Immobilization of urease in metal–organic frameworks via biomimetic mineralization and its application in urea degradation

Journal Pre-proof Immobilization of urease in metal–organic frameworks via biomimetic mineralization and its application in urea degradation

Xiao Liang, Qing Li, Zhiyuan Shi, Shaowei Bai, Quanshun Li PII:

S1004-9541(20)30057-4

DOI:

https://doi.org/10.1016/j.cjche.2020.01.014

Reference:

CJCHE 1633

To appear in:

Chinese Journal of Chemical Engineering

Received date:

18 October 2019

Revised date:

20 January 2020

Accepted date:

27 January 2020

Please cite this article as: X. Liang, Q. Li, Z. Shi, et al., Immobilization of urease in metal–organic frameworks via biomimetic mineralization and its application in urea degradation, Chinese Journal of Chemical Engineering(2020), https://doi.org/10.1016/ j.cjche.2020.01.014

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© 2020 Published by Elsevier.

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Immobilization of urease in metal-organic frameworks via biomimetic mineralization and its application in urea degradation

Xiao Liang,† Qing Li, † Zhiyuan Shi, Shaowei Bai, Quanshun Li*

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Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education,

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School of Life Sciences, Jilin University, Changchun 130012, China

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*Corresponding author.

E-mail: [email protected]. †

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Tel.: +86-431-85155201; Fax: +86-431-85155200.

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These authors contributed equally to the work.

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Abstract Enzyme immobilization has been accepted as an efficient technique for improving the stability and recyclability of enzymes. Herein, biomimetic mineralization strategy was employed to achieve the immobilization of urease in a type of metal-organic frameworks (zeolite imidazolate framework-8, ZIF-8), and the immobilized enzyme

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urease@ZIF-8 was systematically evaluated for its structure, activity, stability and recyclability, using the hydrolysis of urea as a model. The entrapment of urease was

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found to be realized in a synchronous manner with the formation of ZIF-8 crystal. The

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loading of urease in ZIF-8 was measured to be ca. 10.6% through the bicinchoninic acid

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(BCA) protein assay. The encapsulated urease could efficiently maintain its native conformation, which endowed the immobilized urease with excellent activity and

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stability, even in harsh conditions (e.g., in the presence of trypsin, acidic or alkali

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conditions, or at high temperature). Further, urease@ZIF-8 exhibited good recyclability

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during the degradation of urea, in which it could keep 58.86% of initial activity after being used for 5 cycles. Thus, biomimetic mineralization could be potentially utilized as a promising method to prepare immobilized ureases with superior activity, stability and recyclability, thereby facilitating the construction of efficient catalysts for industrial biocatalysis and biosensing. Keywords:

Urease;

Metal-organic

frameworks;

Immobilization;

Biomimetic

mineralization; Stability; Recyclability

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1. Introduction Urea (NH2CONH2) is a nitrogenous organic compound and has been widely used as a fertilizer, which is estimated to be produced with an annual global production of 9.3 billion tons by 2050 [1]. However, excessive urea not only decreases the pH of soil but also easily converts to ammonia and nitric acid, which will cause serious environmental

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pollution [2]. In addition, urea is a component in blood and other body fluids owing to the metabolism of proteins in kidney and liver [3]. The urea level in urine and blood has

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been identified to be a useful marker for evaluating the function of kidney and liver

dehydration,

burns

and

obstruction

in

urinary system

[6].

Thus,

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failure,

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[4,5], and increased urea level in blood will cause a series of diseases such as renal

human health.

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the decomposition of excess urea is an urgent problem to be solved in agriculture and

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Urease (urea amidohydrolase, EC 3.5.1.5) is an effective enzyme which can catalyze

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the decomposition of urea to ammonia and carbon dioxide [7]. It plays a key role in determining the urea content in blood, urine and wastewater, thereby facilitating the removal of urea from blood in the treatment of uremia in a dialysis manner [8]. Moreover, ureases have been found in various bacteria, fungi and plants, which possess an important function in the circulation of nitrogen in nature [9]. However, the activity, stability and recyclability of enzymes are easily affected by high temperature, extreme pH and long-term storage, which will limit their applications in health and environment

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fields. Thus, it is of inevitable quest to develop an appropriate strategy for the immobilization of ureases. Immobilized enzymes have been widely accepted to possess multiple advantages over free enzymes, such as the convenience in handling, the improved stability and recyclability, and the easy separation of enzymes from the reaction mixture [10-14]. A

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series of immobilization techniques have been successfully developed including physical adsorption, covalent coupling and entrapment in solid matrices [15-18]. For the

