A Multi-Metal Based InorganicâProtein Hybrid System for Enzyme

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A Multi-Metal Based Inorganic–Protein Hybrid System for Enzyme Immobilization Sanjay Kumar Singh Patel, Hyunsoo Choi, and Jung-Kul Lee ACS Sustainable Chem. Eng., Just Accepted Manuscript • Publication Date (Web): 24 Jul 2019 Downloaded from pubs.acs.org on July 24, 2019

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ACS Sustainable Chemistry & Engineering

A Multi-Metal Based Inorganic–Protein Hybrid System for Enzyme Immobilization

Sanjay K. S. Patel, Hyunsoo Choi, Jung-Kul Lee*

Department of Chemical Engineering, Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul 05029, Republic of Korea

*Corresponding

author

E-mail: [email protected] (Jung-Kul Lee, Fax: +82-2-458-3504)

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ABSTRACT Here, a novel multi-metal based inorganic-protein hybrid was developed to immobilize laccase as a model enzyme using copper (Cu) and zinc (Zn) metal ions in phosphate-buffered saline. The synthesized multi-metallic Cu3/Zn3(PO4)2-laccase hybrid (Cu/Zn-Lac) showed a significantly higher encapsulation yield (EY) of 96.5% compared with 87.0% with Cu3(PO4)2-laccase (CuLac) and 90.2% with Zn3(PO4)2-laccase (Zn-Lac), respectively. The relative activity (RA) of Cu/Zn-Lac was 1.2, 1.5-, and 2.6-fold higher than Zn-Lac, Cu-Lac, and free enzyme, respectively. The catalytic efficiency of Cu/Zn-Lac was 3.2-fold higher than the free enzyme (71.0 s-1 µM-1). The anodic peak current for the oxidation of 2,2’-azino-bis(3ethylbenzothiazoline-6-sulfonic acid by Cu/Zn-Lac was 2.0- and 2.7-fold higher than Zn-Lac (1.05 µA) and Cu/Lac (0.77 µA), respectively. Remarkably, Cu/Zn-Lac displayed 2.1- and 2.7fold lower charge transfer resistance compared with Zn-Lac (112 Ω) and Cu/Lac (145 Ω), respectively. Under repeated batch conditions, the residual activity of multi-metal hybrids to degrade bisphenol A was 1.9- and 5.1-fold higher than Zn-Lac (43.7%) and Cu-Lac (16.5%), respectively, even after ten cycles of reuse. The multi-metallic system exhibited higher enzyme efficiency, electrochemical substrate oxidation, and degradation potential for bisphenol A compared with individual metal-based hybrid systems. This approach to synthesizing multimetallic based protein hybrids could be extended to enhance the catalytic properties and reusability of enzymes.

KEYWORDS: Bisphenol A; Immobilization; Inorganic-protein hybrid; Laccase; Stability; Reusability


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INTRODUCTION Over the past few years, controllable immobilization of enzymes through encapsulation as inorganic–protein hybrids has received considerable attention due to various structures and properties of the hybrids.1-4 Additionally, recovery of immobilized enzymes during the biotransformation process is economical compared with single use of expensive free enzymes, which is a major limiting factor for their effective implementation in industries.5,6 Immobilization of enzyme through metals as inorganic components and proteins as organic components exhibits variable influences on enzyme properties.1-3 Generally, inorganic–protein hybrids are synthesized through simple three-step processes including nucleation, aggregation, and anisotropic growth to develop a well-ordered flower like structure.1 These nanoflower (NF) based systems have been proved to be more effective in immobilization compared with various types of solid supports due to their large surface area, highly porous nature, and favorable confining environments. In contrast, immobilization on solid supports mostly results in lower efficiency due to their strong interactions, which may lead to significant adverse structural influences or mass transfer limitations.1,3,5,7,8 The structural properties of the metal–protein hybrids were reported to vary with their conditions of synthesis such as use of enzymes and metal ions including copper (Cu), calcium, cobalt (Co), and zinc (Zn) in phosphate buffer.1,2,9-11 Efficient immobilization of various enzymes has been reported through inorganic–protein hybrid systems.1,3, 12-15 Laccase (EC 1.10.3.2) has broad industrial applications including degradation of toxic phenolic compounds such as bisphenol A and synthetic dyes.16-18 Use of immobilized laccase for the degradation of phenolic compounds has been suggested as an effective approach due to its higher stability compared with the free enzyme. Primarily, lower efficiency after immobilization 3 ACS Paragon Plus Environment


