Immobilized carbonic anhydrase on mesoporous

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May 25, 2018 - mizing the performance of [email protected]MOF composites [30,38]. ...... laccase via adsorption onto bimodal mesoporous Zr-MOF, Process Biochem.

International Journal of Biological Macromolecules 117 (2018) 189–198

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Immobilized carbonic anhydrase on mesoporous cruciate flower-like metal organic framework for promoting CO2 sequestration Sizhu Ren, Yuxiao Feng, Huan Wen, Conghai Li, Baoting Sun, Jiandong Cui ⁎, Shiru Jia ⁎ Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University of Science and Technology, No 29, 13th, Avenue, Tianjin Economic and Technological Development Area (TEDA), Tianjin 300457, PR China

a r t i c l e

i n f o

Article history: Received 14 April 2018 Received in revised form 23 May 2018 Accepted 23 May 2018 Available online 25 May 2018 Keywords: CO2 sequestration Carbonic anhydrase Immobilization ZIF-8 CaCO3

a b s t r a c t CO2 capture by immobilized carbonic anhydrase (CA) has become an alternative and environmental friendly approach in CO2 sequestration technology. However, the immobilized CA usually exhibits low CO2 sequestration efficiency due to no gas adsorption function for the conventional CA supports. Metal organic frameworks (MOFs) are an excellent material for gas adsorption and enzyme immobilization. Herein, a combined immobilization system of CA and ZIF-8 with cruciate flower-like morphology for CO2 adsorption was prepared for the first time by adsorbing CA onto ZIF-8. The immobilization efficiency was greater than 95%, and the maximum activity recovery reached 75%, indicating the highly efficient immobilization process. The resultant [email protected] composites exhibited outstanding thermostability, the tolerance against denaturants, and reusability compared with free CA. Furthermore, we demonstrated for the first time that the shape of ZIF-8 could be controlled by adjusting concentrations of Zn2+ ions at the high concentration of 2-methylimidazole (1 M). More importantly, we also demonstrated the applicability of the [email protected] composites to the sequestration of CO2 in carbonate minerals. The yields of the CaCO3 obtained by using [email protected] composites were 22-folds compared to free CA. Thus, this [email protected] composite can be successfully used as a robust biocatalyst for sequestration of CO2. © 2018 Elsevier B.V. All rights reserved.

1. Introduction Nowadays, global warming has becomes one of the major problems to the earth. One of the major contributors of the greenhouse gases is carbon dioxide (CO2). In order to solve this problem, various technologies, including CO2 capture and storage; and converting CO2 into fuels and chemicals, have been proposed [1]. Among these approaches, turning CO2 into fuels and chemicals offers a win-win strategy to both decrease atmospheric CO2 and efficiently exploit carbon resources [2,3]. Carbonic anhydrase (CA, EC4.2.1.1) is a category of zinc-containing metalloenzymes, which containing zinc ligands and accelerate the hydration of CO2 to bicarbonate and protons [4]. Recent reports showed that CA could be effectively used for CO2 capture [5,6]. However, application of free CA is strongly limited due to its low stability under the extreme condition and poor reusability [7]. In the past twenty years, immobilization technology was applied to overcome these problems [8–11]. Generally, the stability of enzyme could be improved by multipoint or multisubunit immobilization due to preventing subunit dissociation [12], decreasing aggregation, autolysis or proteolysis [13], enhancing enzyme rigidification and resistance to inhibitors or chemicals [14,15] and producing favorable microenvironments [16]. ⁎ Corresponding authors. E-mail addresses: [email protected], (J. Cui), [email protected] (S. Jia).

https://doi.org/10.1016/j.ijbiomac.2018.05.173 0141-8130/© 2018 Elsevier B.V. All rights reserved.

