Preparation of a Flowerlike Nanobiocatalyst System via Biomimetic

Research Note pubs.acs.org/IECR

Cite This: Ind. Eng. Chem. Res. 2017, 56, 14923−14930

Preparation of a Flowerlike Nanobiocatalyst System via Biomimetic Mineralization of Cobalt Phosphate with Enzyme Yang Song, Jing Gao, Ying He, Liya Zhou, Li Ma, Zhihong Huang, and Yanjun Jiang* School of Chemical Engineering and Technology, Hebei University of Technology, 8 Guangrong Road, Tianjin, 300130, People’s Republic of China S Supporting Information *

ABSTRACT: This study reported a novel and facile method for the spontaneous synthesis of flowerlike cobalt phosphate nanocrystals (Co3(PO4)2 nanoflowers) without adding any templates, and the growth mechanism was further studied. Subsequently, an excellent nanobiocatalyst system was established via the biomimetic mineralization of cobalt phosphate with Co-type nitrile hydratase (NHase). Because of the interactions between Co ions in the microenvironment of cobalt phosphate and enzyme active site, the encapsulated NHase (NHase@Co3(PO4)2) exhibited high catalytic efficiencies and desirable stabilities. The enzymatic activity of encapsulated NHase was 238.4 U/g and the protein loading amount was 210.73 mg/g. Compared with free NHase, the optimum temperature of NHase@Co3(PO4)2 was 40 °C, which was higher than that of the free NHase (30 °C). The thermal, pH, antiproteolytic, and storage stability of NHase@Co3(PO4)2 were all improved. Furthermore, NHase@Co3(PO4)2 could be applied in the production of nicotinamide (NAM) with a satisfying yield and it could be reused seven times. All these results in this work clearly confirmed that the cobalt phosphate nanocrystals, as an original nanocarrier, would have promising applications for constructing nanobiocatalyst systems.

1. INTRODUCTION Enzymes are recognized as the best present from natural biosome and are endowed with ultrahigh catalytic activity, specificity, and selectivity. However, enzymes will face harsh conditions, including high temperature, strong acid and alkali, etc., if they are applied directly into the industrial process. Hence, immobilized enzyme, which can improve enzyme stability and recycle several times for industrial process, has been widely used and studied.1−3 Traditional methods are generally immobilizing target enzymes on the pre-existing solid carriers via adsorption, cross-linking, or covalent binding approach.4,5 Recently, biomimetic mineralization has been considered as an outstanding process to fabricate advanced materials with diverse morphologies,6−9 and it is worth mentioning that the gel diffusion is an excellently controllable method for mimetic biomineralization. 10−15 Thus, the application of a biomineralization process in enzyme immobilization has gained much attention, because of the particularly mild reaction conditions and ultrahigh enzyme activity recovery.16−18 Moreover, thanks to the mild process of biomineralization, enzyme−inorganic hybrid composites could provide reliable stabilization effect for incorporated enzymes and let them exhibit plentiful biological functions. Currently, the biomineralization of metal phosphates has been proven to be an outstanding approach to synthesize nanobiocatalysts. Ge et al. took the lead in synthesizing copper phosphate nanoflowers with the immobilization of enzymes, which exhibited pretty appearance and excellent catalytic properties.19 Subsequently, a series of hybrid bioinorganic nanoflowers was © 2017 American Chemical Society

created and various biocatalyst systems were established, based on the metal-phosphates biomineralization.20−26 Nevertheless, studies about the preparation of cobalt phosphate nanoflowers for immobilizing enzyme have rarely been reported. LópezGallego et al. had reported that cobalt phosphate sponges were only formed in the presence of ploy-histidine tagged proteins at their N-terminus, and they believed that the His-tag would selectively drive the mineralization, acting as a nucleation site for Co3(PO4)2.24,27 Similarly, Hu et al. had also synthesized flower-like hierarchical Co3(PO4)2·8H2O via the microwaveassisted method, and the flowerlike shape could appear only by using hexamethylenetetramine (HMTA) as a template;28 Kim et al. presented a protein-directed assembly method to prepare BSA−cobalt phosphate hybrid flowers and demonstrated that BSA worked as the template to interact with cobalt and phosphate.29 However, different from the above studies, we found that the cobalt phosphate crystals could autogenously assemble into a flowerlike shape in a mild aqueous solution without adding any organic macromolecule as the assistant template. To our knowledge, this is the first time that Co3(PO4)2 nanoflowers were synthesized just depending on the cobalt salt and phosphate buffer solution (PBS). In addition, to establish an efficient nanobiocatalyst system, the component of excellent enzyme is also an essential part in Received: Revised: Accepted: Published: 14923

