Biomimetic Mineralization of Cytochrome C Improves the Catalytic Efficiency and Confers a Functional Multi-Enzyme Composite

catalysts

Article Biomimetic Mineralization of Cytochrome c Improves the Catalytic Eﬃciency and Confers a Functional Multi-Enzyme Composite

Xiao-Qing Gong 1, Chuan-Wan Wei 1, Jia-Kun Xu 2, Xiao-Juan Wang 1,*, Shu-Qin Gao 3 and Ying-Wu Lin 1,3,4,*

1 School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China 2 Key Lab of Sustainable Development of Polar Fisheries, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences; Laboratory for Marine Drugs and Byproducts of Pilot National Lab for Marine Science and Technology, Qingdao 266071, China 3 Laboratory of Protein Structure and Function, University of South China, Hengyang 421001, China 4 Hunan Key Laboratory for the Design and Application of Actinide Complexes, University of South China, Hengyang 421001, China * Correspondence: [email protected] (X.-J.W.); [email protected] or [email protected] (Y.-W.L.); Tel.: +86-743-8578079 (X.-J.W.); +86-734-8282375 (Y.-W.L.) Received: 4 July 2019; Accepted: 25 July 2019; Published: 29 July 2019

Abstract: The encapsulated enzyme system by metal-organic frameworks (MOFs) exhibits great potential in biofuel cells, pharmaceuticals, and biocatalysis. However, the catalytic efficiency and the enzymatic activity are severely hampered due to enzyme leaching and deficiency of storage stability. In this study, we immobilized cytochrome c (Cyt c) into dimethylimidazole-copper (Cu(Im)2) by biomimetic mineralization, and constructed a bioinorganic hybrid material, termed Cyt c@Cu(Im)2. Encapsulated Cyt c in Cu(Im)2 with a nanosheet structure exhibited significantly improved catalytic efficiency, enzymatic activity and kinetic performance. The catalytic efficiency (kcat/Km) for Cyt c@Cu(Im)2 was ~20-fold higher compared to that of free Cyt c. Moreover, the increased activity was not affected by long-term storage. Based on this system, we further constructed a multi-enzyme composite with glucose-oxidase (GOx), termed GOx-Cyt c@Cu(Im)2, which exhibited greatly improved enzymatic activity, stability, and excellent selectivity for the detection of low concentrations of glucose. This strategy may provide new insights into the design of enzymes with high activity and stability, as well as the construction of multi-enzyme systems.

Keywords: enzymatic immobilization; biomimetic mineralization; multi-enzyme; bioinorganic hybrid material; biosensors

1. Introduction Enzymes have been widely applied as catalysts inbiological systems and chemical systems, in an efficient and green manner [1–4]. Compared to synthetic catalysts, enzymes are often of high selectivity (i.e., chemo-, region-, stereo-) and minimal by-product generation [5–8]. However, the instability and poor reusability of natural enzymes restrict their potential applications. Enzyme encapsulation within a protective shell is a simple and efficient method to improve the stability and reusability of naturalenzymes [9–11]. Metal-organic frameworks (MOFs) are usually constructed by linking organic small molecules and metal ions, which can offer superior thermal and chemical protection for biomacromolecules such as proteins, as compared to other materials such as mesoporous silica and calcium carbonate [12]. However, MOFs usually possess a pore with dimensionssignificantly smaller than those of biomacromolecules, which may block the mass transfer of the enzyme and decrease the