Furthermore, the immobilized enzymes could be recovered to reuse. Among enzyme immobilization methods, the most important and useful method is physical adsorption. Enzymes can be immobilized on the carrier through weak interactions (such as hydrogen bonding, electrostatic interactions, Van der Waals forces, etc.). The weak binding does not change the native structure of the enzyme. This prevents the active sites of the enzyme from disturbance and allows the enzyme to retain its activity [17]. However, this methodology has the risk of enzyme desorption from carriers. Therefore, it may be necessary to use highly activated supports to adsorb most enzymes [18,19]. In order to improve application of CA, CA was immobilized onto various support materials. For example, CA enzyme was immobilized in alginate beads, the immobilized CA showed better operational stability by retaining nearly 67% of its initial activity even after six cycles [20]. Bovine carbonic anhydrase (BCA) was covalently immobilized onto OAPS (octa(aminophenyl) silsesquioxane)-functionalized Fe3O4/SiO2 nanoparticles by using glutaraldehyde as a spacer [21]. The immobilized CA retained nearly 82% of its activity after 30 days. CA was immobilized within polyurethane (PU) foam. Thermal stability of immobilized CA was improved significantly [22]. However, the conventional supports for enzymes exhibit non-uniformity and long-range ordering from the atomic to microscale regime, and require harsh conditions and extended periods of time for either material preparation or enzyme immobilization, thus leading to low immobilization efficiency. Especially, the

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immobilized CA usually exhibits low CO2 sequestration efficiency due to no gas adsorption function for the conventional CA supports. Therefore, it is necessary to develop new supports with diversified structures and porosity. Metal organic frameworks (MOFs) are highly ordered crystalline materials in which metallic canters are bridged via organic multitopic ligands to yield low density network structures of diverse topologies [23,24]. Due to their large specific areas, identical and adjustable pore sizes, and excellent adsorption affinities, MOFs have been extensively studied for various applications, such as gas adsorption, separation, catalysis, and drug delivery [25,26]. Recently, some enzymes have successfully immobilized onto MOFs, for example, Cytochrome c [27], lipase [28], and catalase [29]. Among various MOFs, ZIF-8 is porous crystalline materials with open-framework zeolite structures, which composed of imidazolate-derived ligands and transition metal ions [30,31]. Furthermore, ZIF-8 was used to carry out the capture of CO2 due to its sodalite structure of large specific area and the kinetic diameter [32,33]. However, for industrialized application, the adsorption efficiency of pure ZIF-8 for CO2 is still low. As mentioned before, CA can accelerate the hydration of CO2. Therefore, we suppose that the separation performance for CO2 would be improved by immobilization CA in ZIF-8. Recently, successful immobilization of CA molecules into ZIF-8 with polyhedrons has been reported [34]. The resulting [email protected] composites exhibited excellent stability under extremely harsh conditions due to the protection of the enzyme by the frameworks. However, morphology of [email protected] composites has largely remained limited to polyhedrons with nanometer size and nanopore [35–37]. A lack of good size and morphological control over the [email protected] composites might limit their practical applications. Recent years, some researches demonstrated that particle morphology and crystal size of MOFs are the important factors for optimizing the performance of [email protected] composites [30,38]. Furthermore, in our previous work, a novel mesoporous [email protected] composite with cruciate flower-like morphology was prepared by embedding catalase molecules into uniformly sized ZIF-8 crystals instead of ZIF-8 with the standard rhombic dodecahedral morphology [30]. The resulting [email protected] composites with cruciate flower-like morphology exhibited 400% higher activity than that of [email protected] composites with standard rhombic dodecahedral morphology. These results showed that particle morphology and crystal size are the important factors for improving the catalytic performances of [email protected] composites. In this study, CA was immobilized for the first time onto a novel ZIF-8 with cruciate flower-like morphology instead of ZIF-8 with the standard polyhedron morphology. As we expected, the resultant [email protected] composites with cruciate flower-like shape exhibited higher activity than that of [email protected] composites with standard polyhedron morphology, and pretty capture performance for CO2. In addition, effects of precursors (Zn2+ ions and 2-methylimidazole) on the crystal morphology of ZIF-8 and [email protected] composites activity were systematically investigated. 2. Materials and methods 2.1. Chemicals and materials