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immobilization of ureases, all these techniques have been employed using different

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supports to facilitate their applications in biocatalysis and biosensing [19-23]. However,

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no matter physical adsorption, covalent coupling or entrapment, immobilized enzymes suffered from several shortcomings such as the leaking, and partial inactivation of

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enzymes [24]. In contrast to these conventional methods, biomimetic mineralization

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could achieve the encapsulation of bioactive molecules within protective exteriors based

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on the concept of adopting and translating the natural self-assembly process in biological systems, thereby maintaining their functions in harsh environment [25-29]. Recently, metal-organic frameworks (MOFs) have been successfully developed to be useful carriers for the immobilization of enzymes in a biomimetic mineralization manner [30-37]. Compared to other materials, MOFs offered stronger thermal and chemical protection for biomacromolecules, and meanwhile promoted the selective diffusion of substrates in the porous network. In our previous reports, thermophilic lipase and deuterohemin-peptide enzyme mimic were successfully immobilized in

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MOFs through biomimetic mineralization, and these immobilized enzymes exhibited favorable catalytic activity, stability and recyclability in the kinetic resolution of sec-alcohols and atom transfer radical polymerization (ATRP) for the synthesis of biomedical polymers [38,39]. In the present study, urease was encapsulated in zeolite imidazolate framework-8

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(ZIF-8) under mild conditions using biomimetic mineralization strategy, and the immobilized enzyme was named as urease@ZIF-8. In the synthetic process, zinc acetate

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(Zn(OAc)2) solution was directly added into an aqueous mixture of urease and 2-methyl

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imidazole (HMeIM), and the sample was incubated overnight at room temperature (25 o

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C). The obtained urease@ZIF-8 was systematically evaluated for its structure, activity,

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2.1. Materials

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2. Materials and methods

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stability and recyclability, using the hydrolysis of urea as a model.

Zn(OAc)2 and HMeIM were purchased from Sigma-Aldrich. Urease was obtained from Tokyo Chemical Industry (Tokyo, Japan). Urea and citric acid were purchased from Aladdin (Shanghai, China). Mesoporous SBA-15 (aperture: 6-11 nm; specific surface area: 550-600 m2/g) was purchased from XFNANO Co. (Nanjing, China). Phenol was provided by Tianjin Chem. Co. (Tianjin, China). The bicinchoninic acid (BCA) protein assay kit was purchased from Promega (Madison, WI). All other reagents were obtained with the highest grade available and used as received.

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2.2. Construction of urease@ZIF-8 Urease@ZIF-8 was prepared in a biomimetic mineralization manner. Briefly, 10 mL of HMeIM solution (156.80 mg/mL) was mixed with 5 mL of urease solution (1 mg/mL), and then 10 mL of Zn(OAc)2 solution (8.76 mg/mL) and 1 mL methanol were added dropwise into the mixture. The sample was incubated at room temperature for 12 h, and

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the precipitate was collected by centrifugation (6000 r/min, 5 min) and then washed with distilled water three times. The obtained urease@ZIF-8 was subjected to lyophilization,

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and the urease loading in ZIF-8 was detected using BCA protein assay kit. For the

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prepation of urease@SBA-15, 1 mL of urease solution (1 mg/mL) was mixed with 10

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mg SBA-15, and the mixture was stirred at room temperature for 24 h (150 r/min); the precipitate was collected by centrifugation (6000 r/min, 5 min), washed with distilled

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water three times and then lyophilized.

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2.3. Characterization of urease@ZIF-8 The scanning electron microcopic (SEM) images and corresponding energy dispersive X-ray (EDX) mapping were carried out on a XL 30 ESEM-FEG scanning electron microscope (FEI, Hillsboro, OR) at an acceleration voltage of 20 kV. The transmission elctron microcopic (TEM) analysis was conducted on a JEM-2100F field emission electron microscope (JEOL, Tokyo, Japan) at an acceleration voltage of 200 kV. The powder X-ray diffraction (PXRD) patterns were captured on a wide-angle X-ray diffractometer (D8 ADVANCE, Bruker, Germany) from 5 to 50° at a scanning rate of 15

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°/min. The Fourier-transform infrared spectra (FT-IR) and thermogravimetric analysis (TGA) were conducted on a synchronous thermal analysis-mass spectrometer-infrared combined instrument (NETZSCH, Bruker, Germany). Nitrogen adsorption and desorption isotherms were performed on an ASAP 2020 system (Micromeritics, Norcross,

GA)

and

specific

surface

areas

were

calculated

by

the

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Brunauer-Emmett-Teller (BET) equation. Far ultraviolet circular dichroism (CD) spectra were obtained on a JASCO-810 circular dichroism spectrometer (JASCO Inc., Tokyo,

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Japan) with a scanning speed of 100 nm/min, in which urease@ZIF-8 was firstly

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urease concentration used was 100 μM.