Catalysts 2019, 9, 648; doi:10.3390/catal9080648 www.mdpi.com/journal/catalysts Catalysts 2019, 9, 648 2 of 10 enzymatic activity [13,14]. Therefore, a general method to allow biomacromolecules to fully display their biological functions is still a challenge in the field of enzyme immobilization. Natural organisms have developed bio-mineralization strategies to deposit minerals in their structural frameworks. The bio-minerals can both provide effective stabilization for embedded enzymes and allow them to fully display biological functions [15–19]. Inspired by the sophisticated bio-inorganic hybrid structures in nature, it is desirable to prepare enzyme-inorganic mineral hybrid composites with both high enzyme activity and stability by biomimetic mineralization. Cytochrome c (Cyt c), as a model enzyme, is widely used in the field of biochemical analysis [20–23], as well as for the preparation of enzyme-MOF composites, due to its inherent peroxidase activity [16,24–26]. Therefore, we aimed to construct a Cyt c-MOF analog by biomimetic mineralization to obtain an efficient catalytic activity and stability. Moreover, multi-enzyme systems are a series of cascade enzymatic reactions completed by the synergistic effect of multiple enzymes, which have attracted great attention in recent years because of their important applications, such as in biosensors [27,28], biocatalysis [29,30], and pharmaceuticals, etc. [31,32]. As shown in this study, we constructed Cyt c@Cu(Im)2 by biomineralization of Cyt c into dimethylimidazole-copper (Cu(Im)2). The assembly of Cyt c and glucose-oxidase (GOx) formed a multi-enzyme of GOx and Cyt c encapsulated into Cu(Im)2. The prepared Cyt c@Cu(Im)2 composite exhibited an excellent stability and catalytic efficiency. Moreover, the encapsulated multi-enzyme GOx-Cyt c@Cu(Im)2 can be used to detect low concentrations of glucose with excellent selectivity.

2. Results and Discussion

2.1. Preparation and Characterization of Enzyme@Cu(Im)2 The preparation process of the composite material containing enzymes was shown in Figure S1. The synthesis of enzyme@Cu(Im)2 was by mixing enzyme, dimethylimidazole (Im) and CuSO4, and the incubation time was no less than 8 h to ensure adequate enzyme biomimetic mineralization. Using this procedure, we constructed both Cyt c@Cu(Im)2 and GOx-Cyt c@Cu(Im)2. To remove non-encapsulated enzymes, all composite materials were centrifuged and washed with double distilled water for several times before vacuum freeze-drying. From Figure S2, it was observed that the encapsulated efficiency of enzymes was more than 99%. The as-prepared materials were characterized by various experimental methods. The scanning electron microscopy (SEM) was applied to obtain the morphology of the prepared materials. The microstructure of Cu(Im)2 presented interlaced sheets (Figure1a), which were somewhat similar to previously reported cupric phosphate nano-flowers [33]. Moreover, these sheets slightly bended to form petals like in Cyt c@Cu(Im)2 (Figure1b), similar to that of the pure Cu(Im) 2. These observations suggest that Cytc has little effect on the structure and morphology of Cu(Im)2. In contrast, GOx-Cyt c@Cu(Im)2 showed an uneven and porous morphology, similar to GOx@Cu(Im)2 (Figure S3), which is due to the coherent interactions of co-assembly between GOx and Cyt c. The X-ray diffraction (XRD) was performed for Cu(Im)2, and Cyt c@Cu(Im)2. As shown in Figure S4, the results showed that the characteristic diffraction peaks of prepared Cyt c@Cu(Im)2 were almost the same as that of Cu(Im)2, indicating that the structure of Cu(Im)2 was almost not affected by the encapsulation of Cyt c, which is in agreement with the results of SEM (Figure1). It is well known that protein molecules have typical Fourier transform infrared spectroscopy (FTIR) 1 1 stretch characteristics at 1640–1660 cm− and 1510–1560 cm− , which correspond to the C=O stretching mode, NH bending and CN stretching modes on the peptide chain [16,26]. These characteristic peaks can be clearly identified in the Cyt c and other composites of mineralizing enzymes (Figure2a), but were absent in the material of Cu(Im)2 prepared in the absence of enzymes, indicating the incorporation of enzymes in the matrix of Cu(Im)2. Thermal gravimetric analysis (TGA) under nitrogen atmosphere also confirmed the embedment of protein into the Cu(Im)2 (Figure2b). In the range of less than 200 ◦C, the mass loss is mainly due to Catalysts 2019, 9, x FOR PEER REVIEW 3 of 10

