fei, Han L, Chen Q, Zhi G-Ying, Zhang D-Hao, PEI-crosslinked lipase on the surface of magnetic microspheres and its characteristics, Colloids and Surfaces B: Biointerfaces (2020), doi: https://doi.org/10.1016/j.colsurfb.2020.110874

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.

PEI-crosslinked lipase on the surface of magnetic microspheres and its characteristics

Yi-Ping Cao†, Yu-Pei Xia†, Xiao-Fei Gu†, Li Han†, Queting Chena, Gao-Ying Zhi‡, Dong-Hao Zhang†§* †

College of Pharmaceutical Science, Hebei University, Baoding, 071002, China; Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmaceutical Science, Hebei University, Baoding, 071002, China; ‡ Computer Center, Hebei University, Baoding, 071002, China; a Affiliated Hospital of Hebei University, Baoding, China

ro of

§

*To whom correspondence should be addressed. Tel: +86 312 5971107

-p

Fax: +86 312 5971107

re

E-mail: [email protected], [email protected]

Jo

ur

na

lP

Graphical abstract

1

Highlights: 

PEI-crosslinked lipase was designed on the surface of magnetic microspheres.



PEI-crosslinked lipase could bind carrier firmly.



PEI-crosslinked lipase performed high activity and stability.



PEI-crosslinked lipase retained its natural structure well.

ro of

Abstract Here, PEI@PMMA microspheres were prepared by grafting polyethyleneimine

(PEI) on poly(methyl methacrylate) (PMMA) magnetic microspheres and successfully

-p

used to immobilize lipase. The results showed that PEI@PMMA microspheres had

strongly adsorbed lipase (49.1 mg/g microsphere) via electrostatic attraction. To

re

prevent lipase shedding, the adsorbed lipase was further crosslinked with PEI on

lP

microspheres using glutaraldehyde as crosslinker. Consequently, PEI-crosslinked lipase (2.14 U/mg) exhibited 2.6 times and 1.4 times higher activity respectively than

na

the directly covalent lipase (0.82 U/mg) and the crosslinked lipase aggregates (1.57 U/mg), which was close to the activity of adsorbed lipase (2.20 U/mg).

ur

Conformational analysis from FTIR spectroscopy showed that PEI-crosslinked lipase

Jo

retained its natural structure well. And the α-helix structure seemed to play a key role in enhancing lipase activity. Furthermore, the effects of various parameters on crosslinking reaction were investigated. Also, PEI-crosslinked lipase revealed higher pH and thermal stability. The Michaelis constant (Km) was increased and the optimum temperature of lipase was widened observably after crosslinking with PEI on PEI@PMMA magnetic microspheres. 2

Keywords: magnetic microspheres; polyethyleneimine; lipase; crosslink; conformation.

1. Introduction Lipase, one of the hydrolases, can catalyze hydrolysis of ester, esterification and

ro of

transesterification reaction with many natural and non-natural substrates. It is broadly applied in chemical industries, food and pharmaceutical industry, environmental protection, and so on [1]. Unfortunately, free enzyme is easily inactivated in

-p

application and cannot be repeatedly used. Enzyme immobilization provides a good

re

strategy to improve enzyme activity, stability, reusability, and even selectivity [1,2]. Particularly, magnetic carriers have drawn much attention with the advantages of

lP

magnetic separation [3-5]. And several methods, including adsorption [6], covalence

na

[7], encapsulation [8,9], and crosslinking aggregation [10], are frequently used to immobilize enzyme. However, enzyme immobilization often comes at the cost of

ur

decreasing enzyme activity [11]. Therefore, it is still a challenge to reduce structural damage of enzyme and improve enzyme activity in the process of enzyme

Jo

immobilization.

Polyethyleneimine (PEI) contains a large amount of primary, secondary and tertiary

amine functional groups across the polymer chain. Moreover, PEI chains possess good biocompatibility and water-solubility [12]. Recently, these merits have attracted a wide interest because the high positive charge density from amino groups implies 3

that PEI is an ideal chain for adsorbing the negative-charged enzyme by electrostatic attraction, as well as because the good hydrophilicity of PEI facilitates its flexibility and stretch in water solution after grafting on carrier [13,14,15]. In this study, we designed a potential way for lipase immobilization on PEI-grafted microspheres. Firstly, poly(methyl methacrylate) (PMMA) magnetic microspheres were synthesized. Then, PEI was grafted on PMMA microspheres to prepared

ro of

PEI@PMMA microsphere. Subsequently, Candida rugosa lipase was adsorbed on

PEI@PMMA microsphere. Finally, glutaraldehyde (GA) was used to crosslink lipase with PEI.

-p

2. Materials and methods

re

2.1. Materials

Divinylbenzene (DVB) and methylmethacrylate (MMA) were from Guangfu

lP

Reagent Company (Tianjin, P. R. China). Polyvinyl alcohol 1788 (PVA, ~88%

na

hydrolyzed) was from Fuchen Reagent Company (Tianjin, P. R. China). Glutaraldehyde (GA) and Azobisisobutyronitrile (AIBN) were purchased from

ur

Kemiou Chemical Co. (Tianjin, China). Candida rugosa lipase and Bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO). Polyethyleneimine (PEI,