September 14, 2017 November 25, 2017 December 5, 2017 December 5, 2017 DOI: 10.1021/acs.iecr.7b03809 Ind. Eng. Chem. Res. 2017, 56, 14923−14930

Research Note

Industrial & Engineering Chemistry Research

acetonitrile/water (3:7, v/v), and the flow rate was 1 mL/min. The UV detector absorbance wavelength was fixed at 230 nm. 2.3. Characterization. Scanning electron microscopy (SEM) images were recorded on a FEI NanoSEM450 microscope. Fourier-transform infrared (FT-IR) spectra were recorded on a Bruker Vector 22 FT-IR spectrophotometer using the KBr pellets method. Powder X-ray diffraction (XRD) patterns were recorded using a Bruker AXS D8 Discover X-ray diffractometer with a Cu Kα anode (λ = 0.15406 nm) at 40 kV and 40 mA. Confocal laser scanning microscopy (CLSM) images were taken with a Leica TCS SP5 confocal microscope with excitation wavelength of 488 nm and emission wavelength of 525 nm. The thermogravimetric analysis (TGA) results were measured with a Mettler Toledo TGA/DSC 1 thermogravimetric analyzer, under N2 atmosphere and a heating rate of 10 °C/min.

the entire process. Nitrile hydratase (NHase; EC 4.2.1.84), as a soluble metalloenzyme, has been considered as an excellent biocatalyst to efficiently catalyze the hydration of a nitrile to an amide.30−32 In recent years, NHase has been successfully used in the production of acrylamide (AM), nicotinamide (NAM), 5-cyanovaleramide, etc.30 However, free NHase is always too fragile to have a wonderful tolerance against extreme industrial environments, leading to the limitation of the industrial applications. There are two types of NHase: one contains the Fe ion at its catalytic center, and the other has the Co ion at its catalytic center.33−35 In this work, the Co-type NHase, which contains a noncorrin Co ion, was selected as the model enzyme for immobilization via the cobiomimetic mineralization process with cobalt phosphate. In the preliminary jar test, the enzymatic activity of free NHase was obviously increased after adding a hint of cobalt salts, which could be attributed to the interactions of the Co ion between Co-type NHase and the microenvironment of Co3(PO4)2. Based on this, taking the advantage of the cobalt phosphate nanocrystals to immobilize Co-type NHase will be a better way to obtain a quite stable and superb biocatalyst.

3. RESULTS AND DISCUSSION In a typical synthesis, the rigid granules of Co(NO3)2·6H2O were directly added into the PBS instead of the aqueous solution of Co(NO3)2, which was different from the method previously proposed.19−26 The entire process could be considered that the Co(NO3)2 was dissolved in the phosphate buffer solution first and then spontaneous crystallized into cobalt phosphate nanoflowers. Hence, a preliminary formation mechanism for the flowerlike Co3(PO4)2 nanocrystals was speculated, and the schematic diagram is proposed in Figure 1a.