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and low enzyme loading on the supports have been recognized as critical issues for its potential applications.17 Laccase activity is highly influenced by the presence of metals ions; laccase exhibits broad substrate specificity.17, 19 In previous studies, laccase was effectively immobilized using the metal–protein hybrid nanoflower system using Cu ions to retain higher relative activity (RA) compared with the free enzyme.1,11 Here, the RA of immobilized laccase was highly influenced by the type of substrate, metal ion concentration, and synthesis conditions used to prepare the inorganic–protein hybrids.1,11,20 Similarly, structural morphology, size, and rigidity of the synthesized inorganic–protein hybrid NF has a significant influence on enzyme immobilization, including loading and RA through variations in the type of metal ions, protein concentration, and synthesis conditions.1-3 Moreover, the soft nature of NF-based enzyme immobilization systems is associated with critical disadvantages including lower stability and reusability for their potential application despite better RA compared with the free enzyme.9,11 Improvement in reusability of enzyme-inorganic hybrids has been demonstrated by altering metal ions such as Cu to Zn using cross-linking approaches to achieve better structural stability of the synthesized NF.6,21 Immobilization of laccase has been largely established as Cu3(PO4)2laccase (Cu-Lac) NFs. Single metal-based metal/protein hybrid assemblies containing Cu or Zn formed NF with soft (Cu) or compact (Zn) structures.11,22 We hypothesized that multi-metal based hybrid NF containing both Cu and Zn might improve the hybrid NFs’ structural morphology, protein loading, and RA, through synergistic effects of Cu and Zn. However, no multi-metal containing metal/protein hybrid NFs have been reported to date. Many amide groups present in proteins are involved in complex formation with metal ions during NF synthesis using a single metal.1,22,23 Thus, simultaneous complex formation of different metals with the amide groups of a protein might be feasible through a self-assembly process in which multiple metals 4 ACS Paragon Plus Environment

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catalyze formation of metal/protein hybrid assemblies. The resulting hybrids are likely to have structural properties that will increase RA and reusability. In the present investigation, we synthesized a unique multi-metal based inorganic–protein hybrid system for the first time, using Cu and Zn metal ions in phosphate buffered saline (PBS) for immobilization of a model enzyme laccase. The novel multi-metal-based Cu3/Zn3(PO4)2laccase (Cu/Zn-Lac) exhibited significant enhancement in RA after immobilization. Cu/Zn-Lac showed high catalytic properties and potential for repeated degradation of bisphenol A compared with synthesized individual metal-based Cu-Lac and Zn-Lac NFs.

RESULTS AND DISCUSSION

Scheme 1 Schematic illustration of multi-metal (copper and zinc) and protein assembly for laccase immobilization as an inorganic–protein hybrid.

The schematic diagram for the synthesis of the novel multi-metal based laccase hybrid system is presented in Scheme 1. Immobilization of laccase at various concentrations was performed in PBS (10 mM) containing CuSO4 and ZnSO4 (each 1 mM) for 24 h at 4°C (Table 1). As controls, free enzyme exhibited 1.2, 1.3, and 1.4-fold higher activity in the presence of Cu (2 mM), Zn (2 mM), and their mixture (1 mM) using 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid 5 ACS Paragon Plus Environment


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(ABTS) as a substrate under the assay conditions (Figure S1).5 Similarly, the RA of free laccase was varied within the range of 108-139%, to give different concentration ratios with 0.5-2.0 mM Cu and Zn metal ions (Table S1). The optimum Cu/Zn ratio was observed to be 1.0/1.0 (mM) to achieve maximum RA for the laccase. An increase in the protein concentration from 0.05 to 1.0 mg mL-1 resulted in lowered encapsulation yields (EY) from 96.5% to 29.1%. In contrast, the weight contents of enzyme in the synthesized multi-metal laccase hybrid as Cu/Zn-Lac showed an increase from 11.4% to 26.2% with an increase in protein concentration from 0.05 to 1.0 mg mL-1. The relative activity (RA) of immobilized laccase was observed in the range of 92.3-256%. In contrast, the synthesized individual metal hybrid of Cu-Lac and Zn-Lac exhibited lower RA of 78.3-172% and 80.4-220% with an EY of 17.4-87.0% and 27.0-90.2%, respectively (Table S2). These results suggested that the multi-metal system of Cu and Zn is more effective for laccase immobilization compared with the mono-metal system. Here, higher RA might be associated with better enzyme activity in the presence of these metal ions because of their synergistic influence. Overall, these synthesized Cu/Zn-Lac hybrids retained significantly higher RA compared with the immobilization of laccase on solid support systems with different covalent and adsorption methods, and nanoparticle-based Cu3(PO4)2-laccases hybrid composites using ABTS as reported previously. 17,24


