Fast Purification and Immobilization of His- and ELP-Tagged Enzyme

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Novel Synthesis Strategy for Biocatalyst: Fast Purification and Immobilization of His- and ELP-Tagged Enzyme from Fermentation Broth Man Zhao,† Junhui Rong,† Juan Han,‡ Yang Zhou,§ Chunmei Li,† Lei Wang,† Yanli Mao,∥ and Yun Wang*,†

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†

School of Chemistry and Chemical Engineering, ‡School of Food and Biological Engineering, and §Institute of Life Science, Jiangsu University, Zhenjiang, Jiangsu Province 212013, China ∥ Henan Province Key Laboratory of Water Pollution Control and Rehabilitation Technology, Henan University of Urban Construction, Pingdingshan, Henan Province 467036, China S Supporting Information *

ABSTRACT: Inspired by natural biomineralization process, inorganic phosphates system has been selected as a candidate for the encapsulation of enzyme; however, during the long-term fabrication process, the loss of enzyme activity is unavoidable, and the biomimetic mineralization mechanism is still poorly understood. Meanwhile, the purification process plays a key role in the preparation of immobilized enzyme with high enzyme loading and activity, while the rapid, low-cost, and eco-friendly purification of biocatalyst from crude fermentation broth remains a critical challenge in biochemical engineering. Here, a binary tag composed of elastin-like polypeptide (ELP) and His-tag was presented for the first time to be fused with β-glucosidase (Glu) to construct a recombinant Glulinker-ELP-His (GLEH) with the aim of developing a fast synthesis strategy combining purification and immobilization processes for a biocatalyst with better stability and recyclability. The purification fold and activity recovery of GLEH reached 18.1 and 95.2%, respectively, once a single inverse transition cycling was conducted at 25 °C for 10 min. Then, efficient biomineralization of hybrid enzymeCu3(PO4)2 nanoflowers was realized in 15 min by the action of His-tag and ultrasonic-assisted reaction method. The activity recovery and relative activity reached the maximum at 90.3 and 111.0%, respectively. We demonstrate that the crystal growth process of a hybrid nanoflower involves obvious nucleation, self-assembly, and the Ostwald ripening process, and the enzyme GLEH acts as a “binder” to assemble Cu3(PO4)2 nanoflakes. The immobilized GLEH nanoflowers show outstanding operation stability and recyclability, and their catalytic efficiency is close to that of free Glu. KEYWORDS: biomimetic mineralization, purification, enzyme immobilization, elastin-like polypeptides, biocatalyst and entrapment.10−13 However, one major limitation of the conventional immobilization support and method is activity loss caused by restricted mass transfer or leaching or denaturation of enzyme. Therefore, various efforts have been made to develop novel solid matrix support and strategy for enzyme immobilization to prevent enzyme inactivation. Inspired by natural biomineralization process, inorganic phosphates system has been selected as a candidate for the encapsulation of enzyme through a de novo approach.14,15 The enzyme molecules are encapsulated within the nanoscale hierarchical petals, which agglomerate into micrometer-sized “nanoflowers” with nanoscale features.14 The spatial confinement, high specific surface area, and mesoporous structures of the organic−inorganic hierarchical nanoflower makes the

1. INTRODUCTION Enzymatic catalysis in vitro has indeed generated enormous interest owing to its merits of high catalytic efficiency, high specificity, low energy consumption, and green manufacturing feature over the past decades.1−3 As a compelling alternative to chemical catalyst, the practical application of native enzymes in industrial biocatalytic processes is hindered by enzyme’s poor stability and high production cost.4,5 Moreover, the homogeneous biocatalyst in the reaction solution is desired to be separated to avoid the contamination of the final product. Enzyme immobilization is acknowledged to be highly effective in minimizing contamination and enhancing the robustness and recyclability of the enzyme.6 A series of nanoparticles such as mesoporous silica,7 hydrogels,8 and magnetic composite particles9 have been used as a supporting matrix to achieve high loading amount, high stability, and reusability. The preparation method mainly includes physical adsorption, covalent bonding, cross-linking, © XXXX American Chemical Society

Received: May 24, 2019 Accepted: August 9, 2019

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DOI: 10.1021/acsami.9b09071 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

hybrid enzyme-Cu3(PO4)2 nanoflowers with a high encapsulation yield (EY) and activity recovery was realized in 15 min by the crucial synergetic action of His-tag and ultrasound technique. Furthermore, the biomimetic mineralization mechanism is also discussed in detail for controlled crystal growth and aggregation.