Jo

Mw = 1800 and 70000) and fluorescein isothiocyanate (FITC) were purchased from Aladdin Reagent Inc. (Shanghai, China). N-hydroxysusccinimide (NHS) (97%) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were obtained from Sinopharm Chemical Reagent Co. (Beijing, China). 2.2. Poly(methyl methacrylate) (PMMA) magnetic microspheres preparation and PEI 4

graft Magnetic particles modified with oleic acid were synthesized using chemical coprecipitation method [13]. And magnetic PMMA microspheres were prepared by using magnetic particles as cores [13]. Briefly, magnetic particles (90 mg) were mixed with methyl methacrylate (MMA, 3.5 mL) and of divinylbenzene (250 µL). Then the mixture was ultrasonicated for 5 min and swelled it for 12 h, and AIBN (130 mg) was

ro of

added into the mixture. Subsequently, the mixture was transferred into 15 mL of PVA water solution (18 mg/mL). After ultrasonicating for 15 min, the mixture was stirred in a three-necked flask for 1 h at 60 °C and 150 rpm, subsequently at 70 °C for 3

-p

hours. Finally, magnetic PMMA microspheres were obtained by magnetic separation

re

and cleaned using water and alcohol for several times.

After that, the magnetic PMMA microspheres were hydrolyzed by adding 60 mg of

lP

microspheres into 7.5 mL sodium hydroxide (2 M) and shaking for 24 h at 110 rpm

na

and 45 °C. And these microspheres were washed using dilute HCl and water for several times. Subsequently, the hydrolyzed microspheres with carboxyl groups were

ur

activated, as described previously [16], by mixing 20 mg of microspheres with 1 mL of buffer (20 mM phosphate, pH 5.0) containing 6.25 mg of EDC and 3.2 mg of NHS

Jo

for 1 h at 25 °C and 160 rpm. The activated microspheres were washed several times with water and alcohol. Then, 20 mg of microspheres was mixed with 1 mL of PEI solution (1%, w/v), and the reaction was conducted at 25 °C and 160 rpm for 4 hours. Finally, the resulting PEI@PMMA magnetic microspheres were washed several times with deionized water and dried under vacuum. 5

2.3. Lipase adsorption and desorption In the adsorption experiments, 20 mg of PEI@PMMA microspheres was mixed with 2 mL of 2 mg/mL lipase solution (pH 7.0) at 35 °C and 110 rpm. After 2 h of incubation, microspheres were magnetically isolated. The adsorption amount was determined by detecting the initial and final concentration of lipase in supernatant [13].

ro of

In the desorption experiments, 20 mg of microspheres loading with lipase was

added into 2 mL of buffer (pH7.0, 20 mM phosphate) containing 1.5 M NaCl, and incubated for 4 h at 35 °C and 110 rpm. Then, these microspheres were cleaned using

-p

NaCl solution (2 M), buffer (pH=7.0, 20 mM phosphate) and water till there was no

ratio of lipase from microsphere.

lP

Desorption ratio (%) = S/I × 100%

re

protein in supernatant. The following equation was used to calculate the desorption

na

where S and I were respectively the shedding amount of lipase and the initial loading amount of lipase.

ur

2.4. Lipase crosslinking with PEI and its activity assay Lipase was firstly adsorbed on PEI@PMMA magnetic microspheres. Then, 20 mg

Jo

of lipase-adsorbed microspheres was mixed with 2 mL of 0.75 ‰ glutaraldehyde solution, and the crosslink reaction started at 112 rpm at 35 °C and went on for 1 hour. The amount of PEI-crosslinked lipase was determined by detecting the amount of lipase in supernatant via Bradford method [13]. Lipase activity was determined by titrating the fatty acid which came from the 6

hydrolysis of olive oil at 37°C. One unit of lipase activity (U) was defined as the amount of lipase that hydrolyzed olive oil liberating 1µmol of fatty acid per minute under the assay conditions. All experiments were conducted in triplicate. The mean values were presented, and standard deviations were given as error bars in figures. 2.5. Characterization of microspheres and the immobilized lipase Scanning electron microscopy (SEM, Phenom pro, USA) was used to characterize

ro of

microsphere size and morphology. The sample was tested at 10 kV accelerating voltage by coating with gold.

Fluorescence labeling lipase was conducted as follows. Briefly, 1 mg of FITC in

-p

0.2 mL of DMSO was mixed with 1 mL of lipase solution (10 mg/mL, 30 mM PBS

re

buffer), and the mixture was incubated at room temperature for 2 h. Then, FITC labeled lipase was precipitated by adding 2 mL of acetone and immobilized on

lP

PEI@PMMA microspheres by crosslinking with PEI. Fluorescence observation was

Japan).

na

performed with confocal laser scanning microscopy (CLSM, Olympus, FV 3000,

ur

2.6. Preparation of crosslinked lipase aggregates and directly covalent lipase on PMMA microspheres (without spacer-arm)

Jo

To obtain crosslinked lipase aggregates, lipase solution was firstly prepared by

dissolving 20 mg of lipase in 1 mL phosphate buffer (pH 7.0, 30 mM). Then, 3 mL acetone was dropped in 1 mL lipase solution at 4 °C. Subsequently, the crosslinking reaction was conducted by adding 333 μL glutaraldehyde (25%) at 200 rpm at 4 °C for 2 h. After that, the aggregates were collected by centrifugation (17000 × g, 10 min) 7

and washed several times with phosphate buffer (pH 7.0, 20 mM) and water. The obtained crosslinked lipase aggregates were lyophilized and stored at 4 °C. To directly immobilize lipase on microspheres by covalent coupling, 20 mg magnetic microspheres with -COOH were firstly dispersed in 1 mL phosphate buffer (pH 5.0, 20 mM). Subsequently, 6.25 mg EDC and 3.2 mg NHS were added and the mixture was left in a temperature-controlled incubator at 160 rpm at 25 °C for 1 h.

