Novel Cell–Inorganic Hybrid Catalytic Interfaces with Enhanced

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Novel Cell−Inorganic Hybrid Catalytic Interfaces with Enhanced Enzymatic Activity and Stability for Sensitive Biosensing of Paraoxon Lei Han† and Aihua Liu*,‡,§,∥ †

College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, 700 Changcheng Road, Qingdao 266109, China Institute for Biosensing & In-Vitro Diagnostics and College of Chemistry & Chemical Engineering, Qingdao University, 308 Ningxia Road, Qingdao 266071, China § Joint Key Laboratory for Biosensors of Shangdong Province, Qingdao University, 308 Ningxia Road, Qingdao 266071, China ∥ College of Medicine, Qingdao University, Qingdao 266021, China ‡

S Supporting Information *

ABSTRACT: To improve the biosensing performance of organophosphorus hydrolase (OPH), the novel bioinorganic hybrid catalysts were facilely explored by biomineralization and cell surface display technology. During biomineralization, cobalt phosphate crystals were deposited onto the surface of OPH-fused bacteria, and the inorganic crystals at middle of cell collapsed inwardly to form the final spindle morphology because of the lowest energy principle and the mechanics principle. OPH would show the allosteric effect from “inactive” form to “active” form, and the “active” form was “fixed” when OPH was embedded into cobalt phosphate. Therefore, the activity of mineralized OPH-fused cells was greatly enhanced about 3 times in comparison with original OPH-fused cells. Additionally, the stability of the novel hybrid catalysts was also significantly improved. Further, the as-synthesized bioinorganic hybrid catalysts were applied to sensitive paraoxon biosensing, which exhibited lower limit of detection than that of the original counterpart. Thus, this hybrid biocatalytic system would provide a model to develop a wide range of biocatalysts and find a wide range of applications in industrial catalysis, analytical chemistry, and environmental engineering. KEYWORDS: biomineralization, cell surface display, bioinorganic hybrid interfaces, organophosphorus hydrolase, paraoxon, biosensing biodegradation of OPs.25,26 So far, researchers had fabricated OPH-fused whole-cell biocatalysts by microbial cell surface display technology.1,9 For example, in our previous work, OPH was displayed on the surface of E. coli using ice nucleation protein (INP) as anchoring motif for the biosensing of OPs.9 However, the activity and stability of cell-displayed OPH still need to be improved for more sensitive detection and rapid biodegradation of OPs. With the growing attention to biomineralization, various proteins such as bull serum albumin27,28 and lysozyme29 as well as phage30 and purple membrane31 have been used as biotemplates for the synthesis of bioinorganic hybrid materials. It is reported that the activities and stabilities of some enzymes can be dramatically enhanced by using pure enzymes as biotemplates to prepare enzyme−inorganic hybrid materials.32 Wang and co-workers reported that α-amylases embedded in specific calcium phosphate (CaHPO4) showed the enhanced activity due to the “allosteric effect” and the stability also be improved.33 In addition, the phenomenon was also discovered in some other enzyme-incorporated transition metal phosphate

1. INTRODUCTION For conventional whole-cell biocatalysts, the reaction substrates generally need to go across the cytomembrane and get into the cell to realize the catalytic reaction based on intracellular enzyme, which greatly limits the reaction efficiency and the application of whole-cell biocatalysts.1,2 As an important alternative technology, microbial cell surface display technology permits foreign enzymes to be displayed onto surface of host cell with the aid of anchoring motif for novel whole-cell biocatalysts.3−6 Because the cell-displayed enzymes can directly contact the reaction substrates, the reaction efficiency can be greatly enhanced. In addition, compared with the pure enzyme catalysts and immobilized enzyme catalysts, the cell-displayed enzymes eliminate tedious enzyme purification process and enzyme immobilization process.1,7−14 However, like the enzyme immobilization technology, the microbial cell-surface display generally reduces the activity of enzymes, due to unfolded or misfolded structure and steric hindrance.15−17 Organophosphates (OPs) are world widely used as pesticides in agriculture.18−22 Unfortunately, OPs remain in the environment for a long time and cause severe damage to the ecosystem.23 Therefore, organophosphorus hydrolase (OPH, EC 3.1.8.1), a kind of enzyme which can hydrolyze the OPs,24 has been extensively studied for the biosensing and © 2017 American Chemical Society