resultant immobilized biocatalyst superior in stability and recyclability.16 The Cu3(PO4)2 nanoflowers can act as artificial enzyme with a peroxidaselike activity, The Cu 3 (PO 4 ) 2 nanoflowers can act as artificial enzyme with peroxidase-like activity, showing an enhanced activity of the hybrid nanoflower. An increment of 506% was observed compared with the free HRP in the solution.17 However, it always takes one day or more to fabricate a nanoflower exhibiting high enzyme loading. During the long-time formation process, the loss of free enzyme’s original activity is unavoidable; unfortunately, the biomimetic mineralization mechanism remains poorly understood. A fast synthesis process is required to ensure the high recovery of the original activity. Purification process plays a key role in the preparation of immobilized enzyme with high enzyme loading and activity, since the active site of the solid support would be occupied by a contaminant. The rapid, low-cost, and eco-friendly purification of a biocatalyst from crude fermentation broth remains a critical challenge in biochemical engineering. Up to now, considerable efforts have been made to explore novel purification strategies for enzyme.18,19 The immobilized metalaffinity chromatography (IMAC) method is highly applied in purifying his-tagged proteins.20 Nevertheless, it is costprohibitive for production on an industrial scale. Elastin-like polypeptides (ELPs) are a kind of artificial biopolymer. It consists of a repetitive sequence Val-Pro-Gly-Xaa-Gly, where Xaa represents amino acid, apart from proline.21 By the aid of the thermally responsive property of ELP, the ELP-tagged proteins can undergo precipitation out of fermentation broth above the phase transition temperature (Tt). This novel purification approach, termed as inverse transition cycling (ITC), has superiority in terms of purification performance compared with the commonly used IMAC approach or many reported studies.22−25 The ELP-tag has also been developed to be a hydrophobic domain to enable the immobilization of recombinase onto the hydrophobic surface in one step.26,27 Due to the thermally induced hydrophobicity of the ELP-tag, the hydrophobic binding and catalytic reactions should be conducted above Tt by adjusting the temperature or salt concentration. The graphene-binding peptide (GBP), HNWYHWWPH, can bind firmly onto some carbon-based materials in its natural state because the GB-tag contains a large amount of aromatic amino acids with hydrophobic nature.28,29 In our recent studies, the binary tags composed of ELP and GBP were fused with β-glucosidase (Glu) for building Glu-linker-ELP-GB with the aim to simplify both purification and immobilization processes.30 The resultant immobilized enzyme on magnetic graphene oxide retained around 70% of its initial activities when it was recycled for eight rounds, but an obvious activity loss was observed with more cycles. We are eager to improve the recyclability of immobilized enzyme by fabricating organic−inorganic hybrid nanoflowers. Herein, to develop a fast synthesis strategy combining the purification and immobilization processes for a biocatalyst with enhanced stability and recyclability, the target enzyme was designed to incorporate a promising ELP and His tags. With regard to the enzymatic hydrolysis of biomass, Glu is deemed essential to the complete degradation of cellulose, so Glu was selected as a model enzyme to construct the recombinant Glulinker-ELP-His (GLEH) in this paper. The well-designed strategy includes two steps: first, the purification of enzyme was achieved in 10 min by the action of a temperatureresponsive ELP-tag; second, the biomimetic mineralization of

2. EXPERIMENTAL SECTION 2.1. Reagents and Materials. The Glu clone genes have their origin in cDNA of Coptotermes formosanus. The recombinase plasmid vector pET28a-GLEH was transfected into the receptor strain BL21(DE3). Ammonium sulfate, cupric sulfate, sodium dihydrogen phosphate, sodium acetate, acrylamide glycerinum, hydrochloric acid, glacial acetic acid, isopropanol, methanol, sodium chloride, ammonium persulfate, calcium chloride, sodium carbonate, and dipotassium phosphate were from Sino pharm Chemical Reagent Co., Ltd. Other reagents used are same as those illustrated in our recent studies.30 2.2. Construction and Expression of Fusion Protein. The construction of the recombinant plasmid, pET-Glu-linker-ELP-6His (pET-GLEH), was consistent with what we reported previously.30 The elastin-like polypeptide genes was designed to encode fifty repeated sequences of VPGVG. The recombinase expression was conducted in Escherichia coli BL21 (DE3). Cells culture was processed overnight in 5 mL of Luria−Bertani (LB) medium containing 50 mg/ L kanamycin. The obtained starter culture was then diluted with 200 mL of LB supplemented medium at a ratio of 1:100 to express recombinase GLEH. The LB medium was cultured under the condition of 200 rpm (37 °C) till OD600nm was in the range of 0.4− 0.6, followed by a 20 min ice immersion treatment and addition of IPTG (0.2 mM). The fusion protein was expressed at 25 °C with shaking (200 rpm) for consecutive 12 h. Then, the culture solution was centrifuged at 4 °C for 10 min (4500g). After washing twice using phosphate-buffered saline (PBS) (pH 7.4), BL21 cell pellets were collected and kept at 80 °C below zero. 2.3. Purification of Fusion Protein. About 0.15 g of frozen cell pellets from 100 mL of fermentation liquid was thawed and suspended after PMSF (0.05 mL, 100 mM) and PBS (10 mM, pH 7.4) with a total volume of 5.00 mL. There is around 1.47 g of frozen cell pellets that contains 4.8 mg of expression amount of proteins in one-liter of fermentation liquid. E. coli was fragmented by ultrasound for 30 min with 6 s of continuous ultrasound at an interval of 6 s in an ice bath. After refrigerated centrifugation (12 000g, 10 min), the supernatant was collected and preserved at eighty degrees below zero. Then, the recombinase GLEH in the supernatant was purified according to our previous study.30 The activity recovery and purification fold were calculated as described below.