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degraded by EDTA or citrate buffer (200 mM, pH 7.0) to release the urease and the

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2.4. Stability analysis after the trypsin treatment

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Urease@ZIF-8 and free urease were first treated with 1.25% trypsin for 1.5 h. Then

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the immobilized enzyme urease@ZIF-8 was collected by the centrifugation (5000 r/min, 10 min) and washed with distilled water three times. Afterwards, 1 M HCl solution was used to destroy the ZIF-8 layer to release free urease. Finally, all the samples were subjected to SDS-PAGE analysis.

2.5. Activity and stability analysis of urease@ZIF-8 First, 100 L of urea solution (1 mg/mL) was mixed with 200 L of citrate buffer (pH 6.7) in a 1.5-mL Eppendorf tube. Then 20 L of urease or urease@ZIF-8 suspension

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(urease concentration of 1 mg/mL) was added into the tube. After the reaction at 37 oC for 1 h, the samples were centrifuged at 8000 r/min for 3 min to obtain the supernatant. Then the sodium phenol (40 μL, 1.35 mol/L) was added into 100 µL of supernatant, and 30 μL of sodium hypochlorite (0.9%) was subsequently added. After the incubation in the dark for 20 min, the sample was detected on a HBS-1096A microplate reader

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(Nanjing, China) to monitor the absorbance at 578 nm which could be used to determine the relative activity. The background was measured using the reference sample of

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identical composition except for no addition of enzyme solution. Subsequently, effects

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of temperature and pH on the enzymatic activity and stability of urease@ZIF-8 were

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systematically evaluated in the same way.

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2.6. Recyclability of urease@ZIF-8

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The urea solution (10 mg/mL) and urease@ZIF-8 or urease@SBA-15 suspension (1

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mg/mL) were mixed together, and the reaction was conducted at 37 °C for 1 h. After the reaction, urease@ZIF-8 was recovered through the centrifugation at 10,000 rpm for 5 min and then the enzymatic activity was measured. The collected enzymes continued to react with the urea solution as described above to evaluate the recyclability. After 3 cycles, the crystal form and morphology of urease@ZIF-8 were detected by PXRD and SEM, respectively.

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3. Results and discussion 3.1. Construction and characterization of urease@ZIF-8 The biomimetic mineralization method was employed to achieve the immobilization of urease in a porous nanomaterial ZIF-8. In the synthetic process, Zn(OAc)2 solution was added into a mixture of urease and HMeIM, and then the sample was incubated at

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room temperature for 12 h. The morphology of urease@ZIF-8 was characterized by SEM and TEM. As shown in Figure 1, both blank ZIF-8 (me-ZIF-8) and urease@ZIF-8

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exhibited a uniform dodecahedral structure with smooth surface, which suggested that

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the loading of urease did not affect the morphology of ZIF-8. Meanwhile, the regular

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dodecahedral structure of urease@ZIF-8 demonstrated that the entrapment of urease was achieved in a synchronous manner with the formation of ZIF-8 crystal. However,

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the particle size increased from ca. 300 nm (me-ZIF-8) to appropriately 1 m

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(urease@ZIF-8), indicating the successful immobilization of urease in ZIF-8. Similarly,

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the regular morphology of me-ZIF-8 and urease@ZIF-8 could be observed in TEM images, and the particle size of urease@ZIF-8 was also much higher than that of me-ZIF-8 (Figure 2). These results were consistent with SEM images, which provided direct evidence for the successful loading of urease in ZIF-8. In addition, EDX mapping and elemental analysis clearly showed the presence of S element in urease@ZIF-8 while no S element was observed in me-ZIF-8, suggesting the encapsulation of urease in ZIF-8 (Figure S1 and S2). There were no significant changes in the intensity of C, H, O

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and N elements after the loading of urease, indicating that the introduction of urease

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through biomimetic mineralization did not alter the structure of ZIF-8.

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Figure 1. SEM images of urease@ZIF-8 (a) and me-ZIF-8 (b). The scale bar is 1 m.

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Figure 2. TEM images of urease@ZIF-8 (a) and me-ZIF-8 (b). The scale bar is 1 m.