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Table 1. Immobilization of laccase as a Cu3/Zn3(PO4)2-enzyme hybrid protein (mg ml-1)

EYa (%)

weightb (%)

RAc (%)

0.05

96.5 ± 3.2

11.4 ± 0.9

188 ± 16

0.10

94.1 ± 3.4

16.2 ± 1.5

224 ± 21

0.25

90.0 ± 3.9

21.3 ± 2.1

256 ± 23

0.50

58.5 ± 5.1

23.7 ± 2.5

137 ±12

1.00

29.1 ± 3.0

26.2 ± 2.3

92.3 ± 8.4

a Encapsulation

yields (amount of enzyme immobilized/amount of initial enzyme × 100).

bWeight percentage of enzyme in the nanoflower hybrids. cRelative

activity (total specific activity of immobilized enzyme/total specific activity of free enzyme × 100).

Field emission scanning electron microscopy (FE-SEM) analysis of immobilized laccase as a Cu/Zn-Lac hybrid showed a spherical highly porous nature with petal-like structures (Figure 1ab), whereas Cu-Lac and Zn-Lac hybrids exhibited a flower-like morphology with soft and compact structure in FE-SEM images, respectively (Figure S2). Elemental mapping was evaluated to confirm the components of the synthesized Cu/Zn-Lac hybrids (Figure 1c-g). Here, the presence of Cu, Zn, C, and P suggests effective formation of the Cu3/Zn3(PO4)2 and laccase hybrid. Energy dispersive spectroscopy analysis suggested that the Cu/Zn-Lac hybrid contained Cu and Zn at about 7.95% and 6.08%, respectively (Table S3). The average sizes of 8, 9, and 18 µm were observed for Cu-Lac, Zn-lac, and Cu/Zn-Lac hybrids, respectively. Further, efficient immobilization of laccase was validated using fluorescein isothiocyanate-labelled enzyme-based synthesis of the Cu/Zn-Lac hybrid through confocal laser scanning microscopy analysis (Figure 1h-i). In contrast, FE-SEM analysis of control hybrid synthesis without laccase enzyme exhibited metal aggregates with irregular morphology and an average size of 0.05-0.5 µm (Figure S3). FESEM images of synthetic Cu/Zn-Lac, taken over an incubation period of up to 24 h suggest that multi-metal hybrids utilize a similar mechanism of nanoﬂower self-assembly involving 7 ACS Paragon Plus Environment


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nucleation, aggregation, and anisotropic growth of individual Cu or Zn metal ions (Figure S4).1,22,23 Here, protein molecules form complexes simultaneously with Cu2+ and Zn2+ ions, mainly coordinated by the backbone amide groups of the protein.1 Metal/phosphate nuclei continuously grew through incorporation of enzyme into Cu/Zn-laccase hybrid assemblies. Finally, larger multi-metal/laccase hybrid nanoflowers formed with incubations longer than 24 h. High-resolution transmission electron microscopy images of the hybrids suggested the formation of a crystal lattice structure in the petals of the hybrid structure (Figure S5). The X-ray diffraction (XRD) patterns of Cu-Lac and Zn-Lac showed sharp peaks like the standard Cu3(PO4)2·3H2O (JCPDS 00-022-0548) and Zn3(PO4)2·4H2O (JCPDS 33-1474), respectively (Figure S6).6 The XRD patterns of the Cu/Zn- Lac exhibited sharp and strong peaks like those of both standards, confirming the synthesis of the multi-metal based hybrid (Figure S6).6 In addition, its chemical structure was evaluated by Fourier transform infrared analysis (Figure S7). The sharp peaks observed at 950–1100 and 630 cm-1 may be associated with P-O vibrations of the phosphate groups, and the reduced typical peaks of peptide bonds at 1550–1650 cm-1 agree with the C=O stretching (amide I band, 1650 cm-1) and N-H bending vibrations (amide II band, 1550 cm-1).21 The catalytic efficiency (kcat Km-1) values of free and immobilized laccases as Cu-Lac, Zn-Lac, and Cu/Zn-Lac are presented in Table S4. The Cu-Lac, Zn-Lac, and Cu/Zn-Lac showed lower Km values of 22.9, 22.0, and 18.7 µM, respectively, compared with the free enzyme value of 29.3 µM for ABTS. The kcat Km-1 was enhanced 3.2-fold through the multi-metal hybrid Cu/Zn-Lac compared with values of 2.1 and 2.4-fold with Cu-Lac and Zn-Lac over the free enzyme (71.0 s-1 µM‑1).