Briefly, 2-methylimidazole solution (1 M, 10 mL) and zinc nitrate solution (0.1 M, 1 mL) were mixed together and stirring at room temperature for 30 min. White precipitates were collected after centrifugation and dried under vacuum at room temperature. For ZIF-8 nanoparticles with cruciate flower-like shape, 2-methylimidazole water solution (1 M, 10 mL) were added into Zn(NO3)2 water solution (0.5 M, 1 mL). After the mixture was stirred for 30 min, the product was collected by centrifuging, washed with DI water for three times and dried. For [email protected] ZIF-8 composites, a certain amount of the ZIF-8 (0.1 g) was immersed in 1 mL of CA solution (1 mg/mL) for 3 h with stirring at 25 °C. After centrifugation at 6,000 ×g for 10 min, the resulting precipitate was washed with DI water for three times, and lyophilized. In addition, effects of Zn (NO3)2 concentration (0.05–1.5 M) and 2-methylimidazole concentration (0.1 and 1 M) on the crystal morphology of ZIF-8 were investigated, respectively. 2.3. Characterization methods Scanning electron microscope (SEM) was taken by Hitachi S4800, and the acceleration voltage was 15 kV. Transmission electron microscope (TEM) images were obtained on JEOL JEM2100 operated at 120 kV. Fourier transform infrared (FTIR) spectra were obtained using a NEXUS870 infrared spectrometer (Thermo Nicolet Corporation, Madison, WI) using the standard KBr disk method. FT-IR measurements were conducted in the region of 400–4000 cm−1. Powder X-ray diffraction (PXRD) patterns were recorded using a X-ray powder diffraction (D/Max-2500 diffractometer, Shimadzu, Japan) at 40 kV and 40 mA. The elemental composition was obtained by using energy-dispersive spectrometer (EDS) (S2 Ranger, Bruker, Germany). Thermal gravimetric analysis (TGA) measurements were performed on SDT Q600 (TA Instruments-Waters LLC, USA). The samples were filled into an alumia crucibel and heated in a continuous flow of nitrogen gas with a ramp rate of 10 °C/min from 25 up to 800 °C. Confocal laser scanning microscopy (CLSM) was used to investigate the distribution of CA on ZIF-8. Prior to observation, the CA samples were mixed with fluorescamine solution (50 mg/mL, Fluorescamine was soluble in acetone) for 3 min to form highly fluorescent product by the reaction between primary amines in proteins and the fluorescamine [39]. CLSM observation was performed with a Leica TCS SP5 microscope (Leica Camera AG, Germany). The samples were excited at 390 nm and the emitted fluorescent light was detected between 460 and 480 nm. 2.4. CA activity assay The activities of free CA and immobilized CA were assayed as previously described by Zhang et al. [40]. Briefly, a certain amount of enzyme sample was added to a solution containing 4.6 mL of Na2HPO4–NaH2PO4 buffer solution (0.2 M, pH 7.0) and 0.2 mL of p-NPA (2 × 10−4 M). The CA enzyme's activity was determined by measuring the p-NP concentration in the hydrolysis solution in a 5 mL UV cuvette at 25 °C for 3 min, using a UV–visible spectrophotometer (Shimadzu UV-1800) at 400 nm wavelength. CA activity is defined as the hydrolysis of p-NPA per minute. The activity recovery of CA in [email protected] were calculated as given in Eq. (1):

Zinc Nitrate hexahydrate(Zn(NO3)2.6H2O) and 2-Methylimidazole were purchased from Adamas Reagent Co., Ltd. (Tianjin, China). 4Nitrophenyl acetate (p-NPA), p-nitrophenol (p-NP), and Carbonic Anhydrase from bovine (CA, EC4.2.1.1) were obtained from SigmaAldrich. All other chemicals and reagents are of analytical grade without further purification.