Further, the PXRD analysis was conducted to monitor the structure of ZIF-8. As shown in Figure 3, the peaks of urease@ZIF-8 matched well with those of urease@ZIF-8, indicating that the ZIF-8 crystal was formed in the biomimetic mineralization, and the loading of urease did not affect the structural integrity of ZIF-8. In the FT-IR spectra, characteristic peaks of urease at 1400-1500 cm-1 could be obviously observed in urease@ZIF-8 group, attributing to the presence of amide I and II bands of

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protein component (Figure 4). Meanwhile, there were no new peaks or significant peak shifts after the loading of urease, which demonstrated that no covalent bonding was formed and the immobilization of urease in ZIF-8 was achieved in a self-assembly method. The nitrogen sorption isotherms of me-ZIF-8 and urease@ZIF-8 showed a classical type I pattern, and the surface area values were calculated to be 961.0 and

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1021.7 m2/g, respectively (Figure S3). The slightly increased specific surface area

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did not affect the crystal integrity of ZIF-8.

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suggested that the immobilization of urease in ZIF-8 through biomimetic mineralization

Figure 3. PXRD patterns of urease@ZIF-8 and me-ZIF-8.

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Figure 4. FT-IR spectra of urease, me-ZIF-8 and urease@ZIF-8.

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After the successful loading of urease in ZIF-8, the enzyme loading of urease@ZIF-8 was measured through TGA curves and BCA protein assay. As shown in Figure 5,

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stronger mass loss could be observed in urease@ZIF-8 than ZIF-8 in the range of

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100-250 oC, which was attributed to the decomposition of urease molecules. Based on

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the curves, the urease loading was calculated to be 8.93%, which was close to that measured by BCA method (10.6%). For urease@SBA-15, the urease loading was much lower (5.9%). In comparison to free urease, the relative activity of urease@ZIF-8 was determined to be 78.0%, which was in the range of 74.0-89.0% when carboxymethyl cellulose, graphene oxide core@shell heparin-mimicking polymer beads, oxide-chitosan composite beads or cross-linked enzyme lyophilisates were employed as the supports or methods for the urease immobilization [20,23,40-42].

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The secondary structure of urease was assayed using CD spectra, in which urease@ZIF-8 was digested with EDTA or citrate buffer (200 mM, pH 7.0) to destroy ZIF-8 structure to release free urease (Figure 6 and S4). Both free urease and urease in urease@ZIF-8 showed the existence of typical -helix conformation, with two wave troughs at 209 and 222 nm. Meanwhile, there were no significant differences between

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these two curves, indicating that the immobilization process in a biomimetic mineralization did not affect the conformation of urease. The phenomenon was

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attributed to the mild conditions in the synthetic process of urease@ZIF-8. The

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immobilization technique could favorably maintain the original conformation urease

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and thus it was beneficial to obtain excellent catalytic activity of urease in the immobilized form. Further, to detect the protection effect of urease by ZIF-8, urease and

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urease@ZIF-8 were treated with 1.25% trypsin for 1.5 h and subjected to the

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SDS-PAGE analysis (Figure S5). The main band could be detected to be ca. 100 kDa

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for free urease, and some minor bands were presented in the image which was caused by the intrinsic characteristics of commercial urease. Meanwhile, the density of major band significantly decreased in free urease group after the trypsin treatment. However, there were no notable changes for all the bands in urease@ZIF-8 after the digestion with trypsin solution. These results provided a clear evidence for that ZIF-8 could protect urease from the degradation of trypsin, probably owing to the restrain of trypsin entering the ZIF-8 interior.

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Figure 5. TGA curves of me-ZIF-8 and urease@ZIF-8.

Figure 6. CD spectra of urease and urease in urease@ZIF-8 after EDTA digestion.

3.2. Activity, stability and recyclability analysis of urease@ZIF-8 As the channel width and cage size of ZIF-8 are smaller than the size of urease, we infer that urease is embedded in ZIF-8 during the biomimetic mineralization process. To understand the mechanism in a deeper way, two samples were prepared: me-ZIF-8 was 14

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physically mixed with urease for 10 min and then washed with distilled water three times (named as complex 1), and urease @ZIF-8 with no addition of HMeIM during the synthesis (denoted as complex 2). As shown in Figure S6, blue color could be clearly observed in urease and urease@ZIF-8 groups owing to the hydrolysis of urea. However, these two complexes could not produce the blue color which demonstrated the absence

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of urease activity for these two samples. Thus, urease was successfully embedded in ZIF-8 only during the crystal growth of ZIF-8, which was mainly attributed to the

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interaction between MOF and enzyme during the biomimetic mineralization.