Received: December 13, 2016 Accepted: February 6, 2017 Published: February 15, 2017 6894

DOI: 10.1021/acsami.6b15992 ACS Appl. Mater. Interfaces 2017, 9, 6894−6901

Research Article

ACS Applied Materials & Interfaces

Figure 1. FE-SEM image (A), TEM images (B, C), and EDX pattern (D) of hybrid spindles after the mineralization for 24 h. Co., Ltd. (Dalian, China). Paraoxon was purchased from Sinopharm Chemical Reagent Corporation (Shanghai, China), prepared into 20 mM stock solution with 20% methanol aqueous solution, and then stored in the darkness to avoid photolysis and diluted ready when it will be used. For safety, direct contact and inhalation of paraoxon should be avoided by taking appropriate precautions and operating in the fume hood. All other reagents are of analytical grade and were used without purification. All aqueous solutions were prepared with Milli-Q water (18.2 MΩ cm). 2.2. Culture of Cell Surface-Displayed OPH. The construction and culture of OPH-fused cell were described in the previous work.9 In brief, E. coli BL21 (DE3) harboring plasmid pTInaPb-N/Oph was grown in Luria-Bertani medium with 50 mg/L kanamycin at 37 °C until OD600 nm reached 0.6. The expression of fusion protein INPOPH was induced with 0.1 mM IPTG at 25 °C for 10 h. Then the cells were harvested by centrifugation (6000 rpm, 5 min), washed, and resuspended in phosphate buffered saline (PBS) solution. 2.3. Synthesis and Characterization of the Mineralized OPHFused Cells. In a typical experiment, 20 μL of CoCl2 aqueous solution (50 mM) was added to 2 mL of PBS (pH 7.4) containing OPH-fused cells (OD600 nm = 6), followed by incubation at 25 °C for 1 day. The resultant materials were centrifuged and washed by water. The amount of cell-Co3(PO4)2·8H2O hybrid spindles was expressed by the corresponding OD600 nm value of original cells, which facilitated the following activity assay experiments. The diluted suspension of the mineralized cells was dropped to a grid and dried at room temperature. Then the morphology of the hybrids was observed by field emission scanning electron microscopy (FE-SEM) (HITACHI S-4800) and transmission electron microscopy (TEM) (HITACHI H-7650). The energy dispersive X-ray spectroscopy (EDX) was recorded on a HORIBA 7593-H spectrometer. The X-ray diffraction (XRD) patterns were recorded on a Bruker-AXS microdiffractometer (D8 ADVANCE) at room temperature.

hybrid biocatalyst, such as carbonic anhydrase−copper phosphate (Cu3(PO4)2·3H2O),32 laccase−Cu3(PO4)2·3H2O,34 and glucose oxidase/horseradish peroxidase−Cu3(PO4)2· 3H2O.35 But seemingly, not all enzymes conform to the phenomenon.32 Therefore, it is necessary to study the bioinorganic hybrid systems based on various enzymes to extend the toolkit of bioinorganic hybrid catalysts and deepen the understanding about the allosteric effect. In addition, although the synthesis method of bioinorganic hybrid materials is green and facile, the use of pure enzyme would lead to the tedious enzyme purification process and expensive cost.1 In this contribution, in order to enhance the activity and stability of OPH-fused whole-cell biocatalysts, we explored a novel cell−inorganic hybrid materials by combination of biomineralization and microbial cell surface display technology. We discoveried that OPH would show the allosteric effect from “inactive” form to “active” form and the “active” form would be “fixed” after OPH was embedded into cobalt phosphate (Co3(PO4)2·8H2O). Further, the sensitive optical detection of paraoxon was realized by using the mineralized cells, which significantly excelled over original OPH-fused cells. To our knowledge, this work is the first example on the cell surfacedisplayed enzyme−inorganic hybrid biocatalysts and would provide a model method to develop a wide range of whole-cell biocatalysts for the diverse applications on industrial catalysis, analytical chemistry, and environmental engineering.