Activity recovery ð%Þ Total activity of [email protected]‐8ðUÞ ¼ Total free CA activity used for [email protected]‐8 productionðUÞ

2.2. Synthesis of the ZIF-8 and [email protected] composites

The enzyme amounts in solution before and after the immobilization measured with the Bradford method. The immobilization efficiency was defined as the ratio of CA amount absorbed in the [email protected] composites to the initial amount of CA. The immobilization efficiency was calculated

Traditional ZIF-8 nanoparticles with standard polyhedron morphology was synthesized by a modification of the procedure of Cui et al. [29].

ð1Þ

2.5. Protein concentration determination and immobilization efficiency

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added to the solution, the CA concentration of the supernatant, and the volume of the supernatant, respectively.

according to Eq. (2).

immobilization efficiency ð%Þ ¼

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m‐C1 V1  100 m

ð2Þ

where m (mg), C1 (mg/mL), and V1 (mL) are the mass of CA initially

2.6. The kinetic parameters The kinetic parameters (Km and Vmax) of free CA and [email protected] were measured by the Lineweaver-Burk double-reciprocal plot method of

Fig. 1. SEM images showing ZIF-8 obtained using various zinc nitrate concentrations at low concentration 2-methylimidazole (0.1 M): (a) 0.1 M, (b) 0.2 M, (c) 0.3 M, (d) 0.4 M, (e) 0.5 M, (f) 0.6 M, (g) 0.7 M, (h) 0.8 M. (i) 0.9 M. (j) 1.0 M.

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Michaelis-Menten Equation between 0.15 and 0.75 mM 4NPA concentrations at a constant enzyme concentration (0.01 mg/mL). Km and Vmax were calculated using the Lineweaver-Burk equation based on computed linear regression calculations. V max ½S V0 ¼ ðK m þ ½SÞ where V0 is the initial catalytic rate. Vmax is the maximum rate conversion. [S] is the initial substrate concentration, and Km is the MichealisMenten constant. 2.7. Thermal and chemical stability of [email protected] The time courses of thermal inactivation of free CA and the [email protected] composites were measured by incubating them in 0.2 mM NaH2PO4Na2HPO4 buffer solution (pH 7.0) without substrate at 60 °C for 10–60 min, and the enzyme samples were taken out at the indicated time points, the residual CA activities were determined by the same procedure as described above. In addition, free CA and [email protected] composites were further investigated for its stability with respect to the three chemical denaturants, urea (6 M), ethanol (60%, v/v) and sodium dodecyl sulfate (SDS, 2%, w/v). The residual activities of free CA and [email protected] were determined after incubating them in 0.2 mM NaH2PO4-Na2HPO4 buffer solution (pH 7.0) at 30 °C for 30 min.

The residual CA activity of each cycle was measured and calculated by taking the enzyme activity of the first cycle as 100%. 2.9. CO2 sequestration in CaCO3 For free CA, the sequestration of CO2 in the form of CaCO3 was performed in a 50 mL total reaction mixture containing 200 mM glycine sodium hydroxide buffer solution (pH 10.5) and 50 mg free enzyme samples. CO2 gas was introduced into the reaction mixture at a uniform flow rate. The reaction temperature was maintained at approximately 20 °C. The reaction mixture was continuously stirred at a constant rate for 30 min. CaCO3 precipitation was recovered by centrifugation. For [email protected], enzyme sample was similarly diluted using deionized water to have the same protein content as the 1.0× free enzyme. The sequestration of CO2 in CaCO3 was performed as above description. After one cycle of reaction, [email protected] was recovered by centrifugation, and washed with deionized water and resuspended in a fresh reaction mixture to carry out next conversion. After 20 cycles, the supernatant from all the 20 cycles was gathered, and a 10 mL CaCl2 (pH 10, 100 mM) solution was added into the collected supernatant. The precipitation reaction was performed for 2 min. The precipitated solids were collected by filtering through 0.2 um membrane filters (Millipore) and dried. For comparison, sequestration of CO2 into CaCO3 was also carried out by using free CA as described above.