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Effects of temperature and pH on the enzymatic activity of urease@ZIF-8 were

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investigated using urea as the substrate. As shown in Figure 7, the optimal temperature and pH of urease@ZIF-8 were determined to be 50 oC and 7.0, respectively. The values

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were the same as those of free urease, which meant that the biomimetic mineralization

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approach did not alter the characteristic profile of enzyme. Meanwhile, the immobilized

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urease in ZIF-8 showed relatively higher catalytic activity than free urease at high temperature and acidic or alkaline conditions. The results implied that the support ZIF-8 could improve the tolerance of enzyme against harsh conditions, and thus these properties would make the immobilized enzymes beneficial for the applications in future biocatalysis, especially at an industrial scale.

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Figure 7. The optimal temperature (a) and pH (b) of urease and urease@ZIF-8.

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Further, the stability of urease@ZIF-8 under different temperature and pH was

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evaluated through the pre-incubation of enzymes for different time. As shown in Figure

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8, the activity of urease and urease@ZIF-8 did not change with the elongation of incubation time at 30 and 50 oC, whereas urease@ZIF-8 exhibited higher stability than

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free urease at 70 and 90 oC within 40 min. After the incubation at 70 oC for 40 min, only

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33.18% of residual activity could be obtained for urease, while urease@ZIF-8 could

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retain 56.35% of initial activity. Notably, 52.74% of original activity could be achieved for urease@ZIF-8 at 90 oC for 40 min while free urease almost deactivated under the same conditions (only 1.11% of initial activity). The half-time values of urease@ZIF-8 were calculated to be 51.2 (70 oC) and 41.8 min (90 oC), which were much higher than those for free enzyme (32.6 and 7.3 min at 70 and 90 oC, respectively). These results revealed that urease@ZIF-8 possessed much stronger tolerance against high temperature, which would be favorable for achieving the catalytic reactions with high efficiency at high temperatures. Meanwhile, the reactions at high temperatures could be useful to

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avoid the pollution of microbial components. Similar phenomenon could be observed in the pH tolerance assay of urease@ZIF-8, in which urease@ZIF-8 could retain 75% of hydrolytic activity after the incubation at pH of 4.0 and 10.0 for 60 min, as shown in Figure 9. However, only 40.53% and 35.01% of residual activity was detected for urease under pH 4.0 and 10.0, respectively. The half-time values of urease@ZIF-8 were

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calculated to be 125.8 (pH 4.0) and 149.7 min (pH 10.0), which were also much higher than those for free enzyme (42.4 and 44.0 min at pH of 4.0 and 10.0, respectively).

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Meanwhile, there were no significant differences in the relative activity for urease and

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urease@ZIF-8 under the optimal pH (7.0). These results clearly showed that the

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immobilized urease in ZIF-8 possessed higher stability under acidic and alkaline

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conditions.

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Figure 8. Residual activity analysis of urease and urease@ZIF-8 under 30 (a), 50 (b), 70 (c) and 90 oC (d) for different time.

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Figure 9. Residual activity analysis of urease and urease@ZIF-8 under pH of 4.0 (a),

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7.0 (b) and 10.0 (c) for different time.

Moreover, the storage stability of urease and urease@ZIF-8 was assayed in which

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urease@ZIF-8 was incubated in distilled water at room temperature for 12 days and the

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residual activity was monitored. As shown in Figure 10, the activities of both urease and

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urease@ZIF-8 exhibited the decreased tendency with the incubation time. After the

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incubation for 12 days, urease@ZIF-8 could keep 54.07% of initial activity, but only 16.55% of activity was obtained for free urease, indicating the good storage stability of immobilized enzyme. The enhanced storage stability could be useful for improving the stability of enzymes during the transportation and storage, and thus it was favorable for improving the cost-effectiveness of enzymatic reactions. In conclusion, urease embedded in ZIF-8 possessed stronger stability against temperature, acidic or alkaline conditions and long-term storage, owing to the conformational stabilization of urease molecules in ZIF-8.

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Figure 10. Residual activity analysis of urease and urease@ZIF-8 at room temperature

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(25 oC) for different time.