2. EXPERIMENTAL SECTION 2.1. Preparation of Flowerlike Co3(PO4)2 Nanocrystals and Immobilized NHase. 2.1.1. Preparation of Pure Co3(PO4)2 Nanoflowers. In a typical synthesis, 0.291 mg of Co(NO3)2·6H2O was added to a flask with well-prepared phosphate buffer solution (10 mM, 100 mL, pH 7.4), and the molar ratio of Co2+ to PO43− was 1:1. After fully oscillating, cobalt nitrate was dissolved rapidly and the solution turned purple, while purple floc began to precipitate at the same time. The precipitates then were generated and incubated at 25 °C for 48 h. Finally, the products (pink precipitates) were obtained by centrifugation at 6500 rpm for 5 min and then washed with ultrapure water three times to remove the impurity. The product was dried at 50 °C for 10 h and then the flowerlike Co3(PO4)2 nanocrystals were successfully synthesized. 2.1.2. Preparation of NHase@Co3(PO4)2. To fabricate immobilized NHase, 10 mL NHase (2 mg/mL) was added to 90 mL well-prepared phosphate buffer solution (10 mM, pH 7.4), and then 0.291 mg of Co(NO3)2·6H2O was added subsequently. In accordance with the experimental operation described above, the mixture was incubated at 25 °C for 48 h. The immobilized NHase (NHase@Co3(PO4)2) was finally obtained by centrifugation at 6500 rpm for 5 min and washed with ultrapure water three times. 2.2. Enzyme Activity Assay. 2.2.1. Definition of Activity. One unit of activity (U) was defined as the amount of NHase that catalyzed acrylonitrile converting to 1 μmol acrylamide per minute. Relative activity was a percentage, representing the ratio of observed activity to the initial activity (the maximum activity). 2.2.2. Activity Assay Method. In a typical test tube, 1 mL of free NHase or suspension of immobilized NHase was incubated at 30 °C for 5 min, and then 1 mL of acrylonitrile solution (250 mM) was added and the reaction was initiated under magnetic stirring conditions (200 rpm) for 5 min. Sequentially, 200 μL of NaOH (2 M) was added to stop the reaction. The resultant of acrylamide was determined by high-performance liquid chromatography (HPLC), which was performed with an Agilent 1200LC system equipped with an Eclipse Plus C18 column (5 μm, 4.6 mm × 250 mm). The mobile phase was

Figure 1. (a) Schematic illustration of the crystallization process of cobalt phosphate nanoflowers. (b) SEM of Co3(PO4)2 nanoflowers. (c) SEM of NHase@Co3(PO4)2 (scale bars of all SEM images = 10 μm).

Once the granules of Co(NO3)2·6H2O were contacted with PBS, they would be dissolved rapidly and release plenty of Co2+ around them. In the meantime, the free PO43− in PBS would combine with the Co ion as much as possible to self-assemble into Co3(PO4)2 nanosheets. As the time was prolonged, there was continued growth of crystals, based on the previous 14924

DOI: 10.1021/acs.iecr.7b03809 Ind. Eng. Chem. Res. 2017, 56, 14923−14930

Research Note


Figure 2. (a) XRD of Co3(PO4)2 nanoflowers and NHase@Co3(PO4)2, (b) FT-IR spectra of Co3(PO4)2 nanoflowers and NHase@Co3(PO4)2, (c) TGA curve of Co3(PO4)2 nanoflowers and NHase@Co3(PO4)2, and (d) CLSM image of NHase@Co3(PO4)2.

NHase was successfully immobilized in cobalt phosphate crystals. This phenomenon could be interpreted as the biomineralization in nature, where organic macromolecules such as protein could play a pivotal role in determining the morphology of composites.36 Organic macromolecules could stabilize the transient phases, influence the shape, and overcome the intrinsic brittleness of the crystalline phases.37 Furthermore, the growth process of pure Co3(PO4)2 nanoflowers and NHase@Co3(PO4)2 in different periods (Figure S1 in the Supporting Information) was also investigated, which was very consistent with the schematic illustration in Figure 1a. For NHase@Co3(PO4)2, obvious naonosheets could be easily observed in the first 24 h, and there was a tendency of continued growth. With an increasing time, different from the pure Co3(PO4)2, NHase@Co3(PO4)2 gradually turned more circular and the petals were centralized together from every direction, which could be attributed to the influence of enzymes. The NHase@Co3(PO4)2 did not stabilize until 48 h later and the morphologies finally remained constant. Besides, several characterization methods were used to confirm that NHase had been indeed incorporated in cobalt phosphate crystals. First, energy-dispersive X-ray analysis (EDAX) was used to analyze the elemental composition (Figure S2 in the Supporting Information). Obviously, there were two additional elements in NHase@Co3(PO4)2namely, carbon and nitrogenthat were not available in pure cobalt phosphate nanoflowers, verifying the success of enzyme immobilization. N2 adsorption−desorption analysis was also performed, and the results are shown in Figure S3 in the Supporting Information. As can be seen from the N 2 adsorption−desorption isotherms, both the pure Co3(PO4)2 nanoflowers and NHase@Co3(PO4)2 were representative type-