Catalysts 2019, 9, 648 3 of 10 the loss of water and unreacted reagent [14]. The second decomposition stage was observed for all enzyme@Cu(Im)2 samples, 25.39% (Cyt c@Cu(Im)2), 25.76% (GOx-Cyt c@Cu(Im)2) between 200 ◦C Figure 1. SEM images of (a) Cu(Im)2 and (b) Cyt c@Cu(Im)2. and 370 ◦C (Table1). This is attributed to thermal decomposition of the encapsulated enzymes [ 34], which further indicates the successful enzyme encapsulation. CatalystsIt is2019 well, 9, xknown FOR PEER that REVIEW protein molecules have typical Fourier transform infrared spectroscopy3 of 10 (FTIR) stretch characteristics at 1640–1660 cm−1 and 1510–1560 cm−1, which correspond to the C=O stretching mode, NH bending and CN stretching modes on the peptide chain [16,26]. These characteristic peaks can be clearly identified in the Cyt c and other composites of mineralizing enzymes (Figure 2a), but were absent in the material of Cu(Im)2 prepared in the absence of enzymes, indicating the incorporation of enzymes in the matrix of Cu(Im)2. Thermal gravimetric analysis (TGA) under nitrogen atmosphere also confirmed the embedment of protein into the Cu(Im)2 (Figure 2b). In the range of less than 200 °C, the mass loss is mainly due to the loss of water and unreacted reagent [14]. The second decomposition stage was observed for all enzyme@Cu(Im)2 samples, 25.39% (Cyt c@Cu(Im)2), 25.76% (GOx-Cyt c@Cu(Im)2) between 200 °C and 370 °C (Table 1). This is attributed to thermal decomposition of the encapsulated enzymes [34], which further indicates the successful enzyme encapsulation. FigureFigure 1.1. SEMSEM imagesimages ofof ((aa)) Cu(Im)Cu(Im)22 and (b)) CytCyt c@Cu(Im)2..

It is well known that protein molecules have typical Fourier transform infrared spectroscopy (FTIR) stretch characteristics at 1640–1660 cm−1 and 1510–1560 cm−1, which correspond to the C=O stretching mode, NH bending and CN stretching modes on the peptide chain [16,26]. These characteristic peaks can be clearly identified in the Cyt c and other composites of mineralizing enzymes (Figure 2a), but were absent in the material of Cu(Im)2 prepared in the absence of enzymes, indicating the incorporation of enzymes in the matrix of Cu(Im)2. Thermal gravimetric analysis (TGA) under nitrogen atmosphere also confirmed the embedment of protein into the Cu(Im)2 (Figure 2b). In the range of less than 200 °C, the mass loss is mainly due to the loss of water and unreacted reagent [14]. The second decomposition stage was observed for all enzyme@Cu(Im)2 samples, 25.39% (Cyt c@Cu(Im)2), 25.76% (GOx-Cyt c@Cu(Im)2) between 200 °C and 370 °CFigure (Table 2. (1).a) FTIRThis spectrais attributed of Cyt toc, Cu(Im)thermal, Cytdecompositionc@Cu(Im) and of GOx-Cytthe encapsc@Cu(Im)ulated enzymes;(b) TGA [34], curves which Figure 2. (a) FTIR spectra of Cyt c, Cu(Im)22, Cyt c@Cu(Im)2 and GOx-Cyt c@Cu(Im)2; (b) TGA curves furtherof Cu(Im)indicates, enzyme@Cu(Im) the successful enzymein N atmosphere. encapsulation. of Cu(Im)2, enzyme@Cu(Im)22 in N2 2atmosphere.