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Finally, the recyclability of urease@ZIF-8 was evaluated, since the reusability is an

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important parameter for analyzing the immobilized enzymes. After being used for

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different cycles, the residual activity of urease@ZIF-8 was measured. As shown in

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Figure 11, urease@ZIF-8 exhibited a favorable recyclability in the hydrolysis of urea, in which the activity decreased from 100% to 58.86% after 5 cycles. In contrast, only 9.24% of initial activity could be retained for urease@SBA-15 after being used for 3 cycles (Figure S7). The improved recyclability of urease@ZIF-8 was mainly caused by the decreased enzyme leaking, as ZIF-8 framework and urease molecule formed a strong interaction during the biomimetic mineralization. Meanwhile, after being used for 3 cycles, there were no obvious changes in the PXRD pattern of urease@ZIF-8, and the uniform dodecahedral structure could be observed, as demonstrated by PXRD and SEM analysis (Figure 12). These results meant that the framework structure and crystal form 19

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of urease@ZIF-8 could be well retained, implying the intrinsic stability of urease@ZIF-8 during the reactions. Though the recyclability has been improved after the immobilization of urease in ZIF-8, the cycles were still limited and the effective strategies are needed to be developed for enhancing the recyclability and reducing the

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biocatalysts’ cost in future.

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cycles.

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Figure 11. Residual activity analysis of urease@ZIF-8 after being used for different

Figure 12. The PXRD pattern (a) and SEM image (b) of urease@ZIF-8 after 3 cycles.

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4. Conclusions In this study, urease was successfully immobilized in ZIF-8 in a biomimetic mineralization manner. In comparison to free urease, urease@ZIF-8 has been demonstrated to possess excellent activity and stability under high temperature and acidic or alkaline conditions. Meanwhile, the immobilized enzyme exhibited favorable

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long-term storage stability and recyclability. Thus, the biomimetic mineralization

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biocatalysts with superior characteristics.

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method could be used as a potential tool for entrapping enzymes in MOFs to construct

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Acknowledgments

The authors gratefully acknowledge the supports from National Key R&D Program

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of China (2018YFC1105401), National Natural Science Foundation of China

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(81673502 and 81872928), Science & Technology Department of Jilin Province

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(20190201288JC), Education Department of Jilin Province (JJKH20190010KJ), Province-University Cooperation Project of Jilin Province (SXGJQY2017-4) and the Fundamental Research Funds of the Central Universities, China.

References [1] A. Tarasov, D.W. Gray, M.Y. Tsai, N. Shields, A. Montrose, N. Creedon, P. Lovera, A. O’Riordan, M.H. Monney, E.M. Vogel, A potentiometric biosensor for rapid on-site disease diagnostics. Biosens. Bioelectron. 79 (2016) 669-678.

21

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[2] G. Dhawan, G. Sumana, B.D. Malhotra, Recent developments in urea biosensors. Biochem. Eng. J. 44 (2019) 42-52. [3] S. Jakhar, C.S. Pundir, Preparation, characterization and application of urease nanoparticles for construction of an improved potentiometric urea biosensor. Biosens. Bioelectron. 100 (2018) 242-250.

oo

f

[4] B. Kovacs, G. Nagy, R. Dombi, K. Toth, Optical biosensor for urea with improved response time. Biosens. Bioelectron. 18 (2003) 111-118.

pr

[5] L.F. Wei, J.S. Shih, Fullerene-cryptand coated piezoelectric crystal urea sensor

e-

based on urease. Anal. Chim. Acta 437 (2001) 77-85.

Pr

[6] W.L. Lau, N.D. Vaziri, Urea, a true uremic toxin: the empire strikes back. Clin. Sci. 131 (2017) 3-12.

al

[7] G. Pozniak, B. Krajewska, W. Trochimczuk, Urease immobilized on modified

[8] M.

Jo u

129-134.

rn

polysulphone membrane: preparation and properties. Biomaterials 16 (1995)

Chellapandian,

M.R.V. Krishnan,

Chitosan-poly(glycidyl methacrylate)

copolymer for immobilization of urease. Process Biochem. 33 (1998) 595-600. [9] B. Sahoo, S.K. Sahu, P. Pramanik, A novel method for the immobilization of urease on phosphonate grafted iron oxide nanoparticle. J. Mol. Catal. B: Enzym. 69 (2011) 95-102. [10] R.A. Sheldon, S. van Pelt, Enzyme immobilisation in biocatalysis: why, what and how. Chem. Soc. Rev. 42 (2013) 6223-6235.