nanosheets and gradually turned more substantial and plentiful. Finally, the Co3(PO4)2 nanoflowers were generated maturely, according to the SEM images shown in Figure 1a. Therefore, the initiation of crystallization was attributed to the enrichment of local concentration of Co2+ released by dissolution and could spontaneously formed Co3(PO4)2 nanoflowers without any assistant templates. In contrast, all of the previous reported methods were a mixture of two solutions,19−26 where the concentration distribution was pretty uniform, so the proteins were needed as a template to direct the nucleation and growth of hybrid nanoflowers. Surprisingly, in this work, the Co3(PO4)2 nanoflowers could be fabricated by just depending on the cobalt salt and phosphate buffer solution (PBS), and the forming process was started with Co3(PO4)2 nanosheets and gradually self-assembled into flowers, rather than the assistant nucleation of proteins. After that, the nanobiocatalyst system was fabricated via the biomineralization of Co3(PO4)2 with NHase. As shown in Figures 1b and 1c, the surface morphologies of both pure Co3(PO4)2 nanoflowers and NHase@Co3(PO4)2 were characterized by SEM. Figure 1b showed that the flowerlike Co3(PO4)2 crystals had a very uniform particle size of 20 μm. In addition, from the enlarged image, one could clearly observe that each petal of the cobalt phosphate nanoflowers was utterly smooth and dense. Figure 1c shows the immobilized NHase (NHase@Co3(PO4)2), which presented daisylike shapes 15 μm in diameter. Different from the pure Co3(PO4)2 nanoflowers, the crystal morphology of NHase@Co3(PO4)2 had been changed notably, because of the addition of enzyme in the process of biomineralization. Moreover, the surfaces of NHase@Co3(PO4)2 were completely rougher and cruder than that of the pure Co3(PO4)2, which demonstrated that 14925


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Figure 3. (a) Thermal stability of free NHase and NHase@Co3(PO4)2 at 45 and 55 °C. (b) pH stability of free NHase and NHase@Co3(PO4)2 at pH 4.0 and 10.0. (c) Antiproteolysis tolerance of free NHase and NHase@Co3(PO4)2 incubated in trypsin. (d) Lineweaver−Burk plots for determination of apparent kinetic parameters of free NHase and NHase@Co3(PO4)2.

which demonstrated that the addition of enzymes did not affect the main crystalline structure of Co3(PO4)2. In addition, the samples of Co3(PO4)2 nanoflowers and NHase@Co3(PO4)2 were also characterized by Fourier transform infrared (FT-IR) spectrometry, and the spectra are shown in Figure 2b. Compared with the pure cobalt phosphate, there were several extra characteristic absorption peaks in the spectrum of NHase@Co3(PO4)2. The peaks appeared at 1540, 2066, and 2962 cm−1 were the absorption peaks of −NH2 (bending vibration), −CN (stretching vibration), and −CH3 (stretching vibration), respectively, which could verify the enzyme’s incorporation. Similarly, the TGA results demonstrated the existence of NHase, based on the difference between those two relative gravities (%). As shown in Figure 2c, when the temperature rose above 100 °C, there would be a sudden slope that is due to the loss of crystalline water. The curve of pure Co3(PO4)2 crystals then had a tendency to be steady. However, the curve of NHase@Co3(PO4)2 fell again with the range of 200−350 °C, because of the decomposition of proteins, which confirmed the successful incorporation of NHase. Moreover, according to the TGA curves, the content of protein was calculated to be 13.7 wt %, which was roughly consistent with the value previously measured by Bradford assay (17.67 wt %). Furthermore, the CLSM image was obtained by making enzymes labeled with fluorescein isothiocyanate (FITC) to prepare immobilized enzyme (Figure 2d). The fluorescence from the images was clearly visible and the surface morphologies could be easily observed, definitely indicating that the enzyme had already been immobilized in the composites.