Table 1. Mass loss (%) of Cu(Im)2 and enzyme@Cu(Im)2 composite. Table 1.Mass loss (%) of Cu(Im)2 and enzyme@Cu(Im)2 composite. Mass Loss (%) Mass Loss (%) Sample Mass Loss (%) Mass Loss (%) Total Mass Loss (%) Sample T < 200 ◦C 200 ◦C < T < 370 ◦C Total Mass Loss (%) T ˂ 200 °C 200 °C ˂ T ˂ 370 °C Cu(Im)2 6.99 10.48 32.46 Cu(Im)2 6.99 10.48 32.46 Cyt c@Cu(Im)2 12.53 25.39 52.33 Cyt c@Cu(Im)2 12.53 25.39 52.33 GOx-Cyt c@Cu(Im)2 12.61 25.76 49.58 GOx-Cyt c@Cu(Im)2 12.61 25.76 49.58

To further prove the embedmentembedment of enzymesenzymes intointo Cu(Im)Cu(Im)22, fluorescence ﬂuorescence labeling assay was carried out by Delta Vision Elite high-resolution microscope. The Cyt c and GOx were labeled using FITC and RhB, respectively, and the labeledlabeled enzymes were encapsulatedencapsulated into Cu(Im)2 by using the same procedure. As As shown shown in in Figure Figure 33,, green,green, redred andand yellow-greenyellow-green ﬂuorescencefluorescence waswas observedobserved forfor Figure 2. (a) FTIR spectra of Cyt c, Cu(Im)2, Cyt c@Cu(Im)2 and GOx-Cyt c@Cu(Im)2; (b) TGA curves (FITC-GOx), (RhB-Cyt c) and (GOx-RhB-Cyt c-FITC@Cu(Im)2),), respectively, respectively, which further indicates the successful of Cu(Im) 2encapsulation , enzyme@Cu(Im) of2 singlein N2 atmosphere. enzyme and multi-enzyme.

2.2. Peroxidase ActivityTable Assay 1.Mass of Free loss Cyt (%) c of and Cu(Im) Cyt c@Cu(Im)2 and enzyme@Cu(Im)2 2 composite.

Mass Loss (%)c Mass Loss (%) The peroxidaseSample catalytic activity of Cyt @Cu(Im)2 was eﬀected by variousTotal factors. Mass ForLoss example, (%) the pH value plays an important roleT in ˂ the200 enzymatic°C activity.200 °C ˂ The T ˂ enzymes370 °C were severely inactivated Cu(Im)2 6.99 10.48 32.46 under highly acidic or alkaline conditions. As shown in Figure S5a, Cyt c@Cu(Im)2 showed the highest catalytic activityCyt c@Cu(Im) at pH2 7.4, which is di12.53ﬀerent from that of free 25.39 Cyt c (at ~pH6.5). 52.33 Moreover, Cyt GOx-Cyt c@Cu(Im)2 12.61 25.76 49.58 c@Cu(Im)2 showed a higher catalytic activity compared to that of free Cyt c under the same conditions

To further prove the embedment of enzymes into Cu(Im)2, fluorescence labeling assay was carried out by Delta Vision Elite high-resolution microscope. The Cyt c and GOx were labeled using FITC and RhB, respectively, and the labeled enzymes were encapsulated into Cu(Im)2 by using the same procedure. As shown in Figure 3, green, red and yellow-green fluorescence was observed for (FITC-GOx), (RhB-Cyt c) and (GOx-RhB-Cyt c-FITC@Cu(Im)2), respectively, which further indicates the successful encapsulation of single enzyme and multi-enzyme.

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as a result of biomineralization. For example, the catalytic reaction rate of Cyt c@Cu(Im)2 was faster than that of free enzyme (Figure4a), which is consistent with the chromogenic reaction (Figure 4a, insert.) CatalystsFigure 2019, 3.9, xHigh FOR resolutionPEER REVIEW microscope image of (a) GOx@Cu(Im)2−RhB, (b) Cyt c@Cu(Im)2−FITC and4 of 10 (c) GOx−RhB and Cyt c−FITC@Cu(Im)2 (scale bar is 15 µm).