ro of

The activated microspheres were washed with water for several times to remove any

unreacted chemicals. After that, lipase immobilization was carried out by mixing 20

mg activated microspheres with 2 mL lipase solution (2 mg/mL) at 110 rpm at 35 °C

-p

for 1 h. Then, the immobilized lipase was magnetically separated and lyophilized.

re

2.7. FTIR spectrum and lipase conformation analysis

Fourier transform infrared spectra (FTIR) were used to analyze the secondary

lP

structures of enzyme. The sample was mixed with KBr and pressed into a tablet, and

na

subsequently analyzed using FTIR spectrometer (8400S, Shimadzu, Japan). Software of Omnic 8.2 (from Thermal Fisher) was employed to acquire the infrared spectrum

ur

of the immobilized lipase via spectra subtraction. The infrared spectra of lipase were analyzed by combining second derivative with Gaussian deconvolution. The software

Jo

of PeakFit 4.0 (SeaSolve Inc., U.S.A.) was used to fit the curve. 2.8. pH and thermal stability investigation pH stability of lipase was investigated by incubating a certain amount of lipase in phosphate buffer with different pH values (5.0，6.0，7.0，8.0，9.0) at 35 °C for 1 h and measuring enzyme activity. And thermal stability of lipase was investigated by 8

incubating a certain amount of lipase in phosphate buffer (pH 7.0, 30 mM) at different temperature (25, 30, 35, 40, 50, and 60 °C) for 1 h and measuring enzyme activity. 2.9. Determination of kinetic constants (Km and Vmax) Lineweaver-Burk curve was established from Michaelis-Menten model, and Km and Vmax of free and immobilized lipase were respectively calculated. 1 1 Km 1 = + × V Vmax Vmax [S]

ro of

where V (mmol/L∙min) was the reaction rate, [S] (mg/mL) was the substrate

concentration, Vmax (mmol/L∙min) was the maximum reaction rate, and Km (mg/mL) was the Michaelis-Menten constant.

-p

To determine Km and Vmax, equivalent immobilized or free lipase was added into

re

olive oil emulsion with different concentrations from 5 to 35 mg/mL in phosphate buffer (30 mM, pH 7.0) at 37 °C, and the initial reaction rate was calculated

lP

accordingly.

na

3. Results and discussion

3.1. Characterization of microspheres and immobilized lipase

ur

Fig 2a showed FITR spectra of PMMA and PEI-grafted (PEI@PMMA) magnetic microspheres. In the spectrum of PMMA microsphere, Fe3O4 adsorption at 582 cm-1,

Jo

C=O stretching vibration adsorption at 1725 cm-1, and C-O-C stretching vibration adsorption at 1140-1300 cm-1 confirmed the successful preparation. In the spectrum of PEI@PMMA microsphere, the N-H stretching vibration adsorption peak at 3435 cm-1 further demonstrated the successful modification of PEI. The SEM of PMMA microspheres in Fig. 2b showed a particle size of 10-20 µm. By titration, the amino 9

content on PEI@PMMA magnetic microsphere was determined to be 600.1 µmol/g microspheres. Then, PEI@PMMA microsphere was used to adsorb lipase via electrostatic attraction. Subsequently, lipase was crosslinked with PEI chains on microspheres using GA as crosslinker. The loading amount of lipase crosslinked with PEI chains was evaluated to be 40.5 mg/g microsphere, which was slightly lower than that of

ro of

lipase adsorbed on PEI@PMMA magnetic microspheres (49.1 mg/g). This was attributed to the fact that lipase underwent partial leakage from microsphere during

crosslinking reaction. To the knowledge, PEI possesses abundant positive charges

-p

because of the large number of amino-groups [17,18], leading to that PEI-grafted

re

microspheres (zeta potential = 17.1 mV) will adsorbs more lipase (zeta potential = -10.9 mV).

lP

To ascertain that lipase was crosslinked with PEI@PMMA magnetic microspheres,

na

the immobilized lipase labeled with FITC was examined by CLSM. Before fluorescence detection, PEI-crosslinked lipase was dealt repeatedly with high

ur

concentration salt solution to shed the uncrosslinked lipase as far as possible. In Fig. 2c, the immobilized lipase appeared green fluorescence, which demonstrated that

Jo

lipase was successfully crosslinked with PEI@PMMA magnetic microspheres. 3.2. Detecting the leakage of PEI-crosslinked lipase To study the bond strength (i.e. immobilized stability) of the immobilized lipase, desorption was conducted by incubating the immobilized lipase in high-concentration salt solution. Fig. 3 showed the loading amount of lipase adsorbed or crosslinked on 10

PEI@PMMA magnetic micorspheres before and after desorption, respectively. As shown, the adsorbed lipase amount decreased sharply from 49.1 to 10.5 mg/g microsphere after desorption, which implied that the adsorbed lipase was unstable and easy to fall off. Recently, enzyme shedding under a condition of high-concentration salt was also stated when Vazquez-Ortega et al. used amino agarose microspheres to adsorb glucosidase [19]. By comparison, as seen from Fig. 3, the amount of

ro of

PEI-crosslinked lipase just declined a little after desorption treatment (from 40.5 to 32.9 mg/g microsphere).