22

Journal Pre-proof

[11] R.C. Rodrigues, C. Ortiz, A. Berenguer-Murcia, R. Torres, R. Fernandez-Lafuente, Modifying enzyme activity and selectivity by immobilization. Chem. Soc. Rev. 42 (2013) 6290-6307. [12] L. Zhou, H. Mou, J. Gao, L. Ma, Y. He, Y. Jiang, Preparation of cross-linked enzyme aggregates of nitrile hydratase ES-NHT-118 from E. coli by

oo

f

macromolecular cross-linking agent. Chin. J. Chem. Eng. 25 (2017) 487-492. [13] T.R.B. Ramakrishna, T.D. Nalder, W. Yang, S.N. Marshall, C.J. Barrow, Controlling

pr

enzyme function through immobilisation on graphene, graphene derivatives and

e-

other two dimensional nanomaterials. J. Mater. Chem. B 6 (2018) 3218-3200.

Pr

[14] M. Sharifi, S.M. Robatjazi, M. Sadri, J.M. Mosaabadi, Immobilization of organophosphorus hydrolase enzyme by covalent attachment on modified cellulose

al

microfibers using different chemical activation strategies: characterization and

rn

stability studies. Chin. J. Chem. Eng. 27 (2019) 191-199.

Jo u

[15] M. Hartmann, X. Kostrov, Immobilization of enzymes on porous silicas - benefits and challenges. Chem. Soc. Rev. 42 (2013) 6277-6289. [16] Z. Zhou, M. Hartmann, Progress in enzyme immobilization in ordered mesoporous materials and related applications. Chem. Soc. Rev. 42 (2013) 3894-3912. [17] C. Wang, H. Han, W. Jiang, X. Ding, Q. Li, Y. Wang, Immobilization of thermostable lipase QLM on core-shell structured polydopamine-coated Fe3O4 nanoparticles. Catalysts 7 (2017) 49. [18] W. Jiang, X. Wang, J. Yang, H. Han, Q. Li, J. Tang, Lipase-inorganic hybrid

23

Journal Pre-proof

nanoflower constructed through biomimetic mineralization: a new support for biodiesel synthesis. J. Colloid Interface Sci. 514 (2018) 102-107. [19] C. Veghela, M. Kulkarni, S. Haram, R. Aiyer, M. Karve, A novel inhibition based biosensor using urease nanoconjugate entrapped biocomposite membrane for potentiometric glyphosate detection. Int. J. Biol. Macromol. 108 (2018) 32-40.

oo

f

[20] F.S. Alatawi, M. Monier, N.H. Elsayed, Amino functionalization of carboxymethyl cellulose for efficient immobilization of urease. Int. J. Biol. Macromol. 114 (2018)

pr

1018-1025.

e-

[21] R. Fopase, S. Nayak, M. Mohanta, P. Kale, B. Paramasivan, Inhibition assays of

Pr

free and immobilized urease for detecting hexavalent chromium in water samples. 3 Biotech. 9 (2019) 124.

al

[22] S. Mondal, S. Malik, R. Sarkar, D. Roy, S. Saha, S. Mishra, A. Sarkar, M.

rn

Chatterjee, B. Mandal, Exuberant immobilization of urease on an inorganic

Jo u

SiO2 support enhances the enzymatic activities by 3-fold for perennial utilization. Bioconjug. Chem. 30 (2019) 134-147. [23] L. Yang, X. Liu, N. Zhou, Y. Tian, Characteristics of refold acid urease immobilized covalently by graphene oxide-chitosan composite beads. J. Biosci. Bioeng. 127 (2019) 16-22. [24] S. Garg, A. De, S. Mozumdar, pH-dependent immobilization of urease on glutathione-capped gold nanoparticles. J. Biomed. Mater. Res. A 103 (2015) 1771-1783.

24

Journal Pre-proof

[25] L. Betancor, H.R. Luckarift, Bioinspired enzyme encapsulation for biocatalysis. Trends Biotechnol. 26 (2008) 566-572. [26] J. Shi, L. Zhang, Z. Jiang, Facile construction of multicompartment multienzyme system through layer-by-layer self-assembly and biomimetic mineralization. ACS Appl. Mater. Interfaces 3 (2011) 881-889.

oo

f

[27] J. Shi, Y. Jiang, S. Zhang, D. Yang, Z. Jiang, Biomimetic/bioinspired design of enzyme@capsule nano/microsystems. Methods Enzymol. 571 (2016) 87-12.

pr

[28] Z. Li, Y. Ding, S. Li, Y. Jiang, Z. Liu, J. Ge, Highly active, stable and prepared

by biomimetic

mineralization of

e-

self-antimicrobial enzyme catalysts

Pr

copper hydroxysulfate. Nanoscale 8 (2016) 174401-17445. [29] N. Rauner, M. Meuris, M. Zoric, J.C. Tiller, Enzymatic mineralization generates

al

ultrastiff and tough hydrogels with tunable mechanics. Nature 543 (2017) 407-410.

rn

[30] X. Lian, Y. Fang, E. Joseph, Q. Wang, J. Li, S. Banerjee, C. Lollar, X. Wang, H.C.