2. MATERIALS AND METHODS 2.1. Chemicals and Materials. Isopropyl β-D-thiogalactoside (IPTG) and kanamycin were purchased from Takara Biotechnology 6895

DOI: 10.1021/acsami.6b15992 ACS Appl. Mater. Interfaces 2017, 9, 6894−6901

Research Article

ACS Applied Materials & Interfaces 2.4. Standard Assay for Catalytic Activity. The OPH activity was investigated by the method developed by Shimazu et al.36 In a typical experiment, the reaction system (200 μL) contained paraoxon (1 mM), 50 μM Co2+, and mineralized cells (OD600 nm = 0.1) in phosphate buffer (PB, 50 mM, pH 7.4) at 37 °C. The mineralized cells were finally added to initiate the reaction. After removing the mineralized cells, the maximum absorbance of the product pnitrophenol (PNP) at 410 nm was monitored. 2.5. Catalytic Activity Characterization of Mineralized OPHFused Cells. To investigate the pH or temperature stability of cellCo3(PO4)2·8H2O, it was incubated either in various buffers in 100 mM varying pH 3.0−11.0 at 37 °C for 1 h or in PB solution (pH 7.4) at different temperatures from 4 to 100 °C for 2 weeks, separately. After that, the materials were collected by centrifugation (5000 rpm, 5 min), and then the remaining relative activities were determined by the standard assay. Throughout these experiments, the pH values were controlled within pH 3.0−11.0 by using different buffers in 100 mM: citrate− NaOH buffer buffer (pH 3.0−5.0), phosphate buffer (PB, pH 5.5− 7.5), Tris−HCl buffer (pH 8.0−9.0), and glycine−NaOH buffer (pH 10.0−11.0). The maximum activity in each group of experiments was defined as 100% relative activity. 2.6. Detection of Paraoxon and Assessment of Practicability. The detection of paraoxon was conducted in PB (50 mM, pH 7.4) containing 50 μM CoCl2, mineralized cells (OD600 nm = 0.1), and varying concentrations of paraoxon (0.2−250 μM) at 37 °C for 10 min. After removing the mineralized cells, the absorbance at 410 nm was measured. All experiments were repeated three times. To assess the applicability of mineralized cells, the above standard assay was used to detect paraoxon in real samples (tap water, seawater, and sewage). The samples were filtered through 0.22 μm filter. Prior to measurement, the pH and ionic strength of the samples were adjusted to be consistent with the PBS buffer. Each of the samples was independently measured three times. The recovery of this method was researched by the standard-addition method.

Figure 2. XRD patterns of hybrid spindles and bulky Co3(PO4)2· 8H2O.