3.3. Conformation and activity analysis of immobilized lipase

-p

Next, we analyzed the activity and conformation of lipase crosslinked with PEI on

re

PEI@PMMA magnetic microspheres. It was generally known that the activity of enzyme was closely related to enzyme conformation [20,21]. However, it was

lP

inevitable to destroy, partly or even wholly, the native conformation of enzyme during

na

immobilization because the interactions between enzyme and carrier, including chemical bond, electrostatic interactions, hydrophobic interaction, etc, often had effect

ur

on enzyme. Recently, FTIR spectroscopy, as a powerful conformational analysis tool, was often employed to give the secondary structure composition of protein by

Jo

analyzing the spectral absorption in amide I band from 1600 to 1700 cm-1 [22-24]. Secondary structures, including β-sheets, α-helix, β-turn, and random coil, could be assigned in view of the fact that each secondary structure had a different C=O stretching frequency in amide I band [25,26]. Generally, absorptions at 1650~1660 cm-1 were assigned to α-helix. Absorptions at 1610~1640 cm-1 and 1680~1700 cm-1 11

were assigned to β-sheet. Absorptions at 1662~1680 cm-1 were assigned to β-turn. And absorptions at 1640~1650 cm-1 were assigned to random coil. Here, to analyze the conformational transitions of PEI-crosslinked lipase on PEI@PMMA magnetic microspheres, FTIR spectra of lipase were obtained and the secondary structures were determined. Fig. 4 showed the absorptions of free lipase and PEI-crosslinked lipase on PEI@PMMA magnetic microspheres in amide I band,

ro of

and Table 1 gave their compositions of secondary structure. In Fig. 4, several bands

were obtained from the absorptions of free and PEI-crosslinked lipase by means of

second derivative and Fourier self-deconvolution. The contents of β-sheet (red line),

according

to

the

areas

of

multicomponent

peak.

Comparing

re

determined

-p

random coil (green line), α-helix (blue line) and β-turn (purple line) separately were

PEI-crosslinked lipase with free lipase, it could be found that the contents of α-helix,

lP

β-sheet and random coil slightly decreased by respective 0.7%, 0.9% and 0.4% after

na

crosslinking lipase with PEI on PEI@PMMA magnetic microspheres. And the content of β-turn increased by 2%. Moreover, the specific activity of lipase decreased from

ur

2.58 U/mg to 2.14 U/mg as listed in Table 1. To further compare with the conformations and activities of lipase immobilized via

Jo

other methods, several ways, including lipase adsorption on PEI@PMMA magnetic microspheres, lipase directly covalent bonding on PMMA magnetic microspheres without spacer-arm, and lipase crosslinking aggregation, were also employed to immobilize lipase. These results were also shown in Fig. 4 and Table 1. Firstly, comparing the adsorbed lipase with the free lipase in Table 1, it seemed that some 12

β-sheet structures in free lipase were turned into β-turn structures after adsorption. Meanwhile, the structures were also accompanied by little change in α-helix and random coil. After adsorbing on PEI@PMMA microspheres, the activity of lipase decreased from 2.58 to 2.20 U/mg. Secondly, comparing the crosslinked lipase aggregates with the free lipase in Table 1, the β-sheets structures remained unchanged. However, the contents of α-helix and random coil appeared to decrease clearly. As a

ro of

result, the specific activity of crosslinked lipase aggregates decreased significantly

from 2.58 to 1.57 U/mg. Thirdly, comparing the covalently immobilized lipase with the free lipase in Table 1, the α-helix structures in free lipase were destroyed in a large

-p

amount and the content of random coil grew significantly, which induced a serious

re

drop in lipase activity from 2.58 to 0.82 U/mg when covalently immobilizing lipase on PMMA microsphere. Therefore, the free lipase performed the highest activity (2.58

lP

U/mg), followed by the adsorbed lipase (2.20 U/mg), subsequently followed by

na

PEI-crosslinked lipase (2.14 U/mg), then followed by the crosslinked lipase aggregates (1.57 U/mg), and the directly covalent lipase performed the lowest activity

ur

(0.82 U/mg). Taking these changes in lipase activity as well as these changes in secondary structure of lipase together, it seemed that lipase activity was independent

Jo

of the contents of β-sheets, β-turn, and random coil structure within limits. The results seemed to suggest that lipase activity was dependent of the α-helix content because the order of α-helix content, i.e. free lipase (17.6%) > adsorbed lipase (17.1%) > PEI-crosslinked lipase (16.9%) > crosslinked lipase aggregates (16.3%) > covalently immobilized lipase (13.3%), was coincident with the order of lipase activity. In 13

another word, the α-helix structures had indispensably contributed to lipase activity, which agreed to the previous reports on lysozyme [27], phospholipase A2 [20], and catalase [28]. Generally, physical adsorption is often considered to be the least structural damage and the highest activity retention in various enzyme immobilization, while covalent immobilization has done great damage to enzyme activity, especially directly covalent

ro of

immobilization of enzyme on carrier without spacer-arm [11]. In Table 1, the activity

of PEI-crosslinked lipase (2.14 U/mg) was closest to that of the adsorbed lipase (2.20 U/mg). Moreover, PEI-crosslinked lipase exhibited obviously higher activity

-p

(increased by 36-161%) than the crosslinked lipase aggregates (1.57 U/mg) as well as

re

the directly covalent lipase (0.82 U/mg). These observations have inspired us since

damage to enzyme activity.