2.8. Reusability of [email protected]

3. Result and discussion

The reusability of [email protected] for the hydrolytic application was evaluated. 0.5 mM 4NPA was added to 10 mL of NaH2PO4-Na2HPO4 buffer solution (pH 7.0) containing 50 mg of the [email protected] This reaction mixture was incubated at 25 °C for 5 min to hydrolyze 4NPA. Upon completion of one cycle, the immobilized enzyme was then separated by centrifugation. The recovered [email protected] was washed three times with deionized water and then suspended again in a fresh reaction mixture.

3.1. Effects of 2-methylimidazole and zinc nitrate concentration on the ZIF8 with cruciate flower-like shape Although ZIF-8 has been used as a excellent support for enzyme immobilization, shape control in ZIF-8 still remains a challenge [41,42]. Moreover, current regulation of ZIF-8 morphology is mainly achieved by employing various additives during the synthesis [43–45]. Recently,

Fig. 2. Schematic illustration of the synthesis of [email protected] and conversion of carbon dioxide into CaCO3.

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we demonstrated that the crystal morphology of ZIF-8 had primary dependence on concentrations of 2-methylimidazole and Zn2+ ions, and could be directly controlled by adjusting concentrations of Zn2+ ions while keeping the high concentration of 2-methylimidazole [30]. However, influence of 2-methylimidazole and zinc nitrate concentration on the ZIF-8 with cruciate flower-like shape still remain poorly understood. To further understand the effect of 2-methylimidazole and zinc nitrate, in this study, the experiments were conducted with low/high concentration of 2-methylimidazole (0.1 M/1 M), and zinc nitrate concentration were varied from 0.05 M to 1.25 M. The results showed that nanosheets appeared at low concentration of 2-methylimidazole (0.1 M) and zinc nitrate concentration (0.1 M and 0.2 M) (Fig. 1a and b). Upon the increase zinc nitrate concentration to 0.3 M, inhomogenous polyhedrons were observed (Fig. 1c and d). A further increase in the zinc nitrate concentration (0.5 M) lead to mixed ZIF-8 crystals of polyhedrons and nanosheets (Fig. 1e and f). With the increase in the zinc nitrate to 0.7 M and 0.8 M, 2D multilay nanosheets structure (Fig. 1g and h) was formed. High zinc nitrate concentrations (0.9 M and 1.0 M) promoted the synthesis of ZIF-8 crystals but with heterogeneous sizes (Fig. 1i and j), indicating a low crystallization level. Furthermore, no uniform truncated rhombic dodecahedral particles with the

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typical ZIF-8 topology were obtained at low concentration of 2methylimidazole (0.1 M) when zinc nitrate concentration were varied from 0.1 M to 1.0 M. On the contrary, uniform rhombic dodecahedral morphology with the typical ZIF-8 topology were observed at high concentration of 2-methylimidazole (1 M) when zinc nitrate concentration were varied from 0.05 M to 0.3 M (Fig. S1a–d). An increase in the concentration of zinc nitrate to 0.5 M resulted in flowerlike polyhedra instead of cruciate flower-like (Fig. S1e and f). With increasing zinc nitrate concentrations to 0.6 M, ZIF-8 with cruciate flower-like started to appear (Fig. S1g). A further increase in the zinc nitrate concentration to 0.8 M lead to integrated ZIF-8 with cruciate flower-like shape (Fig. S1i). Changing the zinc nitrate concentration between 0.9 M and 1.25 M caused the formation of more integrated ZIF-8 with cruciate flower-like shape (Fig. S1j–l). These results indicated that the 2methylimidazole concentrations have strong influence on the morphology of ZIF-8 crystals. Moreover, the shape of ZIF-8 could be controlled by adjusting concentrations of Zn2+ ions at the high concentration of 2methylimidazole (1 M). PXRD measurements (Fig. S2) also confirmed that the crystalline form of ZIF-8 with cruciate flower-like are gradually grown with the increase of zinc nitrate concentration at high concentration of 2-methylimidazole (1 M). When zinc nitrate concentration was