2.2. Peroxidase Activity Assay of Free Cyt c and Cyt c@Cu(Im)2

The peroxidase catalytic activity of Cyt c@Cu(Im)2 was effected by various factors. For example, the pH value plays an important role in the enzymatic activity. The enzymes were severely inactivated under highly acidic or alkaline conditions. As shown in Figure S5a, Cyt c@Cu(Im)2 showed the highest catalytic activity at pH 7.4, which is different from that of free Cyt c (at ~pH6.5). Moreover, Cyt c@Cu(Im)2 showed a higher catalytic activity compared to that of free Cyt c under the same conditions as a result of biomineralization. For example, the catalytic reaction rate of Cyt c@Cu(Im)2 was faster than that of free enzyme (Figure 4a), which is consistent with the chromogenic Figure 3. High resolution microscopemicroscope imageimage ofof (a(a)) GOx@Cu(Im) GOx@Cu(Im)2−RhB,RhB, ((bb)) CytCyt cc@Cu(Im)@Cu(Im)2−FITC and reaction (Figure 4a, insert.) 2− 2− (c) GOxGOx−RhB and Cyt c−FITC@Cu(Im)FITC@Cu(Im)2 (scale(scale bar bar is is 15 15 µm).µm). − − 2

2.2. Peroxidase Activity Assay of Free Cyt c and Cyt c@Cu(Im)2

FigureFigure 4.4. ((aa)) TheThe kinetic kinetic analysis analysis of of free free Cyt Cytc and c and Cyt Cytc@Cu(Im) c@Cu(Im)2, the2, the chromogenic chromogenic reaction reaction of free of Cytfree

cCyt(insert c (insert A) and A) Cytand cCyt@Cu(Im) c@Cu(Im)2 (insert2 (insert B); ( bB)); The (b) activityThe activity of (1) of free (1) Cytfree cCyt, (2) c Cyt, (2) cCyt/copper c/copper ion. (3)ion. Cyt (3)

cCyt/Cu(Im) c/Cu(Im)2, (4)2 Cyt, (4) cCyt/Im c and/Im (5)and Cyt (5) cCyt@Cu(Im) c@Cu(Im)2, respectively.2, respectively.

Moreover, we prepared several non-mineralization composites, including Cyt c, Cyt c/CuSO , Cyt Moreover, we prepared several non-mineralization composites, including Cyt c, Cyt c/CuSO4 4, c/Cu(Im) and Cyt c/Im. The experimental results showed these materials had poor catalytic eﬃciency Cyt c/Cu(Im)2 2 and Cyt c/Im. The experimental results showed these materials had poor catalytic comparedefficiency tocompared biomimetic to mineralizationbiomimetic mineralization under the same under experimental the same conditions,experimental which conditions, elucidated which that theelucidated mineralization that the process mineralization was important process to was improve important the catalytic to improve activity the ofcatalytic Cyt c. Thisactivity observation of Cyt c. wasThis consistentobservation with was that consistent reported with by Jianget that reported al. [26]. by The Jianget reason al. for [26]. the catalyticThe reason eﬃ forciency the (catalytickcat/Km) increased from 35.7 to 705 M 1s 1 (Table2) might be the formation of a bimetallic center provided efficiency (kcat/Km) increased from− − 35.7 to 705 M−1s−1 (Table 2) might be the formation of a bimetallic by Cu(Im) and the ferric porphyrin of Cyt c [5,35], or a new catalytic site formed by Cu(Im) and center provided2 by Cu(Im)2 and the ferric porphyrin of Cyt c [5,35], or a new catalytic site formed2 by coordinating amino acids on the protein surface [5]. Cu(Im)Figure2 and 4. coordinating(a) The kinetic amino analysis acids of free on Cytthe cprotein and Cyt surface c@Cu(Im) [5].2, the chromogenic reaction of free Cyt c (insert A) and Cyt c@Cu(Im)2 (insert B); (b) The activity of (1) free Cyt c, (2) Cyt c/copper ion. (3) Table 2. Kinetic parameters for free enzyme Cyt c, and Cyt c@Cu(Im) . Cyt c/Cu(Im)2, (4)Table Cyt 2.c/Im Kinetic and (5)parameters Cyt c@Cu(Im) for free2, respectively. enzyme Cyt c, and Cyt c@Cu(Im)22.