lP

PEI-crosslinked lipase not only made enzyme immobilize tightly, but also had little

na

3.4. Effect of crosslinking time, temperature and pH on PEI-crosslinked lipase The effect of crosslinking time on PEI-crosslinked lipase was investigated and the

ur

results were shown in Fig. 5a. As shown, lipase loading amount increased to the maximum as increasing crosslinking time to 60 min, and then followed a slight

Jo

decrease. Meanwhile, the specific activity of PEI-crosslinked lipase fluctuated slightly near 2.1 U/mg with reaction time below 60 min. Subsequently, the specific activity declined to 1.46 U/mg as reaction time increased to 120 min, indicating that the crosslinking reaction should be controlled within 60 min. Recently, the study by Wang et al. also showed that PEI could quickly crosslink with glutaraldehyde in a short time 14

[29]. Fig. 5b showed the effect of crosslinking temperature on PEI-crosslinked lipase. As shown, the specific activity of PEI-crosslinked lipase remained rough constant at approximate 2.0 U/mg regardless of reaction temperature. While lipase loading amount increased observably with temperature from 26.5 to 35 °C, and subsequently decreased at 40 °C. The decline in lipase loading amount under high temperature was

ro of

ascribed to two aspects. Firstly, glutaraldehyde was easy to self-polymerization under

high temperature [30], which reduced the amount of lipase crosslinked with PEI.

Secondly, the imine bond formed here was instable and easy to be split as temperature

-p

increased [31], resulting in the destruction of glutaraldehyde crosslinking.

re

Fig. 5c showed the effect of crosslinking pH on PEI-crosslinked lipase. As can be seen, the specific activity of lipase seemed to be independent of pH in the process of

lP

crosslinking. While lipase loading amount increased gradually to the maximum at pH

na

7.0, and then remained unchanged. A possible reason for the low amount of lipase loading at acidic pH could be attributed to that the Schiff base formed from

ur

PEI/glutaraldehyde was unstable at acidic condition and easy to be decomposed, which thus reduced the amount of PEI-crosslinked lipase. Another possible reason

Jo

was that when pH value was near to the isoelectric point of lipase (pI=5.0), lipase was easy to fall off from microsphere in the process of crosslinking due to the weakened electrostatic attraction between lipase and PEI. 3.5. pH and thermal stability of PEI-crosslinked lipase The stability of PEI-crosslinked lipase was further studied. Fig. 6a showed pH 15

stability of the free, PEI-crosslinked, and adsorbed lipase. As shown, the residual activity of free lipase retained only 51% after incubating in pH 9.0 for 1 h, and 63% after incubating in pH 5.0 for 1 h. These results indicated that the stability of free lipase was poor under alkaline or acid condition. Compared with free lipase, the adsorbed lipase displayed improved pH stability under the same conditions. Moreover, it was exciting that PEI-crosslinked lipase showed a better improvement in stability

ro of

than the adsorbed lipase in Fig. 6a. Moreover, the thermal stability study in Fig. 6b

also showed that, after incubating at the given temperature for 1 h, PEI-crosslinked lipase retained higher residual activity than free or adsorbed lipase. These results

-p

suggested that the stability of lipase was improved after crosslinking with PEI on

re

PEI@PMMA microspheres.

3.6. Kinetic parameters, the optimum temperature and reuse of PEI-crosslinked lipase

lP

The classical Michaelis-Menten model was often used to describe the kinetic

na

behavior of the immobilized lipase [32,33]. Kinetic parameters of free lipase and PEI-crosslinked lipase were investigated and the Lineweaver-Burk plots were shown

ur

in Fig. 7a. From the Lineweaver-Burk plots, the Km values of free and PEI-crosslinked lipase were calculated respectively to be 4.39 and 5.68 mg/mL. The later was higher

Jo

than the former, indicating that the substrate affinity of PEI-crosslinked lipase was lower than that of free lipase. Furthermore, the Vmax values of free and PEI-crosslinked lipase were calculated respectively to be 0.37 and 0.22 mmol/L∙min. These results could be ascribed to the restricted flexibility of enzyme conformation and the diffusion limitation caused by PEI chains. 16

We further studied the effect of temperature on the activity of PEI-crosslinked lipase and tried to obtain the optimum temperature. In Fig. 7b, free lipase performed the highest activity at 37 °C, and its activity showed a sharp peak in the range from 30 to 40 °C. Moreover, Fig. 7b revealed that the optimum temperature of lipase was widened observably after crosslinking with PEI on PEI@PMMA magnetic microspheres.

ro of

Reuse of PEI-crosslinked lipase was shown in Fig. 7c. As can be seen, after

recycling 4 times, PEI-crosslinked lipase still retained more than 80% residual activity, whereas, the adsorbed lipase just remained about 60% residual activity. The adsorbed

-p

lipase lost its activity quickly with the increase in recycle times, which was ascribed

re

to that lipase had fallen off from carrier seriously.

lP

4. Conclusions

In conclusion, an efficient enzyme immobilization was developed by means of

na

crosslink with PEI on PEI@PMMA microsphere. Compared with other immobilized lipase, PEI-crosslinked lipase retained enzyme natural structure well. Particularly,

ur

PEI-crosslinked lipase (2.14 U/mg) has performed 2.6 times and 1.4 times higher catalytic activity respectively than the directly covalent lipase (0.82 U/mg) and the

Jo

crosslinked lipase aggregates (1.57 U/mg), which was close to the activity of adsorbed lipase (2.20 U/mg). Moreover, PEI-crosslinked lipase revealed higher pH and thermal stability. After 4 cycles of reuse, it still retained more than 80% residual activity. Therefore, crosslinking lipase with PEI on carrier was proposed to be a potential tool in enzyme immobilization. 17

Notes There are no conflicts of interest to declare.