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Figure 1. FE-SEM (a), high resolution (b) images, and elemental mapping images of carbon, oxygen, copper, phosphorus, and zinc (c-g) in the multi-metal based Cu3/Zn3(PO4)2-laccase hybrid (Cu/Zn-Lac) synthesized using 0.25 mg mL-1 of total protein, 1 mM of CuSO4, and 1 mM of ZnSO4 in 50 mL of phosphate-buffered saline (10 mM, pH 7.4) incubated for 24 h at 4°C. Confocal laser scanning microscopy images of the Cu/Zn-Lac hybrid synthesized with the fluorescein isothiocyanate labeled laccase in the green channel (h) and in bright field (i).



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Figure 2. Electrochemical properties of immobilized laccase. (a) Cyclic voltammogram curves of bare glassy carbon electrode (GCE), GCE/Cu/Zn-Lac, GCE/Cu-Lac, and GCE/Zn-Lac, and (b) Nyquist plots of immobilized laccase hybrids. Here, enhancement in the kcat Km-1 value for Cu/Zn-Lac might be associated with either a cooperative effect of the immobilized enzymes, increased affinity towards the substrate or suitable confinement of the enzyme in the hybrids.1,11,20 Circular dichroism analysis suggested that the assembly of laccase as Cu-Zn-Lac did not significantly alter the secondary structure of the enzyme after immobilization (Figure S8). Further, to confirm the high catalytic potential of Cu/Zn-Lac, cyclic voltammogram analysis was performed using ABTS. The cathodic and anodic peak currents were detected for the reduction and oxidation of ABTS in the two-step reaction (ABTS to ABTS+• and ABTS+• to ABTS2+).25 Cu/Zn-Lac exhibited higher oxidation and reduction peak currents, with potentials of 0.56 and 0.49 V, respectively, for ABTS to ABTS+• compared with Cu/Lac and Zn-Lac (Figure 2a). The maximum anodic peak currents of Cu/Lac, Zn-Lac, and Cu/Zn-Lac were 0.77, 1.05, and 2.09 µA for the catalytic oxidation of ABTS, respectively. Further, electrochemical impedance spectroscopy (EIS) analysis was performed to monitor the charge transfer resistance. The Nyquist plot (-Zim vs. Zre) of the EIS analysis showed 10 ACS Paragon Plus Environment

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the charge transfer resistance of 145, 112, and 54 ohms for the Cu/Lac, Zn-Lac, and Cu/Zn-Lac, respectively (Figure 2b). Overall, these results suggested that Cu/Zn-Lac showed significantly higher catalytic oxidation of ABTS and lower charge transfer resistance compared with both CuLac and Zn-Lac hybrid NFs.

Figure 3. Degradation of bisphenol A (100 µM) by free enzyme (FE) or laccase hybrids. Profile (a), degradation in the presence of HBT (0.1 mM) as a mediator upon incubation for 6 h (b), repeated batch degradation of bisphenol A by laccase hybrids (c), and current for the oxidation of ABTS (0.25 mM) at 0.56 V (d).



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Bisphenol A is a well-known toxic compound for aquatic fauna through a variable influence on their reproductive functions.5,26 It is employed widely for making plastics and epoxy resins. To investigate the potential application of immobilized laccase as Cu/Zn-Lac, degradation of bisphenol A was performed (Figure 3). Compared with 54.2% degradation by free laccase, CuLac, Zn-Lac, and Cu/Zn-Lac showed high amounts of degradation (72.8, 79.2 and 89.4%, respectively) after 12 h of incubation (Figure 3a). Bisphenol A degradation was also influenced by the pH of the medium.26 Therefore, the degradation was evaluated in different buffers of pH 3-7 by incubation for 6 h (Figure S9). The optimum pH value for the effective degradation of bisphenol A was 5.0 with degradation efficiencies for free laccase, Cu-Lac, Zn-Lac, and Cu/ZnLac of 46.4, 65.9, 72.6, and 85.5%, respectively. Overall, Cu/Zn-Lac exhibited broad pH stability for bisphenol A degradation. Remarkably, the bisphenol A degradation potential of Cu/Zn-Lac was 4.3-fold higher than that of the free enzyme at pH 7, which might be correlated with higher enzyme stability. As the degradation of phenolic compounds is significantly influenced by the presence of mediators,5 the degradation was performed in the presence of laccase mediator 1-hydroxy benzotriazole (HBT) at a concentration of 0.1 mM. After 6 h of incubation, Cu/Zn-Lac exhibited almost complete degradation (99.2%), whereas free laccase, Cu-Lac, and Zn-Lac resulted in 61.9, 82.1, and 85.3% degradation, respectively (Figure 3b). Here, Cu/Zn-Lac resulted in higher degradation potential than the maximum with up to 80.0% degradation by multi-walled carbon nanotubes-modified laccase-carrying electrospun fibrous membranes.26 After storage at 25 °C, Cu-Lac, Zn-Lac, and Cu/Zn-Lac showed residual bisphenol A degradation activities of 91.7, 95.3 and 97.8%, respectively, whereas free laccase lost more than 91% of its activity after 5 days of incubation (Figure S10). To evaluate the robustness of synthesized Cu/Zn-Lac NF, repeated batch degradation of bisphenol A was performed (Figure 12 ACS Paragon Plus Environment