mineralization time from 3 to 6 h, the increasing amount of Co3(PO4)2·8H2O crystals was deposited on primary crystals and extended along the surface of cell (Figure 3C,D). As the mineralization time was prolonged to 12 h, plenty of Co3(PO4)2·8H2O crystals were deposited onto the surface of cell, and the spindle structure was beginning to form (Figure 3E,F). As for the spindle structure of the final materials (Figure 1B), we infer that due to the mechanics principle, the crystals at middle of cell collapsed inwardly in order to acquire the lowest energy. Based on this inference, if crystals continued to grow, the crystals at middle of cell would collapse inwardly, and the cell would be torn to two spheres. Therefore, the dosage of CoCl2 was increased to 1 mM for typical material synthesis, and the corresponding FE-SEM image was recorded. As shown in Figure S1 (Supporting Information), cells were torn and the avulsions were irregular, supporting the above inference. In addition, for the nondisplayed cell, the biomineralization still occurred (Figure S2), indicating that it is common biomolecules on cytomembrane rather than displayed OPH to provide nucleation sites of inorganic crystals. According to the above results, the cell-templated growth could be divided into three steps (Scheme 1). (1) Since the biomolecules (such as proteins and phospholipids) on the surface of cell formed complexes with Co2+ by electrostatic adsorption and metal complexation,37 a mass of Co2+ gathered onto the surface of cell, and then the primary crystals of Co3(PO4)2·8H2O was formed. (2) The increasing amount of Co3(PO4)2·8H2O crystals was deposited on primary seed crystals and extended along the surface of cell. (3) On the basis of the lowest energy principle and the mechanics principle, the crystals at middle of cell collapsed inwardly to form the final spindle structure. 3.3. Enhanced Enzymatic Performance of the Mineralized OPH-Fused Cells. To illuminate the impact of allosteric regulation on catalytic activity of OPH-fused cell, we used the hydrolysis of paraoxon by OPH-fused cell as a model reaction to evaluate the catalytic activities of OPH-fused cell before and after the biomineralization. The OPH is capable of facilitating the hydrolysis of PNP-substituted OPs, and the resultant PNP exhibits typical absorption peak at 410 nm.36 In the case of mixture of OPH-fused cell and 0.5 mM paraoxon, the absorbance at 410 nm (A410) was 0.8 (Figure 4, yellow line). Surprisingly, the A410 value reached 3.2 for the mixture of OPH-fused cell-Co3(PO4)2·8H2O and 0.5 mM paraoxon (Figure 4, red line), which was 3 times higher than that value of OPH-fused cell. It should be mentioned here that the OPH-

3. RESULTS AND DISCUSSION 3.1. Synthesis and Structure Characterization of Materials. Cell-Co3(PO 4) 2·8H2O hybrid spindles were obtained by adding CoCl2 into PBS solution containing OPH cells. After 3 days of incubation at room temperature, a purple precipitate appeared with spindle-like morphology (Figure 1). As shown in the FE-SEM images (Figure 1), mineralized cells presented spindle-like morphology with the length of 1 μm and diameter of 450 nm (Figure 1A). The TEM images further confirmed the spindle-like morphology with lamellar structure (Figure 1B,C). The EDX analysis of mineralized cells indicated the presence of cell and Co3(PO4)2·8H2O in the bioinorganic hybrid (Figure 1D). As shown in XRD patterns of as-prepared cell-Co3(PO4)2·8H2O hybrids and bulk Co3(PO4)2·8H2O (Figure 2), all diffraction peaks were consistent with the characteristic peaks of cell-Co3(PO4)2·8H2O (JCPDS 41-0375), indicating the formation of Co3(PO4)2·8H2O on the mineralized cells. It is noteworthy that bulky Co3(PO4)2·8H2O was synthesized with higher concentration of CoCl2 (10 mM) in PBS solution without cells, while bulky Co3(PO4)2·8H2O was not obtained with low concentration of CoCl2 (0.5 mM). Thus, we could deduce that the surface of cells provided nucleation sites for Co3(PO4)2·8H2O crystals in the presence of Co2+. 3.2. Formation Mechanism of Mineralized OPH-Fused Cells. To study the formation mechanism of OPH-cellCo 3 (PO 4 ) 2·8H2 O hybrid spindles, FE-SEM analysis of mineralized OPH cells at different growth stages was conducted. As shown in Figure 3A,B, the biomolecules on the surface of cell provided the nucleation sites and the primary crystals of Co3(PO4)2·8H2O was formed. With the increase of 6896

DOI: 10.1021/acsami.6b15992 ACS Appl. Mater. Interfaces 2017, 9, 6894−6901

Research Article

ACS Applied Materials & Interfaces

Figure 3. TEM images of the OPH-cell-templated mineralization after 3 (A), 6 (C), and 12 h (E). (B), (D), and (F) are the enlarged view of the pane-marked areas of (A), (C), and (E), separately.