activity recovery (%) =

purification fold =

purified enzyme activity × 100 crude enzyme activity

specific activity of purified enzyme specific activity of crude enzyme

(1)

(2)

2.4. Synthesis of Hybrid GLEH Nanoflower (GLEH-NF). Hybrid GLEH-NF was prepared in accordance with previous research with few alterations. Typically, the formation of hybrid nanoflowers was started by adding copper sulfate solution to PBS buffer (10 mM, pH 7.4) containing various concentrations of GLEH, and the reaction lasted for 24 h at 25 °C, followed by centrifugation. The resultant precipitate was washed twice by PBS and dried through the vacuum drying process (25 °C). 2.5. Stability of Hybrid GLEH-NF. The reusability, thermal stability, and storage stability of immobilized GLEH-NF were studied. The thermal stability was determined after half an hour of incubation (20−70 °C). A 30-day storage stability experiment was carried out at four degrees Celsius. The reusability of the hybrid GLEH-NF cultured under optimized reaction condition was assessed by repeated hydrolysis of p-nitrophenylglycerol (p-NPG) experiments. The B


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ACS Applied Materials & Interfaces Scheme 1. Recombinase Construction, Expression, Purification, and Immobilization Processesa

a (a) Recombinase construction strategy. (b) Purification by inverse transition cycling (ITC). (c) Enzyme immobilization by biomimetic mineralization.

Figure 1. (a) SDS-PAGE analysis of samples by one-round inverse transition cycling. Lanes: M, marker; 1, clarified lysate of E. coli (pET-28a); 2, clarified lysate of E. coli (Glu); 3, clarified lysate of E. coli (GLEH); 4, precipitate of clarified lysate for Glu (0.5 M (NH4)2SO4); 0.1−1.0 M, precipitate of clarified lysate for GLEH. (b) Purification fold and activity recovery as functions of ammonium sulfate concentrations. was stirred for 1 h. Then, another 4 h of stirring at 4 °C away from light was conducted with the addition of 63 μg of FITC. The mixture underwent dialysis (MWCO: 7000) overnight for the removal of unreacted reagents, and the obtained FITC-GLEH was immobilized by fabricating hybrid NF. The fluorescence images of the immobilized enzyme were observed by an inverted fluorescence microscope (IX73, Olympus, Japan).

immobilized GLEH nanoflowers were rinsed using Tris−HCl (pH 8.0) after each cycle. 2.6. Enzyme Activity Assays. The enzyme activities were measured according to our previous research.30 The calculation formulas for encapsulation yield (EY), activity recovery (AR), and relative activity (RA) are as given below.

AR(%) =

total activity of GLEH‐NF × 100% total activity of free enzyme

(3)

3. RESULTS AND DISCUSSION 3.1. Construction, Expression, and Purification of Recombinase GLEH. In this research, β-glucosidase, composed of 488 amino acids, belongs to Glu1B from C. formosanus (CfGlu1B). The designed ELP consists of 50 repeated pentapeptide VPGVP. The schematic diagram of recombinant plasmid pET-GLEH is shown in Figure S1. The recombinase construction strategy is shown in Scheme 1a. Then, the recombinase GLEH was purified by the ITC method according to our previous research,30 and the purification process is shown in Scheme 1b. (NH4)2SO4 was selected to reduce the phase transition temperature of GLEH. The ITC experiments were operated at various concentrations of (NH4)2SO4, and their purification performances were

EY(%) =

total amount of protein − amount of supernatant protein total amount of protein × 100%

RA =

AR × 100% EY

(4) (5)

2.7. Synthesis of Fluorescently Labeled GLEH-NF. Fluorescein isothiocyanate (FITC) labeling method was used in this study. FITC has green fluorescence that can be used to mark the recombinant enzyme. Two millimeters of GLEH purified by oneround of ITC was added to 10 mL of PBS solution (10 mM, pH 7.4). After the addition of EDC (5 mg) and NHS (2.5 mg), the mixture C


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Figure 2. Effects of various Cu2+ concentrations on the morphologies of hybrid nanoflowers. (a1-2) 1.2 mM; (b1-2) 2.4 mM; (c1-2) 4.8 mM; (d12) 7.2 mM; (e1-2) 9.6 mM; other conditions: 50 μg/mL GLEH, 10 mM PBS (pH 7.4), and 24 h reaction (25 °C).