Fig. 3. SEM images of (a) [email protected] with cruciate flower-like morphology and (b) [email protected] with standard rhombic dodecahedral morphology; TEM images of (c) [email protected] with cruciate flower-like morphology and (d) [email protected] with standard rhombic dodecahedral morphology.

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maintained at low concentration (between 0.2 M and 0.3 M), the product exhibited the notable peak of dia(Zn) for ZIF-8. However, with increasing zinc nitrate concentration to 0.6 M, a cruciate flower-like structure with an unidentified XRD pattern was observed. This result was quite consistent with the our previous report that the increase of zinc nitrate concentration at high concentration of 2-methylimidazole (1 M) significantly accelerated the nucleation process of ZIF-8 crystals, resulting in crystal growth in all directions and yielding larger, wellintergrown crystals with cruciate flower-like shape [30]. Similar results were also found by the previous report [46,47]. 3.2. Synthesis and characterization of the [email protected] composites with cruciate flower-like shape The preparation of the [email protected] composites with cruciate flowerlike shape was shown schematically in Fig. 2. First, the ZIF-8 with cruciate flower-like shape were synthesized by mixing 2-methylimidazole

solution (1 M, 10 mL) and zinc nitrate solution (0.6 M, 1 mL) together and stirring at room temperature, and then the obtained ZIF-8 particles were mixed with CA solution for 3 h. After centrifugation and washing, the [email protected] composites were obtained. The SEM images showed that these [email protected] composite particles exhibited cruciate flower-like shape instead of traditional ZIF-8 nanoparticles with standard polyhedron morphology (Fig. 3a and b). The TEM images of the [email protected] composites also exhibited the same cruciate flower-like morphology (Fig. 3c and d). The ZIF-8 with cruciate flower-like structure was also observed in some reports [29,48]. The formation of the cruciate flower-like structure may be due to the fact that the two ZIF-8 nanoparticles assemble simultaneously during the growth of nanocrystals. FTIR spectrum revealed that stretches characteristic at 1500–1550 cm−1 (1539 cm−1) in the [email protected] composite particles was ascribed to amide II in proteins (Fig. 4b) [49], whereas, the same band was not observed in ZIF-8, indicating the presence of CA in the composites. The absorption band observed at 2934 cm−1 was ascribed to the aliphatic

Fig. 4. Confocal microscope images of (a) [email protected] with cruciate flower-like morphology; FT-IR spectra analysis of (b) [email protected] with cruciate flower-like morphology; TGA curves of [email protected] ZIF-8 with cruciate flower-like morphology.

S. Ren et al. / International Journal of Biological Macromolecules 117 (2018) 189–198 Table 1 Comparison of apparent kinetic parameters of free CA and [email protected] Enzyme

Km (mM)

Vmax (mM/min)

Free CA [email protected] with cruciate flower-like shape [email protected] with standard polyhedron morphology

0.24 0.25 0.43

3.29 3.23 2.68

C\\H stretch of the imidazole, the band at 1593 cm−1 was attributed to the C_N stretching vibrations, indicating the presence of imidazole in the composites [50,51]. Furthermore, two distinct weight loss steps were observed for the [email protected] composite particles by TGA (Fig. 4c). The first weight loss step occurred at 200–400 °C because of the loss of CA protein. The second weight loss step was observed at 500–800 °C, possibly due to decomposition of ZIF-8. In addition, fluorescence micrograph further confirmed immobilization of CA onto ZIF-8 by using fluorescein isothiocyanate (FITC) labeled CA protein because the labeled CA protein exhibited strong green fluorescence (Fig. 4a). The results revealed that [email protected] particles were biocomposites that are composed of CA and ZIF-8. 3.3. The catalytic activity and stability of the [email protected] composites with cruciate flower-like To improve the immobilization efficiency and enhance the stability of adsorbed enzymes, much efforts have been made in exploring