Author contributions

ro of

The study was designed by D.H.Z. and G.Y.Z.. Microspheres preparation and modification were performed and analyzed by Y.P.C., Y.P.X. X.F.G. and L.H.. Lipase

immobilization and conformation analysis were performed by Y.P.X., Y.P.C. and Q.C.. All authors contributed to writing the manuscript. All authors read and approved the

-p

final manuscript.

re

Acknowledgments

lP

The authors thank the financial supports from the National Natural Science Foundation of China (No. 21878065), Natural Science Foundation of Hebei Province,

na

China (No. B2015201167), and Interdisciplinary Research Program of Natural

Jo

ur

Science of Hebei University (2019DJY020).

18

References [1] S.S. Nadar, V.K. Rathod, Immobilization of proline activated lipase within metal organic framework (MOF), Int. J. Biol. Macromol., In press, (2019), doi:10.1016/j.ijbiomac.2019.10.199. [2] W.A.A. Wahab, E.A. Karam, M.E. Hassan, A.L. Kansoh, M.A. Esawy, G.E.A.

ro of

Awad, Optimization of pectinase immobilization on grafted alginate-agar gel beads by 24 full factorial CCD and thermodynamic profiling for evaluating of

operational covalent immobilization, Int. J. Biol. Macromol., 113 (2018)

-p

159-170.

re

[3] S.S. Nadar, V.K. Rathod, Magnetic macromolecular cross linked enzyme aggregates (CLEAs) of glucoamylase, Enzyme Microb. Tech., 83 (2016) 78-87.

lP

[4] U.V. Sojitra, S.S. Nadar, V.K. Rathod, A magnetic tri-enzyme nanobiocatalyst for

na

fruit juice clarification, Food Chem., 213 (2016) 296-305. [5] S.S. Nadar, V.K. Rathod, A co-immobilization of pectinase and cellulose onto

ur

magnetic nanoparticles for antioxidant extraction from waste fruit peels, Biocatalysis and Agricultural Biotechnology, 17 (2019) 470-479.

Jo

[6] T. Jesionowski, J. Zdarta, B. Krajewska, Enzyme immobilization by adsorption: A review, Adsorption-Journal of the International Adsorption Society, 20 (2014) 801–821. [7] D.-M. Liu, J. Chen, Y.-P. Shi, Screening of enzyme inhibitors from traditional Chinese medicine by magnetic immobilized α-glucosidase coupled with capillary 19

electrophoresis, Talanta, 164 (2017) 548-555. [8] S.S. Nadar, V.K. Rathod, Encapsulation of lipase within metal-organic framework (MOF) with enhanced activity intensified under ultrasound, Enzym. Microb. Tech., 108 (2018) 11-20. [9] A.S. Drozdov, O.E. Shapovalova, V. Ivanovski, D. Avnir, V.V. Vinogradov, Entrapment of enzymes within sol–gel-derived magnetite, Chem. Mater., 28

ro of

(2016) 2248–2253.

[10] S.L. Martins, B.F. Albuquerque, M.A.P. Nunes, M.H.L. Ribeiro, Exploring magnetic and imprinted cross-linked enzyme aggregates of rhamnopyranosidase

-p

in microbioreactors, Bioresource Technol., 249 (2018) 704-712.

re

[11] D.-M. Liu, J. Chen, Y.-P. Shi, Advances on methods and easy separated support materials for enzymes immobilization, TrAC-Trend. Anal. Chem., 102 (2018)

lP

332-342.

na

[12] S.F. Motevalizadeh, M. Khoobi, A. Sadighi, M. Khalilvand-Sedagheh, M. Pazhouhandeh, A. Ramazani, M.A. Faramarzi, A. Shafiee, Lipase immobilization

ur

onto polyethylenimine coated magnetic nanoparticles assisted by divalent metal chelated ions, J Mol Catal B: Enzyme, 120 (2015) 75–83.

Jo

[13] Y.-F. Dou, D.-H. Zhang, H. Liu, Y.-P. Xia, G.-Y. Zhi, Polyethyleneimine grafting and Cibacron Blue F3GA modifying poly(methylmethacrylate) magnetic microspheres for protein adsorption, J. Chem. Technol. Biot., 93 (2018) 994-1002. [14] T.-T. Xia, C.-Z. Liu, J.-H. Hu, C. Guo, Improved performance of immobilized 20

laccase on amine-functioned magnetic Fe3O4 nanoparticles modified with polyethylenimine, Chem Eng J, 295 (2016) 201–206. [15] Y. Wang, Q. Wang, X. Song, J. Cai, Hydrophilic polyethylenimine modified magnetic graphene oxide composite as an efficient support for dextranase immobilization with improved stability and recyclable performance, Biochem Eng J, 141 (2019), 163-172.

ro of

[16] R. Raghav, S. Srivastava, Immobilization strategy for enhancing sensitivity of

immunosensors: L-Asparagine–AuNPs as a promising alternative of EDC–NHS

activated citrate–AuNPs for antibody immobilization, Biosens. Bioelectron., 78

-p

(2016) 396-403.

re

[17] C. Schulz, R. Ludwig, L. Gorton, Polyethyleneimine as a promoter layer for the immobilization of cellobiose dehydrogenase from Myriococcum thermophilum

lP

on graphite electrodes, Anal. Chem., 86 (2014) 4256–4263.

na

[18] J.J. Virgen-Ortiz, J.C.S. dos Santos, A. Berenguer-Murcia, O. Barbosa, R.C. Rodrigues, R. Fernandez-Lafuente, Polyethylenimine: a very useful ionic

ur

polymer in the design of immobilized enzyme biocatalysts, J. Mater. Chem. B, 5 (2017) 7461-7490.