Figure 3. Effects of various GLEH concentrations on the morphologies of hybrid nanoflowers. (a−e) 10, 30, 50, 100, and 150 μg/mL; other conditions: 4.8 mM Cu2+, 10 mM PBS (pH 7.4), and 24 h reaction (25 °C).

containing GLEH. The process of enzyme immobilization by biomimetic mineralization is shown in Scheme 1c. To achieve the controllable synthesis of nanoflower morphology and improve the catalytic properties of hybrid GLEH-NF, the effects of different synthesis conditions were investigated. Figure 2 shows the morphology change of GLEH-NF synthesized under different concentrations of Cu2+. As the concentration of copper ions increases, the petals of the nanoflowers become denser and the morphology of the nanoflowers tends to be spherical. In terms of the catalytic performance of GLEH-NF, the activity recovery and relative activity of GLEH-NF have a similar upward and subsequent downward trend along with the rise of copper ion concentration (Figure 5a). The maximum values of AR (87.2%) and RA were achieved at a copper ion concentration of 4.8 mM, and its RA was 123% higher than that of free GLEH. This improvement in the catalytic activity of GLEHNF in comparison with that of free GLEH could be due to its enhanced enzyme stability.31 However, the access of substrate to the active sites of GLEH would be inhibited when the petals of the nanoflowers were too dense, which led to a decrease in the catalytic performance of GLEH-NF when the maximum encapsulation yield (85.1%) was achieved at 7.2 mM copper ion concentration. The enzyme concentration also had a great influence on the morphology and immobilization performance of hybrid nanoflowers, as displayed in Figures 3 and 5b.32 The activity recovery and the encapsulation yield gradually decreased with

evaluated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Figure 1). As can be seen from Lanes 1, 2, and 3 in Figure 1a, the molecular mass of β-glucosidase (54 kDa) and GLEH (78 kDa) are in accordance with their theoretical values. No obvious bands were found in Lane 0.1 M, which showed clearly that the efficient separation of GLEH was a failure, as the ammonium sulfate concentration dropped to 0.1 M. Apparent protein bands were found in the concentration range of 0.3−1.0 M. At the same time, it could be seen from Figure 1b that the activity recovery of enzyme activity gradually increased along with the rise of the concentration of (NH4)2SO4 and achieved an equilibrium at the concentration of 0.5 M; however, the purification ratio started to decrease rapidly when the (NH4)2SO4 concentration was higher than 0.5 M. Thus, 0.5 M ammonium sulfate was selected in the follow-up purification experiments. After the first round of ITC, the activity recovery reached 95.2%; meanwhile, an obvious increase of purification fold (18.1) was achieved. The ITC purification method is not only effective but also fast. The high purity and activity recovery of GLEH were obtained by only one round of ITC in 10 min. After purification, it was directly used for the preparation of the subsequent nanoflower without lyophilization treatment, which further simplified the production process of the immobilized enzyme and reduced the production cost. 3.2. Synthesis and Characterization of Immobilized GLEH-NF. The GLEH−Cu3(PO4)2 nanoflowers (GLEH-NF) were synthesized by adding CuSO4 into the PBS solution D


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Figure 4. Effects of various incubation times on the morphologies of hybrid nanoflowers. (a−l) 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h; other conditions: 4.8 mM Cu2+, 50 μg/mL GLEH, 10 mM PBS (pH 7.4), and 24 h reaction (25 °C).

the increase of enzyme concentration (Figure 5b), while the relative activity of GLEH-NF reached the maximum of 122% at a moderate GLEH concentration of 50 μg/mL. With increasing enzyme concentration, the density of nanoflowers increased gradually; meanwhile, the scattered petals dwindled and the particle size distribution became more uniform. To evaluate the effect of His-tag in GLEH-NF on the biomimetic mineralization of Cu3(PO4)2, the process was conducted with the assistance of Glu and Glu-linker-ELP without His-tag. The encapsulation yields of hybrid Glu-NF and Glu-linker-ELP-NF were dramatically decreased to 15.2 and 20.9%, respectively, which were much lower than that of GLEH-NF containing His-tag. It showed that the His-tag could greatly improve the loading amount of GLEH-NF.33 This might be attributed to the coordination between His-tag and Cu2+, which promoted the aggregation of Cu3(PO4)2 nanoflake unit as the “glue” effect. In addition, the effects of different incubation times (10 min to 96 h) on the synthesis of nanoflowers were also investigated. As seen in Figure 4a,b, the precipitation of copper phosphate gradually accumulated to form petal-shaped crystals in the

early stage of nanoflower formation. The scattered petals gradually form “buds” of nanoflowers through nucleation (Figure 4c,d). Then, the buds of nanoflowers grew up by selfassembly (Figure 4e,f). As can be seen from Figure S2, the hybrid nanoflowers could also be formed by the self-assembly in the absence of GLEH at a high copper ion concentration of 4.8 mM; however, this process took longer than that in the presence of GLEH. The nanoflowers in the initial flowing stage began to appear when the incubation time was 3 h, but the time was shortened to 2 h in the presence of GLEH. More obvious distinction would be observed when the Cu 2+ concentration was reduced to 2.4 mM. As shown in Figure S3, many Cu3(PO4)2 nanoflakes and few aggregates were formed when the incubation time was 4 h, but no obvious nanoflake was observed in the absence of GLEH in Figure S4. These also show that GLEH acts as a glue in the self-assembly course to improve the aggregation rate of the Cu3(PO4)2 nanoflake unit. As displayed in Figure 5c, the activity recovery, encapsulation yield, and relative activity of hybrid nanoflowers increased first, then decreased with the prolongation of incubation time, and reached the maximum values of 83.4, E


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Figure 5. Effects of various cultural conditions on the immobilization performance of hybrid nanoflowers. (a) Copper ion concentration; (b) GLEH concentration; (c) incubation time; (d) temperature; and (e) ultrasonic duration.