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satisfying supports with controllable structure and properties [52–54]. In this study, to explore the catalytic activity of [email protected] composites, different morphologies of ZIF-8 such as cube, sphere and cruciate flower-like shape were prepared with high concentration of 2methylimidazole (1.0 M) and different zinc nitrate concentration (0.05 M–1.5 M), and used as solid supports for CA adsorption. The adsorption capacity (immobilization efficiency) of CA on different ZIF-8 samples was shown in Fig. 3S. It was found that all ZIF-8 exhibited high adsorption capacity for CA. However, the prepared [email protected] composite with cruciate flower-like shape at 0.6 M zinc nitrate concentration showed the maximum activity recovery (75%). In contrast, the prepared [email protected] composite with traditional standard polyhedron morphology at 0.05 M zinc nitrate concentration showed the minimum activity recovery. Furthermore, all [email protected] composite with cruciate flower-like shape displayed higher activity recovery than the [email protected] composite with traditional polyhedron morphology (Fig. S4). The similar results were also observed in the previous reports [30]. In addition, we determined the enzymatic Michaelis-Menten kinetics of CA adsorbed on the [email protected] composites. The results showed that the [email protected] composite with cruciate flower-like shape and free CA exhibited the similar apparent Km values (Table 1), indicating that the ZIF-8 did not cause the increased mass transfer limitation for substrates. Furthermore, apparent Km values of [email protected] composite with cruciate flower-like shape are lower than that of [email protected] composite with standard rhombic dodecahedral morphology, suggesting a higher substrate affinity of [email protected] composite with standard polyhedron morphology

Fig. 5. Stability of free CA and [email protected] with cruciate flower-like morphology; (a) thermostability and (b) stability against chemical denaturants; (c) reusability of [email protected] with cruciate flower-like morphology; (d) comparison of conversion of carbon dioxide into CaCO3.

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compared to [email protected] composite with cruciate flower-like shape. Furthermore, the apparent Vmax value of immobilized CA was lower than that of free CA. It might attribute to the substantial secondary structural perturbation and changes on the enzyme surface upon adsorption [55,56]. It was worth noting that the catalytic efficiency (Vmax/Km) of [email protected] composites was higher than that of free CA (Table 1), which indicated that the adsorbed CA had relatively high catalytic efficiency. The similar results were also observed in previous reports [57]. The difference could be due to the conformational changes of enzymes and/or the partition of substrates between the ZIF-8 and buffer [58,59]. Catalytic stabilities play an important role in a enzymatic reaction. Thus, the thermal stability of the immobilized CA was studied. Fig. 5a showed the thermal stability of the immobilized and the free CA. The activities of the free CA and immobilized CA declined as reaction time increased. It is interesting to note that free CA lost its most activity at 60 °C for 10 min, whereas the immobilized CA retained 40% of its original activity at the same conditions. The results presented that CA immobilized on the ZIF-8 owned a higher thermal stability than that of the free CA. The adsorption of the enzyme on the ZIF-8 might have reduced the molecular mobility and change in the CA conformation, which helped to retain enzyme activity at a higher temperature [60,61]. A similar phenomenon was also observed while assessing the stability of CA against chemical denaturants (Fig. 5b). For example, [email protected] composites still retained 93% of its initial activity in 2%SDS for 30 min. However, free CA only retained 8% of initial activity. Likewise, the improved activity and stability were also observed in the [email protected] composites with cruciate flower-like morphology in our previous study [30]. These results indicated that ZIF-8 with cruciate flower-like shape was a suitable carrier for enzyme immobilization. 3.4. Reusability of [email protected] and conversion of CO2 to CaCO3 Enzyme reusability is an essential fundamental parameter for the CO2 capture application in a power plant since it can reduce the cost