Jo u

Zhou, Enzyme-MOF (metal-organic framework) composites. Chem. Soc. Rev. 42 (2013) 6223-6235.

[31] F. Lyu, Y. Zhang, R.N. Zare, J. Ge, Z. Liu, One-pot synthesis of protein-embedded metal-organic frameworks with enhanced biological activities. Nano Lett. 14 (2014) 5761-5765. [32] X. Wu, J. Ge, C. Yang, M. Hou, Z. Liu, Facile synthesis of multiple enzymecontaining metal-organic frameworks in a biomolecule-friendly environment. Chem. Commun. 51 (2015) 13408-13411.

25

Journal Pre-proof

[33] X. Wu, M. Hou, J. Ge, Metal-organic frameworks and inorganic nanoflowers: a type of emerging inorganic crystal nanocarriers for enzyme immobilization. Catal. Sci. Technol. 5 (2015) 5077-5085. [34] P. Li, S.Y. Moon, M.A. Guelta, L. Lin, D.A. Gomez-Gualdron, R.Q. Snurr, S.P. Harvey, J. T. Hupp, O.K. Farha, Nanosizing a metal-organic framework enzyme

oo

f

carrier for accelerating nerve agent hydrolysis. ACS Nano 10 (2016) 9174-9182. [35] E. Gkaniatsou, C. Sicard, R. Ricoux, L. Benahmed, F. Bourdreux, Q. Zhang, C.

pr

Serre, J.P. Mahy, N. Steunou, Enzyme encapsulation in mesoporous metal-organic

Pr

Int. Ed. 57 (2018) 16141-16146.

e-

frameworks for selective biodegradation of harmful dye molecules. Angew. Chem.

[36] C. Lin, K. Xu, R. Zheng, Y. Zheng, Immobilization of amidase into a magnetic

al

hierarchically porous metal-organic framework for efficient biocatalysis. Chem.

rn

Commun. 55 (2019) 5697-5700.

Jo u

[37] X. Wu, H. Yue, Y. Zhang, X. Gao, X. Li, L. Wang, Y. Cao, M. Hou, H. An, L. Zhang, S. Li, J. Ma, H. Lin, Y. Fu, H. Gu, W. Lou, W. Wei, R.N. Zare, J. Ge, Packaging and delivering enzymes by amorphous metal-organic frameworks, Nat. Commun. 10 (2019) 5165. [38] H. He, H. Han, H. Shi, Y. Tian, F. Sun, Y. Song, Q. Li, G. Zhu, Construction of thermophilic

lipase-embedded

metal-organic

frameworks

via

biomimetic

mineralization: a biocatalyst for ester hydrolysis and kinetic resolution. ACS Appl. Mater. Interfaces 8 (2016) 24517-24524.

26

Journal Pre-proof

[39] W. Jiang, X. Wang, J. Chen, Y. Lu, H. Han, Y. Ding, Q. Li, J. Tang, Deuterohemin-peptide

enzyme

mimic-embedded

metal-organic

frameworks

through biomimetic mineralization with efficient ATRP catalytic activity. ACS Appl. Mater. Interfaces 9 (2017) 16948-16957. [40] B. Somturk, I. Yilmaz, C. Altinkaynak, A. Karatepe, N. Ozdemir, I. Ocsoy,

Enzyme Microb. Technol. 86 (2016) 134-142.

oo

f

Synthesis of urease hybrid nanoflowers and their enhanced catalytic properties.

pr

[41] J. Zhang, Z. Shi, C. He, X. Song, Y. Yang, S. Sun, W. Zhao, C. Zhao,

e-

Urease immobilized GO core@shell heparin-mimicking polymer beads with safe an

Pr

d effectiveurea removal for blood purification, Int. J. Biol. Macromol. (2019) doi: 10.1016/j.ijbiomac.2019.11.197.

al

[42] T. Akkas, A. Zakharyuta, A. Taralp, C.W. Ow-Yang, Cross-linked enzyme

rn

lyophilisates (CLELs) of urease: a new method to immobilize ureases, Enzyme

Jo u

Microb. Technol. 132 (2020) 109390.

27

Journal Pre-proof

Jo u

rn

al

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e-

pr

oo

f

Graphical Abstract

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