Figure 6. (a) Photograph, (b) XRD patterns, (c) FT-IR spectra, (d) EDS spectra, and (e) particle diameter spectra of nanoflowers.

67.2, and 124% at 24 h, respectively. These results indicated that the inactivation of enzymes encapsulated in GLEH-NF happened during the long incubation time. As can be seen from Figure 5d, the encapsulation yield was basically unchanged, which suggested that the temperature produced little influence on the coordination of His-tag and Cu2+. However, the activity recovery and relative activity first increased and then decreased with an increase in temperature. As displayed in Figure S5, the petals of the nanoflowers became more and more sparse as the temperature increased. When the temperature was 55 °C, flaky Cu3(PO4)2·3H2O would not form nanoflowers through self-assembly. This was attributed to the fact that the formation of Cu3(PO4)2 was an

exothermic reaction, and the high-temperature reaction led to the difficulty in the formation of Cu3(PO4)2.34 The GLEH-NF petals formed at 4 °C were the densest, which limited the contact between the substrates and enzyme active sites. So, the activity recovery and relative activity of GLEH-NF synthesized at 25 °C reached the maximum 87.5 and 128%, respectively. As illustrated in the previous discussion of Figures 4 and S2−S4, the rising copper ion concentration in the reaction solution would speed up the nucleation rate of cupric phosphate nanoflake and then accelerate the formation of nanoflowers. So, it can be deduced that the introduction of effective methods to accelerate crystal nucleation will promote the formation of nanoflowers. It has been proved that F


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Figure 7. Imaging under bright field (a, c, e, g) and fluorescence field (b, d, f, h). (a, b) GLEH-NF (4 °C); (c, d) GLEH-NF (25 °C); (e, f) FITClabeled GLEH-NF (4 °C); and (g, h) FITC-labeled GLEH-NF (25 °C).

Figure 8. Scanning electron microscopy (SEM) analysis of the synthesized GLEH-NF (4.8 mM Cu2+, 50 μg/mL GLEH, 25 °C, and 24 h) (a) and elemental mapping images of carbon (b), nitrogen (c), oxygen (d), phosphorous (e), and copper (f).

synthesized under optimal conditions (4.8 mM Cu2+, 50 μg/ mL GLEH, and 24 h) was performed by fluorescence microscope, particle size analyzer, X-ray diffractor (XRD), Fourier transform infrared (FT-IR) spectrometer, and energydispersive X-ray spectroscope (EDS). The recombinase GLEH labeled with FITC was incorporated into hybrid nanoflowers and then analyzed by inverted fluorescence microscope (Figure 7). No fluorescence could be detected for GLEH-NF in the absence of FITC, while green fluorescent spots was observed for the FITC-labeled GLEH-NF. The results confirmed that recombinant GLEH was successfully incorporated into GLEHNF. Meanwhile, the immobilized FITC-GLEH showed a good dispersion in nanoflowers. It could also be seen from Figure 7 that GLEH-NF synthesized at 4 °C was more compact and uniform. The locations and intensities of the diffraction peaks of cupric phosphate in Figure 6b are in good agreement with those given in the JCPDS card (00-022-0548). The sharp diffraction peak in yellow and green curves demonstrated that

ultrasound can accelerate the crystal nucleation and crystal growth rates based on the cavitation effect.35,36 Therefore, ultrasound was introduced into the biomineralization reaction under optimal conditions (4.8 mM Cu2+, 50 μg/mL GLEH, and 25 °C). Figure 5e shows that the ultrasonic-assisted reaction method significantly accelerated the formation of hybrid nanoflowers. When the sonication time was 15 min, the encapsulation yield, activity recovery, and relative activity reached the maximum of 81.2, 90.3, and 111.0%, respectively. As many studies have reported, ultrasound technique can promote the coordination interaction between metal ions and ligands.36 The improved performance of enzyme immobilization compared with that of hybrid nanoflowers synthesized without ultrasonic treatment perhaps is an effect of enhanced coordination interaction between copper ions and enzymes by ultrasonic treatment.35 The resultant hybrid GLEH-NF had a blue appearance as displayed in Figure 6a. The characterization of nanoflowers G


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ACS Applied Materials & Interfaces Scheme 2. Schematic Representation of Formation Mechanism of Hybrid Nanoflowers

Figure 9. Optimum temperatures (a) and pH (b) of free Glu, free GLEH, GLEH-NF (4 °C), and GLEH-NF (25 °C). The maximum activities of enzymes (Glu/GLEH/GLEH-NF) were defined as 100% for the calculation of relative activities.