Jo

[19] P.G. Vazquez-Ortega, M.T. Alcaraz-Fructuoso, J.A. Rojas-Contreras, J. López-Miranda, R. Fernandez-Lafuente, Stabilization of dimeric β-glucosidase from Aspergillus niger via glutaraldehyde immobilization under different conditions, Enzym. Microb. Tech., 110 (2018) 38-45. [20] S. Suladze, S. Cinar, B. Sperlich, R. Winter, Pressure Modulation of the 21

Enzymatic Activity of Phospholipase A2, A putative membrane-associated pressure sensor, J. Am. Chem. Soc., 137 (2015) 12588-12596. [21] D. Oyen, R.B. Fenwick, P.C. Aoto, R.L. Stanfield, I.A. Wilson, H.J. Dyson, P.E. Wright, Defining the structural basis for allosteric product release from e. coli dihydrofolate reductase using nmr relaxation dispersion, J. Am. Chem. Soc., 139 (2017) 11233-11240.

ro of

[22] X. Zhou, Z. He, H. Huang, Secondary structure transitions of Bovine serum albumin induced by trmperature variation, Vib. Spectrosc., 92 (2017) 273-279.

[23] X. Zhou, Z. He, H. Huang, One pot synthesis of α-amylase metal organic

-p

framework (MOF)-sponge via dip-coating technique, Int. J. Biol. Macromol.,

re

138 (2019) 1035-1043.

[24] S.S. Nadar, V.K. Rathod, Sonochemical Effect on Activity and Conformation of

lP

Commercial Lipases, Appl. Biochem. Biotech., 181 (2017) 1435-1453.

na

[25] M. Carbonaro, A. Nucara, Secondary structure of food proteins by Fourier transform spectroscopy in the mid-infrared region, Amino Acids, 38 (2010)

ur

679-690.

[26] C. Guo, J. Zhang, J.S. Jordan, X. Wang, R.W. Henning, J.L. Yarger, Structural

Jo

Comparison of Various Silkworm Silks: An insight into the structure-property relationship, Biomacromolecules, 19 (2018) 906-917.

[27] A.A. Vertegel, R.W. Siegel, J.S. Dordick, Silica nanoparticle size influences the structure and enzymatic activity of adsorbed lysozyme, Langmuir, 20 (2004) 6800-6807. 22

[28] F.Y. Mahlicli, Y. Sen, M. Mutlu, S.A. Altinkaya, Immobilization of superoxide dismutase/catalase

onto

polysulfone

membranes

to

suppress

hemodialysis-induced oxidative stress: a comparison of two immobilization methods, J. Membrane Sci., 479 (2015) 175-189. [29] T. Wang, Y. Chen, J. Ma, Z. Jin, M. Chai, X. Xiao, L. Zhang, Y. Zhang, A polyethyleneimine-modified attapulgite as a novel solid support in matrix

ro of

solid-phase dispersion for the extraction of cadmium traces in seafood products, Talanta, 180 (2018) 254-259.

[30] I. Kovács, A.F. Farkas, A. Szitás, Z. Kónya, J. Kiss, Adsorption, polymerization

re

surfaces, Surf. Sci., 664 (2017) 129-136.

-p

and decomposition of acetaldehyde on clean and carbon-covered Rh(111)

[31] L. Xu, X. Zhou, Y. Yu, W.Q. Tian, J. Ma, S. Lei, Surface-confined crystalline

lP

two-dimensional covalent organic frameworks via on-surface schiff-base

na

coupling, ACS Nano, 7 (2013) 8066–8073. [32] S.S. Nadar, A.B. Muley, M.R. Ladole, P.U. Joshi, Macromolecular cross-linked

ur

enzyme aggregates (M-CLEAs) of alpha-amylase, Int. J. Biol. Macromol., 84 (2016) 69-78.

Jo

[33] U.V. Sojitra, S.S. Nadar, V.K. Rathod, Immobilization of pectinase onto chitosan magnetic nanoparticles by macromolecular cross-linker, Carbohydrate Polymers, 157 (2017) 677-685.

23

Table 1. Secondary structure contents and enzyme activity for free lipase, adsorbed lipase, PEI-crosslinked lipase, crosslinked lipase aggregates, and directly covalent lipase (SD, n=3.) Adsorbed lipase (%)

PEIcrosslinked lipase (%)

Crosslinked lipase aggregates (%)

Directly covalent lipase (%)

1610-1640 1680-1700

35.6±0.1

34.5±0.1

34.7±0.1

35.6±0.2

35.1±0.1

1640-1650

19.0±0.2

18.7±0.1

18.6±0.1

1650-1660 1660-1680

17.6±0.1 27.8±0.1

17.1±0.2 29.7±0.2

16.9±0.1 29.8±0.2

2.58±0.07

2.20±0.04

2.14±0.04

Jo

ur

na

lP

re

Specific activity (U/mg)

ro of

Random coil α-helix β- turn

Free lipase (%)

18.0±0.2

22.9±0.1

16.3±0.1 30.1±0.1

13.3±0.1 28.7±0.2

1.57±0.03

0.82±0.02

-p

β-sheet

Peaks (cm-1)

24

Figure captions: Fig. 1. PEI-crosslinked lipase on PEI@PMMA magnetic microspheres. Fig. 2. a) FTIR spectra of PMMA and PEI@PMMA magnetic microspheres; b) SEM images of PMMA magnetic microspheres; c) CLSM microscope images of lipase crosslinked with PEI on PEI@PMMA magnetic microspheres surface. Fig. 3. The loading amount of lipase via adsorption and PEI-crosslink on

ro of

PEI@PMMA magnetic micorspheres before and after desorption. (SD, n = 3.