IV curves, which were the characteristic of mesoporous structure.38 According to the inset of corresponding pore size distribution, the average pore size of Co3(PO4)2 nanoflowers was 5.88 nm and the NHase@Co3(PO4)2 was 4.37 nm, which calculated by using the Barrett−Joyner−Halenda (BJH) model. The cumulative volumes of pores for Co3(PO4)2 nanoflowers and NHase@Co3(PO4)2 were calculated to be 0.0544 cm3/g and 0.0355 cc/g. The decreased pore size and pores volume of NHase@Co3(PO4)2 also confirmed that the enzymes were immobilized in the Co3(PO4)2 nanoflowers successfully. Besides, zeta potentials had been considered to have an important role in selecting the optimum matrix for enzyme immobilization.20,39−41 In this work, the zeta potential of pure Co3(PO4)2 nanoflowers and NHase@Co3(PO4)2 were measured. The results indicated that the zeta potential value of Co3(PO4)2 nanoflowers was approximately +3.5 mV, and the zeta potential value of NHase@Co3(PO4)2 was −1.7 mV under the neutral conditions. It was known that the isoelectric point of NHase was ∼5.5 and it could be negative under the neutral conditions.42,43 It could be obviously observed that the zeta potential of NHase@Co3(PO4)2 showed a tendency to be negative, because of the addition of NHase. When the matrix and enzyme were oppositely charged, it would have a maximal binding efficiency between the protein and matrix.39 These results further confirmed that the Co3(PO4)2 could be an appropriate matrix for immobilizing NHase. Meanwhile, the samples were characterized by X-ray diffraction (XRD) and the results were corresponded well with the Joint Committee on Powder Diffraction File data for Co3(PO4)2·8H2O (File No. 330432), as shown in Figure 2a. The peaks of Co3(PO4)2 nanoflowers and NHase@Co3(PO4)2 were almost the same, 14926


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environment.48,49 In addition, the stability of antiproteolytic digestion was also examined, as shown in Figure 3c. When they were incubated in the trypsin buffer solution, the residual activity of free NHase was only 44.2% after 1 h, but NHase@ Co3(PO4)2 still remained the equal activity as much as the initial value. It was believed that the Co3(PO4)2 minerals had trapped the enzyme in the interior of particles instead of being bound on the surface, and then it could prevent the NHase from proteolytic digestion.50 All these stabilities had reflected the superiority of NHase@Co3(PO4)2, which was promising for industrial applications with high enzymatic activity and improved stability. The kinetic parameters of free NHase and immobilized NHase were also studied and calculated on the basis of the Lineweaver−Burk plot method. As shown in Figure 3d, the data were fitted well and the kinetic parameters were easily obtained. As shown in Table 1, the Km value of NHase@Co3(PO4)2 was