for the sample synthesized at 4 °C. The enzyme-induced biomimetic mineralization shows an obvious decline in the mean granularity and an increase in the specific area of nanoflowers, which is consistent with some studies. For example, the specific surface area of BSA/zinc phosphate nanoflowers shows an obvious trend of increase after the incorporation of enzyme.37 This could well be because the intrinsic anisotropic crystal growth of petals would be restrained by the coordination of GLEH and Cu ions on the top and bottom surface of Cu3(PO4)2 nanopetals when adding GLEH in the reaction system, resulting in the smaller size of nanopetals and the aggregated nanoflowers.38 Moreover, the specific surface area became larger due to the aggregation of more small-sized petals. According to the above analysis, the crystal growth mechanism of a unique nanoflower hierarchical structure constructed by the decoration of enzyme on the Cu3(PO4)2 nanoflake unit surfaces as a “binder” to assemble nanoflakes was investigated. The SEM images of the samples at different time intervals in Figure 4 suggest that the mechanism involved obvious nucleation, self-assembly, and the Ostwald ripening process. From the SEM images in Figure 4b, the cupric phosphate nanoparticles with low crystallinity were formed after 30 min of reaction. When the reaction time was prolonged to 1 h, the SEM image in Figure 4c displays irregular Cu3(PO4)2 nanoflake units generated via an “initial crystallization” process based on the Gibbs−Thomson law. In this stage, the enzyme was mounted on the Cu3(PO4)2 nanoflake unit surfaces by means of coordination with Cu ions. Further increase of the reaction time to 2 h, the interlaced Cu3(PO4)2 nanoflower hierarchical structure appeared owing to the “self-assembly” process. When the reaction was extended to 3 h, the integrated flowerlike hierarchical structure of Cu3(PO4)2 is formed by the Ostwald ripening process

hybrid nanoflowers also had high crystallinity after the encapsulation of GLEH. The chemical composition of nanoflowers was characterized by FT-IR and EDS. As shown in spectra a and b in Figure 6c, the adsorption bands at 1051 (asymmetric vibration), 992 (symmetric vibration), and 627 cm−1 (bending vibration) were ascribed to P−O vibrations. The characteristic protein peaks of −CONH (1400−1600 cm−1), −CH2, and −CH3 (2800−3000 cm−1) could be found in spectra c and d, indicating the successful encapsulation of GLEH during the biomimetic mineralization process. The EDS curves exhibited the existence of Cu, P, C, and O elements in the resultant GLEH-NF synthesized at 25 °C (Figure 6d). All Cu and P elements were assigned to Cu3(PO4)2, while all of the C element and partial O element were from enzyme. As show in Figure 8, the elemental mapping images further confirmed the distribution of five major elements (Cu, P, C, O, and N) throughout GLEH-NF, instead of adsorption on the surface. It could be seen from the particle diameter spectra (Figure 6e) that the distribution of nanoflowers was relatively uniform. Compared to that of pure Cu3(PO4)2 nanoflowers without GLEH, the average particle size of the hybrid nanoflowers containing GLEH synthesized at 4 and 25 °C was decreased to 20.8 and 25.2 μm, respectively. The analysis of N2 adsorption−desorption isotherms for nanoflowers was conducted (see Figure S6). The specific area of pure nanoflowers synthesized at 4 and 25 °C were 27.04 and 10.36 m2/g, respectively, while that of hybrid nanoflowers synthesized at 4 and 25 °C were 46.44 and 15.21 m2/g, respectively. The specific surface area of nanoflowers declined with increase in temperature, which was consistent with the results in Figure S5 that the petals of nanoflowers became more and more sparse with the increase of temperature. Meanwhile, the specific area of nanoflowers showed an obvious trend of increase after the incorporation of GLEH, especially H


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Figure 10. Effects of temperature (a), incubation time (b), and cycles (c) on the activities of Glu, GLEH, and GLEH-NF (4/25 °C). The original enzyme activities were defined as 100% for the calculation of relative activities.

Table 1. Kinetic Constants for Glu, GLEH, and GLEH-NF in Hydrolyzing p-NPG enzymes Glu GLEH GLEH-NF (4 °C) GLEH-NF (25 °C)

kcat (s−1)