Condition: 20 mg of microsphere, 2 mL of lipase (2 mg/mL), 0.75 ‰ GA, pH=7.0, T=35 °C, R=110 r/min, 2 h of adsorption, crosslinking 1 h, 4 h of desorption.).

-p

Fig. 4. Curve fitting in amide I region of infrared spectrogram for free lipase,

re

PEI-crosslinked lipase, adsorbed lipase, crosslinked lipase aggregates, and directly

lP

covalent lipase.

Fig. 5. Effect of a) crosslinking time, b) crosslinking temperature and c) crosslinking

na

pH on PEI-crosslinked lipase. (SD, n = 3. Condition: 20 mg of microsphere, 2 mL of lipase (2 mg/mL), 0.75 ‰ GA, pH=7.0, T=35 °C, R=110 r/min, 2 h of adsorption,

ur

crosslinking 1 h.)

Fig. 6. a) pH stability, as well as b) thermostability, of the free, PEI-crosslinked and

Jo

adsorbed lipase. (SD, n = 3.) Fig. 7. a) Lineweaver-Burk plots of free and PEI-crosslinked lipase; b) the effect of temperature on the activity of PEI-crosslinked lipase; and c) Reuse of PEI-crosslinked lipase and adsorbed lipase. (SD, n = 3.)

25

ro of

-p

re

lP

na

ur

Jo Fig. 1.

26

Fig. 2.

Transmittance (a.u.)

a)

PMMA microsphere PEI@PMMA microphere

1140-1300 582

1000

1725

2000

3000

Wave number (cm-1)

c)

4000

Jo

ur

na

lP

re

-p

b)

ro of

3435

27

Fig. 3.

50

PEI@PMMA adsorption/PEI-crosslinked PEI@PMMA desorption

40 35 30 25

ro of

CRL loading (mg/g)

45

20 15 10 5 0

PEI-crosslinked

Jo

ur

na

lP

re

-p

Adsorption

28

Fig. 4.

1650

1675

1700

1625

1650

1675

1700

Wavenumber （ cm-1） Total β-sheet Random Coil α-helix β-turn

Crosslinked lipase aggregates

Total β-sheet Random Coil α-helix β-turn

Directly covalent lipase

Absorbance

Absorbance

1625

1600

1700

Absorbance

Total β-sheet Random Coil α-helix β-turn

Adsorbed lipase

1600

1675

1600

1625

1650

1675

Wavenumber （ cm-1）

Jo

ur

na

lP

Wavenumber （ cm-1）

-p

1650

1700

re

1625

Wavenumber （ cm-1）

ro of

Absorbance 1600

Total β-sheet Random Coil α-helix β-turn

PEI-crosslinked lipase

Absorbance

Total β-sheet Random coil α-helix β-turn

Free lipase

29

1600

1625

1650

1675

Wavenumber （ cm-1）

1700

Fig. 5.

35 2.0

30 25

1.5

20 1.0 15 10

0

20

40

60

80

100

120

Loading capacity

30

2.0

25 1.5

20 15

45

35

c)

Loading capacity Specific enzyme activity

2.4 2.0

-p

35 30

1.6

25 20

5

6

re

1.2

15 10

Specific activity (U/mg)

Lipase loading (mg/g)

1.0

30

40

Cross-linking temperature (oC)

Cross-linking time (min)

40

7

Jo

ur

na

lP

Cross-linking pH

30

2.5

35

10 25

0.5

b)

Specific enzyme activity

40

ro of

2.5

8

Specific activity (U/mg)

40

a)

Lipase loading (mg/g)

45

Loading capacity Specific enzyme activity

Specific activity (U/mg)

Lipase loading (mg/g)

45

0.8

Fig. 6.

a)

80

60

40

20

5

6

7

8

100

Free CRL PEI-crosslinked CRL Adsorbed CRL

b)

80 60 40 20 20

9

30

pH

40

50

o

Jo

ur

na

lP

re

-p

Tempertaure( C)

31

60

ro of

Free CRL PEI-crosslinked CRL Adsorbed CRL

Relative activity(%)

Relative activity(%)

100

PEI-crosslinked CRL

8 7 6 5 4 3 2 1

0 -0.25 -0.20 -0.15 -0.10 -0.05 0.00 0.05 0.10 0.15

80 60 40 20 20

40

50

Temperature( C)

c) 100

PEI-crosslinked CRL Adsorbed CRL

80

-p

60 40 20

1

re

Relative activity(%)

30

o

1/[S](mL/mg)

0

Free CRL PEI-crosslinked CRL Adsorbed CRL

b)

100

2

3

Jo

ur

na

lP

Number of reuse

32

60

ro of

Free CRL

Relative activity(%)

a)

1/V(L*min/mmol)

Fig. 7.

4