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3c). After five and ten cycles of reuse, Cu/Zn-Lac showed a relative degradation potential of 91.9% and 84.7%, respectively. In contrast, Cu-Lac and Zn-Lac showed significantly lower relative degradation efficiency of 16.5% and 43.7% after 10 cycles of reuse, respectively. Here, the reduction in bisphenol A degradation efficiency can be correlated with the low current for the oxidation of ABTS to ABTS+• by immobilized laccase after reuse (Figure 3d). Further, the better structural stability of reused Cu/Zn-Lac NF under repeated batch conditions compared with the fragile structure of Cu-Lac was confirmed using FE-SEM analysis (Figure S11). Remarkably, Cu/Zn-Lac showed higher potential for the degradation of bisphenol A under repeated batch conditions compared with laccase immobilized on polyamide 6/chitosan nanofibers and titanium nanoparticles.27,28 Laccase immobilized on these supports lost 40% and 50% bisphenol A degradation ability after only three and five cycles, respectively.27,28 The degradation mechanism of bisphenol A by T. versicolor laccase is well-known, and has been widely reported.29,30 Here, the major observed degradation products of bisphenol A were 4isopropenylphenol and 4-methylstyrene, as described previously (Figure S12).29

CONCLUSIONS In conclusion, for the first time, the multi-metal (Cu and Zn) NF based enzyme assembly system was proposed to improve the properties compared with either the free form of the enzyme or individual metal ion synthesized NF in this study. We observed that RA, electrochemical properties, and reusability of immobilized enzymes were significantly improved through synthesis of Cu/Zn-Lac compared with individual metal ion NF. Overall, Cu/Zn-Lac exhibited 1.9- and 5.1-fold higher potential for bisphenol A degradation compared with that by Zn-Lac and Cu-Lac NF under repeated batch conditions, respectively. These results show that multi-metal 13 ACS Paragon Plus Environment


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NF-based inorganic-protein hybrids are more beneficial than individual metal-based hybrids, and they can be applied to immobilize various enzymes for sustainable applications.

ASSOCIATED CONTENT Supplementary Information The detailed experimental methods, effect of metal ions ratio on the activity of free laccase (Table S1), immobilization of laccase as an inorganic-protein hybrid (Table S2), energy dispersive spectroscopy analysis of inorganic-protein hybrid (Table S3), kinetic parameters of the free enzyme and inorganic-protein hybrids (Table S4), activity of free laccase in the presence of metal ions (Figure S1), FE-SEM images of hybrid NFs synthesized using metal ions (Figure S2), FE-SEM images and elemental mapping of hybrids and controls (Figure S3), proposed selfassembly mechanism of multi-metal based hybrid nanoflowers and FE-SEM images of synthesized hybrids (Figure S4), high resolution TEM images of hybrid NFs (Figure S5), XRD patterns of hybrid NFs (Figure S6), FTIR spectra of hybrid NFs (Figure S7), CD spectra of free and immobilized laccase hybrid (Figure S8), effect of pH on the degradation of bisphenol A by free enzyme or laccase hybrids (Figure S9), stability during room temperature storage of free enzyme and NFs (Figure S10), FE-SEM images of immobilized laccase hybrid NFs after 10 cycles of repeated degradation of bisphenol A (Figure S11), and HPLC analysis of the products of laccase-mediated degradation of bisphenol A (Figure S12).

AUTHOR INFORMATION Corresponding Author E-mail: [email protected] (Jung-Kul Lee) Fax: (+82) 2-458-3504 14 ACS Paragon Plus Environment

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Notes The authors declare no competing interest.

ACKNOWLEDGEMENT This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2017R1A2B3011676, 2017R1A4A1014806, 2019R1C1C1009766). This work was supported by KU Research Professor program of Konkuk University.

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Table of Contents

A novel multi-metal based inorganic–protein hybrid exhibits higher catalytic efficiency and bisphenol A degradation compared with individual metal-based hybrids.


A Multi-Metal Based InorganicâProtein Hybrid System for Enzyme

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A Multi-Metal Based InorganicâProtein Hybrid System for Enzyme