Several catalytic properties of immobilized NHase (NHase@ Co 3 (PO 4 ) 2 ) and free NHase were investigated. First, corresponding to the SEM images in Figure S1, the effect of immobilized time on the specific activity and the loading amount of NHase was studied (Figure S4 in the Supporting Information). With the time prolonging, the catalytic activity and the loading amount of NHase were both gradually increased. When the time was extended to 48 h, the enzymatic activity reached the highest value of 238.4 U/gimmobilized NHase, and the corresponding protein loading amount was 210.73 mg/ gimmobilized NHase. Besides, the effects of different temperature and pH on the activity of free NHase and NHase@Co3(PO4)2 were also investigated (Figures S5 and S6 in the Supporting Information). As shown in Figure S5, the optimum temperature of free NHase was 30 °C. After the process of immobilization, the NHase@Co3(PO4)2 was protected by Co3(PO4)2 minerals and the optimum temperature rose to 40 °C. Similarly, pH had a great impact on enzymatic activities as well, and the extreme pH could make the enzyme inactivated. Figure S6 shows that the optimum pH of free NHase was 7.0 and the enzymatic activities were primarily lower under acidic and alkaline conditions. However, the optimum pH of NHase@ Co3(PO4)2 was shifted to 6.0, which was due to the electrostatic interactions between the matrix and solution, and then leading to the unequal partitioning of H+ and OH− concentrations in microenvironment and bulk solution.44,45 In addition, it was worth mentioning that the NHase@Co3(PO4)2 presented the insensitivity to pH according to the trend of curves. Thanks to the tight protection of Co3(PO4)2 minerals, the immobilized NHase would be possibly applied to the wider range of pH environment. Stabilities (thermal, pH, operational stability, etc.) were pretty important parameters for evaluating immobilized enzyme. Higher temperature could effectively improve the reaction rate, but the excessive temperature would destroy the structure and conformation of the enzyme. As shown in Figure 3a, the thermal stability of free NHase was pretty unsatisfactory under high temperature. When the free NHase was incubated in PBS solution at 45 °C for 30 min, the enzymatic activity rapidly decreased to 30.7% of the initial activity. When the temperature rose to 55 °C, the residual activity of free NHase remain at only 10.3% after being incubated for 1 h. However, NHase@Co3(PO4)2 could still retain 84.2% and 66.5% of original activity after being incubated for 1 h at 45 and 55 °C, respectively. The improved stability of NHase@Co3(PO4)2 was attributed to the stable interactions between enzymes and cobalt phosphate minerals, which prevented the NHase from inactivation due to overheating. Our present finding is similar to the previous results obtained from other protein−inorganic hybrid nanoflowers system. Ge et al. had reported that the copper-containing enzyme incorporated in copper phosphate nanoflowers exhibited much higher catalytic activities and stabilities.19,36,46,47 Similarly, the pH stability of free NHase was also too terrible to resist the strong acid or alkali environment (Figure 3b). In the first 30 min, the activity of free NHase had lost nearly one-half of the initial activity but NHase@ Co3(PO4)2 maintained 95.3% of the original activity. After being incubated for 5 h, 75.6% of the residual activity for NHase@Co3(PO4)2 in pH 4.0 and 63.9% in pH 10.0 could be maintained, respectively, while free NHase lost almost all activity. Consistent with the results of Figures S5 and S6, the immobilized NHase was able to stabilize the conformation of enzymes and retain the activity when facing the extreme

Table 1. Kinetic Parameters of Free NHase and NHase@ Co3(PO4)2 free NHase NHase@Co3(PO4)2

Km (mM)

Kcat (S−1)

Kcat/Km (L mmol S−1)

10.23 34.43

1.993 × 103 1.574 × 103

194.8 45.72

larger than that of free NHase, which indicated that the affinity between the substrate and enzyme had been weakened after immobilization. The ratio of Kcat/Km was usually considered to be an indicator of evaluating the catalytic efficiency. After the process of immobilization, the value of Kcat and Kcat/Km were also decreased, in comparison to that of free NHase, which was due to the mass-transfer resistance and lower possibility of forming a substrate−enzyme complex of immobilized enzyme.51 As the active form of vitamin B3 and an important component of coenzyme NAD (nicotinamide adenosine dinucleotide), nicotinamide (NAM) has a wide range of applications in the medicinal and food industries, such as use in the prevention of diabetes, cancers, leprosy, and acen.52,53 Compared with the harsh and extreme conditions in chemical synthetic method, utilizing NHase to catalyze the biotransformation of 3-cyanopyridine to NAM has been commonly recognized as a quite mild and efficient approach for industrial applications. Therefore, taking the advantage of NHase@ Co3(PO4)2 to prepare NAM will not only maintain admirable stability but also achieve reutilization, in comparison to that observed with the free NHase. Thus, the effect of free NHase dosage on the yield of NAM was investigated, and the results are shown in Figure 4a. When the added amount of free NHase was 3.63 U, only 17.5% of NAM would be converted (as shown in Figure 4a, curve a). It was clear that the yield of NAM obviously increased with the increasing activity of free NHase. When the added NHase was ∼21.75 U, a NAM yield of >87.5% can be obtained just after reaction for 1 h, and the yield would increase to 100% after 3 h. Similarly, the same research was also studied on NHase@Co3(PO4)2 and the result was similar to free NHase (Figure 4b), indicating the immobilized NHase possess the outstanding catalytic properties as free NHase. When the activities were almost the same, the performance of immobilized NHase was more favorable than free NHase in the catalytic system, which was consistent with the previous studies.54−56 This phenomenon could be explained by the fact that the free NHase was more sensitive to the substrate and 14927