Km (mM) 5.77 5.02 7.43 7.39

± ± ± ±

0.65 0.74 0.89 0.78

169.7 180.8 186.1 203.5

± ± ± ±

5.3 7.2 16.3 18.7

kcat/Km (s−1 mM−1) 29.4 36.0 25.0 27.6

± ± ± ±

1.7 1.5 2.1 2.6

The storage stabilities of hybrid GLEH-NF, Glu, and GLEH were evaluated in PBS (pH 5.5) at 4 °C. As shown in Figure 10b, the free Glu and GLEH lost 86.2 and 47.3% of their original activities over 30 days. However, the inactivation of enzyme was significantly decreased by encapsulating GLEH in hybrid nanoflowers. The loss of the enzyme activity of GLEHNF synthesized at 4 and 25 °C was reduced to 24.8 and 35.0%, respectively, under the same conditions. The results indicated that both the ELP-tag and encapsulation of enzyme within the confined space of a nanoflower matrix contributed to the enhancement of enzyme stability. The reusability of GLEH-NF was evaluated for 16 cycles (see Figure 10c). The hybrid GLEH-NF synthesized at 4 and 25 °C retained 75.8 and 69.2% of its original activities when recycled sixteen times. The resultant GLEH-NF shows an outstanding operation stability, which is greatly superior to the reusability of various immobilized Glu onto other support material.30,39 3.5. Kinetic Constants. The Km values of free GLEH and Glu were very close (see Table 1). However, the Km values of the GLEH-NF synthesized at 4 and 25 °C were around 7.43 and 7.39 mM, respectively, both of which were slightly higher than that of the free one. The increase in the Km values of GLEH-NF indicated that the affinity of GLEH to substrates was weakened after the encapsulation of GLEH in the hybrid nanoflower matrix mainly because of the diffusion-controlled mass transfer limitation.40,41 A slight decrease in the kcat/Km value of GLEH-NF was observed relative to that in GLEH, while the value was close to that of free Glu. These data indicated that the catalytic efficiency of GLEH-NF was lower than that of free GLEH-NF but similar to that of free Glu. It could also be deduced that the binary ELP-His tags had no remarkable influence on the affinity of enzyme to the substrate but improved the catalytic efficiency of Glu.

according to the SEM image in Figure 4e,f. In the whole process, the higher the Cu ion concentrations, the denser the nanoflowers were (Figure 2). So, the concentration of cupric ions should mainly affect the nucleation speed. In addition, as shown in Figure 3, as the added amount of enzyme increased, the constructed nanoflake unit decreased until it disappeared during the same reaction time. Therefore, we believed that GLEH played the role of a binder in the Cu 3(PO 4 ) 2 nanoflower-forming process; then, the addition amount of enzyme could affect the self-assembly speed of cupric phosphate nanoflakes. The synthesis mechanism of hybrid nanoflowers is shown in Scheme 2. 3.3. Optimal pH and Temperature of Enzymes. It can be seen from Figure 9a that the activities of GLEH and GLEHNF reached the maximum when pH was 5.5. Meanwhile, Figure 9b shows that the optimum temperature for both GLEH and GLEH-NF was 45 °C. The optimal temperature of free β-glucosidase was increased to 50 °C, which was slightly higher than those of free GLEH and GLEH-NF. 3.4. Enzyme Stabilities. Figure 10a shows that the enzyme activity of β-glucosidase was significantly reduced as the temperature exceeded 45 °C (Figure 10a). A complete inactivation could be observed when it is over 60 °C. It was noteworthy that the free GLEH and immobilized GLEH-NF showed a stronger thermal stability compared with free βglucosidase. The residual activity of GLEH was 45.0% at 60 °C. It had been found in our previous research that ELPs could form a precipitate to wrap linked Glu because the phasechange property of ELPs occurred under elevated temperature, and ELP would become soluble again with the fall of temperature. As a result, the wrapped enzymes were set free, and the recovery of enzyme activity was achieved.30 The hybrid GLEH-NF synthesized at 4 and 25 °C still retained 69.2 and 62.3% of the original enzyme activity after incubation of 30 min at 60 °C. These results demonstrated that the hybrid GLEH-NF synthesized at 4 °C had a higher thermal stability than that synthesized at 25 °C. This might be attributed to the fact that the nanoflower petals synthesized at low temperatures were denser, which limited the structural change of encapsulated enzymes and then maintained their threedimensional structure at high temperatures.

4. CONCLUSIONS The well-designed strategy in the present work includes two steps: first, the purification of enzyme was achieved in 10 min by the action of a temperature-responsive ELP-tag; second, the biomineralization of hybrid enzyme-Cu3(PO4)2 nanoflower with a high encapsulation yield and activity recovery was I