Co3(PO4)2·8H2O, the mixture of paraoxon and cell-Co3(PO4)2· 8H2O (prepared with nondisplayed cell template) exhibited very low absorbance (Figure 4, green line), which was nearly the same value of paraoxon (Figure 4, blue line), indicating that neither Co3(PO4)2·8H2O nor nondisplayed cell had catalytic activity.

bacteria amounts in both cases were the same. In addition, all the reactions were conducted in PBS solution containing 50 μM CoCl2 which had been saturated for Co2+-dependent OPH in the reaction systems, eliminating the possibility that dissociation of Co3(PO4)2·8H2O could promote the activity of OPH. In a control experiment, to eliminate the possibility that the catalytic activity resulted from nondisplayed cell6897

DOI: 10.1021/acsami.6b15992 ACS Appl. Mater. Interfaces 2017, 9, 6894−6901

Research Article

ACS Applied Materials & Interfaces Scheme 1. Schematic Diagram of the Possible Mechanism To Form Hybrid Spindles

Scheme 2. Schematic Diagram of the Enhanced Enzymatic Activity after Mineralization of OPH Cell

efficiency of the biocatalytic system. Thus, we prepared the hybrid materials which had the different thickness of crystal layer by using the different Co2+ concentrations. The catalytic activities of these materials were detected and compared. As the thickness of crystal layer increased, the reaction rate first increased and then decreased, and the mineralized cells in typical synthesis experiment were optimal for catalytic activity (Figure S3). To a certain extent, the amount of Co3(PO4)2· 8H2O could improve the activity of OPH. However, the thicker layer of Co3(PO4)2·8H2O would encapsulate OPH and then reduce the probability of binding of substrates with active sites on account of mass transfer limitation, therefore reducing the apparent catalytic activity. Considering that recombinant OPH in E. coli is hard to be obtained due to the formation of insoluble inclusion bodies within the cells43 and pure OPH is not commercially available, the proposed cell−inorganic hybrid materials can avoid the use of pure OPH−inorganic hybrid materials, indirectly demonstrating the allosteric effect of pure OPH with inorganic crystals. Besides the enhanced activity, the stability of mineralized cells was also improved compared with the original OPH cells. The mineralized OPH cells still remained over 60% of its original activity at pH 6−11, while the OPH cells only remained about 50% of its original activity at pH 7−9 (Figure 5A). On the other hand, the mineralized OPH cells still remained over 90% of original activity at 60 °C and over 50% of original activity at 70 °C, while the OPH cells only remained about 50% of original activity at 60 °C and about 10% of its original activity at 70 °C (Figure 5B). These results were consistent with the usual fact that the stability of enzyme could be enhanced after immobilization. But the conventional immobilization generally reduces the activity of enzymes. Fortunately, both activity and stability were enhanced after mineralization. 3.4. Detection of Paraoxon and Assessment of Practicability. To confirm the significance of the proposed hybrid biocatalytic system, the spectrophotometric detection of paraoxon was conducted by using the prepared hybrid spindles. Paraoxon is effective constituent of common organophosphorus pesticide and is considered to be a kind of environmental pollutant. By measuring the A410 value with varying paraoxon concentration, the calibration curve was plotted using A410 as a function of paraoxon concentration (Figure 6). The regression equation of the calibration curve is y = 0.006x + 0.0203 (R2 = 0.999) with the linear range of 0.2−200 μM paraoxon, which

Figure 4. Absorbance spectra of different mixture in PB solution containing 50 μM CoCl2 after reaction for 10 min.

The above results suggest that the rational design of a bacterium-displayed enzyme−inorganic hybrid biocatalytic system based on allosteric effect had been established. As for enzymology, the allosteric effect is an interesting phenomenon that the binding of a allosteric effector to one site (allosteric site) on an enzyme molecule will bring about conformational change so as to indirectly regulate the property of another specific site (the active site) on the same enzyme molecule.38,39 It has been proven that on account of the interaction with Co2+, the free OPH showed the allosteric effect from the inactive form to the active form.40 However, the free cobalt ion is greatly different from the solid-state cobalt salt. To our knowledge, for some enzymes, both the allosteric effects with ion and crystal can be simultaneously realized. For instance, the α-amylase is a kind of Ca2+-containing enzyme. In aqueous solution, most α-amylases are “active” with the enabled active sites in the presence of Ca2+, whereas most of them are “inactive” with the inhibited active sites in the absence of Ca2+. Ca2+ ions act as the switch to turn the spatial conformation of α-amylase from “inactive” form to “active” form and then improve the catalytic performance of α-amylase.41 For the αamylase-incorporated CaHPO4 crystal, the allosteric effect induced by Ca2+ still existed. It is noteworthy that the “active” form was “fixed” for α-amylase embedded into CaHPO4 crystal, while the allosteric phenomenon between “inactive” form and “active” form is balanced dynamically for α-amylase in an aqueous solution containing Ca2+ ions.33 This contribution is the first example about the allosteric effect of OPH with solidstate cobalt. As shown in Scheme 2, after the deposition of Co3(PO4)2·8H2O onto the surface of cell, effector Co2+ induced the positive modulation of allosteric effect and the displayed OPH kept in active form. The key point of the tremendous activity increase is that the embedment in Co3(PO4)2·8H2O was able to “fix” the allosteric state (active form) of enzyme induced by the allosteric activator (Co2+).42 Evidently, the thickness of crystal layer would influence the 6898