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Figure 4. (a) Effect of free NHase activity and reaction time on yield of nicotinamide (3.63 U (curve a), 7.25 U (curve b), 14.50 U (curve c), 21.75 U (curve d), and 29.00 U (curve e)). (b) Effect of NHase@Co3(PO4)2 activity and reaction time on yield of nicotinamide (1.01 U (curve a), 2.93 U (curve b), 4.04 U (curve c), 7.32 U (curve d), 14.65 U (curve d)). (c) Reusability of NHase@Co3(PO4)2. (d) Storage stability of free NHase and NHase@Co3(PO4)2.

limited by substrate inhibition.56 Hence, it is more appropriate to apply NHase@Co3(PO4)2 to the industrial production of nicotinamide and that will gain the satisfactory yield. Generally, the reusability of immobilized enzyme is considered to have the most commendable superiority over the free enzyme. In order to achieve recycling, the NHase@ Co3(PO4)2 was separated by centrifugation. When the NHase@Co3(PO4)2 was reused for the seventh time, the relative activity would still maintain ∼60% of the initial activity, indicating the feasibility of applying to continuously industrial production and realizing the cost-effective and resource-saving catalytic process (Figure 4c). Moreover, NHase is a type of enzyme that could be broken down easily by microorganisms in the storage process and inactivated within a short time.57 Therefore, the storage stability was also an important parameter for immobilized NHase. As shown in Figure 4d, the activity of NHase@Co3(PO4)2 started to decrease gradually after 15 days but could still maintain 64.28% of the initial activity after 27 days, while the free NHase was only 11.91%. The decline of activity in the long-term storage process might be caused by the change of surrounding environment, the microbial degradation and the destruction of enzyme conformation. Nevertheless, because of the protection of cobalt phosphate minerals, the NHase@Co3(PO4)2 could retain its catalytic ability and have a promising future for industrial applications.

NHase. The NHase was successfully immobilized in the Co3(PO4)2 and exhibited significantly high catalytic efficiencies and desirable stabilities, which could be attributed to the interactions of Co2+ between Co-type NHase and the microenvironment of cobalt phosphate. Therefore, the Co3(PO4)2 nanoflowers, which exhibited the superb protective effect and stabilities, would have a promising application for industrial enzyme immobilization.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.iecr.7b03809. Experimental details, including experimental materials and the experimental methods of the study on the performance of the enzyme; Figures S1−S6 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-22-60204945. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Nature Science Foundation of China (Nos. 21576068, 21276060, 21276062, and 21306039), the Natural Science Foundation of Tianjin, China (No. 16JCYBJC19 800), the Natural Science Foundation of Hebei Province, China (Nos. B2015202082, B2016202027, and B2017202056), the Program for Top 100 Innovative Talents in Colleges and Universities of Hebei Province, China

4. CONCLUSIONS In conclusion, the pure flowerlike Co3(PO4)2 nanocrystals were synthesized for the first time and a preliminary formation mechanism was speculated. After that, an excellent nanobiocatalyst system was established via the biomimetic mineralization of Co3(PO4)2 nanoflowers with Co-type 14928


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(No. SLRC2017029) and Hebei High Level Personnel of Support Program, China (No. A2016002027).

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Preparation of a Flowerlike Nanobiocatalyst System via Biomimetic

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