Research Article


(8) Matsumoto, T.; Isogawa, Y.; Tanaka, T.; Kondo, A. StreptavidinHydrogel Prepared by Sortase A-Assisted Click chemistry for enzyme Immobilization on an Electrode. Biosens. Bioelectron. 2017, 99, 56−61. (9) Han, J.; Wang, L.; Wang, L.; Li, C.; Mao, Y.; Wang, Y. Fabrication of a Core-Shell-Shell Magnetic Polymeric Microsphere with Excellent Performance for Separation and Purification of Bromelain. Food Chem. 2019, 283, 1−10. (10) Liu, D. M.; Chen, J.; Shi, Y. P. Advances on Methods and Easy Separated Support Materials for Enzymes Immobilization. TrAC, Trends Anal. Chem. 2018, 102, 332−342. (11) Kadam, A. A.; Jang, J.; Lee, D. S. Supermagnetically Tuned Halloysite Nanotubes Functionalized with Aminosilane for Covalent Laccase Immobilization. ACS Appl. Mater. Interfaces 2017, 9, 15492− 15501. (12) Han, J.; Wan, J.; Wang, Y.; Wang, L.; Li, C.; Mao, Y.; Ni, L. Recyclable Soluble−Insoluble Upper Critical Solution TemperatureType Poly(Methacrylamide-Co-Acrylic Acid)−Cellulase Biocatalyst for Hydrolysis of Cellulose into Glucose. ACS Sustainable Chem. Eng. 2018, 6, 7779−7788. (13) Wang, L.; Zhi, W.; Wan, J.; Han, J.; Li, C.; Wang, Y. Recyclable β-Glucosidase by One-Pot Encapsulation with Cu-MOFs for Enhanced Hydrolysis of Cellulose to Glucose. ACS Sustainable Chem. Eng. 2019, 7, 3339−3348. (14) Jun, G.; Jiandu, L.; Zare, R. N. Protein-Inorganic Hybrid Nanoflowers. Nat. Nanotechnol. 2012, 7, 428−432. (15) Thawari, A. G.; Rao, C. P. Peroxidase-Like Catalytic Activity of Copper-Mediated Protein−Inorganic Hybrid Nanoflowers and Nanofibers of β-Lactoglobulin and α-Lactalbumin: Synthesis, Spectral Characterization, Microscopic Features, and Catalytic Activity. ACS Appl. Mater. Interfaces 2016, 8, 10392−10402. (16) Jiayu, S.; Jiechao, G.; Weimin, L.; Minhua, L.; Hongyan, Z.; Pengfei, W.; Yanming, W.; Zhongwei, N. Multi-Enzyme CoEmbedded Organic-Inorganic Hybrid Nanoflowers: Synthesis and Application as a Colorimetric Sensor. Nanoscale 2013, 6, 255−262. (17) Lin, Z.; Xiao, Y.; Yin, Y.; Hu, W.; Liu, W.; Yang, H.-H. Facile Synthesis of Enzyme-Inorganic Hybrid Nanoflowers and Its Application as a Colorimetric Platform for Visual Detection of Hydrogen Peroxide and Phenol. ACS Appl. Mater. Interfaces 2014, 6, 10775−10782. (18) Leung, D. W.; Spencer, S. A.; Cachianes, G.; Hammonds, R. G.; Collins, C.; Henzel, W. J.; Barnard, R.; Waters, M. J.; Wood, W. I. Growth Hormone Receptor and Serum Binding Protein: Purification, Cloning and Expression. Nature 1987, 330, 537−543. (19) Rigaut, G.; Shevchenko, A.; Rutz, B.; Wilm, M.; Mann, M.; Séraphin, B. A Generic Protein Purification Method for Protein Complex Characterization and Proteome Exploration. Nat. Biotechnol. 1999, 17, 1030−1032. (20) Wong, J. W.; Albright, R. L.; Wang, N.-H. L. Immobilized Metal Ion Affinity Chromatography (IMAC) Chemistry and Bioseparation Applications. Sep. Purif. Methods 2006, 20, 49−106. (21) Ge, X.; Yang, D. S. C.; Trabbic-Carlson, K.; Kim, B.; Chilkoti, A.; Filipe, C. D. M. Self-Cleavable Stimulus Responsive Tags for Protein Purification Without Chromatography. J. Am. Chem. Soc. 2005, 127, 11228−11229. (22) Gao, H.; Qi, X.; Hart, D. J.; Gao, S.; Wang, H.; Xu, S.; Zhang, Y.; Liu, X.; Liu, Y.; An, Y. Three Novel Escherichia coli Vectors for Convenient and Efficient Molecular Biological Manipulations. J. Agric. Food Chem. 2018, 66, 6123−6131. (23) Banki, M. R.; Feng, L.; Wood, D. W. Simple Bioseparations Using Self-Cleaving Elastin-Like Polypeptide Tags. Nat. Methods 2005, 2, 659−662. (24) Dan, E. M.; Trabbic-Carlson, K.; Chilkoti, A. Protein Purification by Fusion with an Environmentally Responsive ElastinLike Polypeptide: Effect of Polypeptide Length on the Purification of Thioredoxin. Biotechnol. Prog. 2010, 17, 720−728. (25) Drew, D.; Lerch, M.; Kunji, E.; Slotboom, D. J.; de Gier, J. W. Optimization of Membrane Protein Overexpression and Purification Using GFP Fusions. Nat. Methods. 2006, 3, 303−313.

realized in 15 min by the synergistic action between His-tag and ultrasonic technique. For simplified steps, the purified precipitate was first separated from fermentation by centrifugation and then resolubilized directly in PBS for the preparation of nanoflowers, avoiding the intermediate drying operation. The entire process was successfully shortened to 50 min, and the hybrid nanoflower displayed significantly enhanced robustness and recyclability. The results suggest that the development of a recombinant enzyme-linker-ELP-His system for optimizing purification and immobilization processes would be competitive in the field of biochemical industry.

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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/acsami.9b09071.

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Details of some experimental procedures, schematic diagram of recombinant plasmid pET-GLEH, morphology change of nanoflowers over time with and without enzyme, and N2 adsorption−desorption curves of nanoflowers (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86 0511 88790683. Fax: +86 0511 88791800. ORCID

Man Zhao: 0000-0002-7072-8553 Yun Wang: 0000-0003-0024-0824 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 21676124 and 21878131) and the Opening Project of Henan Province Key Laboratory of Water Pollution Control and Rehabilitation Technology (Nos. CJSZ2018001 and CJSZ2018009).

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Fast Purification and Immobilization of His- and ELP-Tagged Enzyme

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