DOI: 10.1021/acsami.6b15992 ACS Appl. Mater. Interfaces 2017, 9, 6894−6901

Research Article

ACS Applied Materials & Interfaces

Table 1. Determination of Paraoxon in Real Samples

a

p-nitrophenyl OPs

tap water

seawater

sewage

detected (μM) added paraoxon (μM) found paraoxon (μM) RSD (%, n = 5) recovery (%)

−a 5.00 4.92 2.7 98.4

−a 5.00 4.89 2.6 97.8

1.06 5.00 6.17 2.9 102.2

Not detectable.

The relative standard deviation (RSD) values were within 3%, indicating high precision and good reproducibility of the proposed method for the practical detection. By the standardaddition method, the recoveries were 97.8−102.2%, demonstrating that the proposed method had excellent accuracy and reliability.

4. CONCLUSIONS In summary, cell surface-displayed OPH−inorganic hybrid materials were rationally fabricated on the basis of the allosteric effect by the biomineralization of Co3(PO4)2·8H2O onto the surface of organophosphorus hydrolase (OPH)-fused cells. The synthetic method was facile, green, and cost-effective, eliminating the separation, purification, and immobilization of enzyme. The as-prepared whole-cell biocatalysts showed enhanced enzymatic activity and stability. On the basis of the experimental results and theory, we provided some explanation about the spindle morphology and enhanced activity. Considering the excellent performance, the bioinorganic hybrid catalysts were applied to the sensitive biosensing of paraoxon and showed lower limit of detection than that of the original counterpart. The as-proposed strategy based on the combination of biomineralization and microbial cell surface display technology would provide a novel model promising to develop a wide range of whole-cell biocatalysts for the applications on industrial catalysis, biosensing, and environmental governance.

Figure 5. Comparison of (A) pH and (B) temperature stability of mineralized cells (a) and original cells (b).

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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.6b15992. Figures S1−S3 and Table S1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (A.L.). ORCID

Lei Han: 0000-0003-1955-0718

Figure 6. Calibration curve for paraoxon using OPH-cell-Co3(PO4)2· 8H2O hybrid biocatalytic system.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge financial support from National Natural Science Foundation of China (Nos. 81673172, 21275152, and 21475144).

was wider than previous reports based on various methods (Table S1, Supporting Information).9,44−46 More importantly, the limit of detection (LOD) in our case was calculated to be 0.08 μM paraoxon (S/N = 3), which was lower than the OPHfused cell-based spectrophotometric method (0.2 μM)9 and purified OPH-based electrochemical sensor (0.12 μM).46 In order to further confirm the practicability, the as-prepared material was applied to the analysis of paraoxon in various real water samples. For each sample, three separate measurements were conducted, and the concentrations of paraoxon were calculated (Table 1) according to the above calibration curve.

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DOI: 10.1021/acsami.6b15992 ACS Appl. Mater. Interfaces 2017, 9, 6894−6901

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DOI: 10.1021/acsami.6b15992 ACS Appl. Mater. Interfaces 2017, 9, 6894−6901

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DOI: 10.1021/acsami.6b15992 ACS Appl. Mater. Interfaces 2017, 9, 6894−6901