of the enzyme driven processes [62,63]. Therefore, the reusability of the [email protected] composites was tested by performing several consecutive operating cycles using 0.5 mM 4NPA solution as the substrate. The [email protected] ZIF-8 composites were collected through centrifugation after each reaction batch, rinsed with water, and utilized for the next reaction cycle. The reusability was defined as the ratio of the activity for the [email protected] composites after recycling to its initial activity. The results showed that the activity of the [email protected] composites with cruciate flower-like shape still retained 85% of its original activity after 9 cycles (Fig. 5c). Furthermore, we found that [email protected] composites with traditional polyhedron morphology lost its most activity after 5 cycles. These results indicated that the [email protected] composites with cruciate flower-like shape have better reusability and potential use in industrial applications. Next, to explore the capture capacity of [email protected] for CO2, we performed the conversion of CO2 into CaCO3 using [email protected] composites. Because CA is known to accelerate only the rate of CO2 hydration, and not to affect the equilibrium of the carbonate system [64]. We expected that the yields of CaCO3 solid obtained by [email protected] composites would be more than that of free CA. The yields of the CaCO3 solid obtained by using [email protected] composites and free CA were shown in Fig. 5d. The results showed that the yields of the CaCO3 solid obtained by using [email protected] ZIF-8 composites were 22-folds compared to free CA. Moreover, free enzyme in solution was difficult to recycle without the operational stability. Such results indicated that [email protected] composites with cruciate flower-like shape had higher efficiency in the conversion of CO2 into CaCO3 than free CA. In addition, to characterize the converted CaCO3, solid precipitants obtained by using free CA and [email protected] composites were identified by SEM, PXRD, and EDS. The results showed that the crystal structures of the resulting precipitates formed by the different catalysts were not significantly different from each other, being mainly comprised of spherical vaterite with diameters ranged from 5 to 10 μm (Fig. 6a and b). PXRD patterns demonstrated that the crystal structures of the resulting precipitants formed by free CA and [email protected] composites were vaterite crystals (Fig. S5a and b). Furthermore, EDS experiment further demonstrated that that the resulting precipitates formed by free CA and [email protected] composites were CaCO3 catalysts (Fig. S6a and b). The results demonstrated that the [email protected] composites could be successfully applied to CO2 sequestration in CaCO3 as an efficient accelerator without affecting the polymorphs of the resulting precipitates. 4. Conclusions In summary, we successfully synthesize a new [email protected] composites with cruciate flower-like morphology instead of ZIF-8 with standard polyhedron morphology. The [email protected] composites exhibited significant improvement in enzyme properties, including activity recovery, catalytic efficiency, thermostability, reusability, and tolerance towards denaturants. Furthermore, we demonstrated for the first time that the crystal morphology of ZIF-8 had primary dependence on concentrations of 2-methylimidazole. More importantly, the [email protected] composites with cruciate flower-like morphology showed efficient application in the sequestration of CO2 into carbonate minerals. These results suggested that the [email protected] composite with cruciate flower-like morphology can be efficiently applied to biomimetic CO2 sequestration. Acknowledgment

Fig. 6. SEM images of calcium carbonate crystals obtained in the presence of (a) Free CA and (b) [email protected] with cruciate flower-like morphology.

This work is partially supported by the National Natural Science Foundation of China (project no. 21676069). Dr. J. D. Cui also thanks supports from the Natural Science Foundation of Hebei Province, China (project no. B2018208041), the Program for Hundreds of Outstanding Innovative Talents in Hebei province (III) under the grant number of SLRC2017036, and the Foundation (No. 2016IM001) of Key Laboratory of Industrial Fermentation Microbiology of Ministry of Education and Tianjin Key Lab of Industrial Microbiology (Tianjin University of Science & Technology).

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Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2018.05.173.

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