1 1 1 Sample kcat (s ) Km (mM) kcat/Km (M s ) Sample −1− − − −1 −1 Moreover, we prepared several non-mikcat (s )neralization K composites,m (mM) includingkcat/K mCyt (M cs, Cyt) c/CuSO4, Cyt c 0.75 0.17 21 5 35.7 Cyt c/Cu(Im)Cyt c 2 and Cyt c/Im. The experimental 0.75 ±± 0.17 results ±showed 21 ± 5 these materials 35.7 had poor catalytic Cyt c@Cu(Im)2 7.05 0.67 10 1 705 efficiency compared to biomimetic mineralization± under± the same experimental conditions, which Cyt c@Cu(Im)2 7.05 ± 0.67 10 ± 1 705 elucidated that the mineralization process was important to improve the catalytic activity of Cyt c. 2.3. Peroxidase Activity Assay of Free GOx and Cyt c, and GOx-Cyt c@Cu(Im) This observation was consistent with that reported by Jianget al. [26]. The2 reason for the catalytic −1 −1 efficiencyBased (k oncat/ theKm) composite increased offrom Cyt 35.7c@Cu(Im) to 7052 ,M wes also (Table constructed 2) might a multi-enzyme be the formation system of a of bimetallic GOx-Cyt ccenter@Cu(Im) provided2 using by asimilar Cu(Im) procedureas2 and the ferric for aporphyrin single enzyme. of Cyt Inc [5,35], addition, or a we new investigated catalytic site its formed enzymatic by activityCu(Im)2 andand cascadecoordinating reaction amino eﬃ ciency,acids on usingABTS the protein as surface a typical [5]. chromogenic substrate. The glucose was catalyzed by GOx to generate H2O2 and gluconic acid, and then ABTS was oxidized by Cytc to + form ABTS · in presenceTable 2. of Kinetic H2O2 parameters. Therefore, for the free enzymatic enzyme Cyt activity c, and Cyt could c@Cu(Im) be detected2. by measuring

Sample kcat (s−1) Km (mM) kcat/Km (M−1s−1) Cyt c 0.75 ± 0.17 21 ± 5 35.7

Cyt c@Cu(Im)2 7.05 ± 0.67 10 ± 1 705

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2.3. Peroxidase Activity Assay of Free GOx and Cyt c, and GOx-Cyt c@Cu(Im)2

Based on the composite of Cyt c@Cu(Im)2, we also constructed a multi-enzyme system of GOx- Cyt c@Cu(Im)2 using asimilar procedureas for a single enzyme. In addition, we investigated its Catalystsenzymatic2019, 9activity, 648 and cascade reaction efficiency, usingABTS as a typical chromogenic substrate.5 of 10 The glucose was catalyzed by GOx to generate H2O2 and gluconic acid, and then ABTS was oxidized +· theby absorbanceCytc to form of ABTS ABTS in+ bypresence UV-vis of spectroscopy H2O2. Therefore, at 420 the nm. enzymatic The influence activity ofcould pH (4.0–10.0)be detected on by the measuring the absorbance of ABTS+ by UV-vis spectroscopy at 420 nm. The influence of pH (4.0–10.0) enzymatic activities of free GOx and Cyt c, and GOx-Cyt c@Cu(Im)2 was investigated to optimize the on the enzymatic activities of free GOx and Cyt c, and GOx-Cyt c@Cu(Im)2 was investigated to reaction conditions. As shown in Figure S5b, the maximal enzymatic activity for pH of GOx and Cyt c, optimize the reaction conditions. As shown in Figure S5b, the maximal enzymatic activity for pH of and GOx-Cyt c@Cu(Im)2 was observed at 6.0 and 7.4, respectively. Since the enzymatic activity and GOx and Cyt c, and GOx-Cyt c@Cu(Im)2 was observed at 6.0 and 7.4, respectively. Since the enzymatic cascade reaction efficiency were influenced by the molar ratio of enzymes, we herein chose the mass activity and cascade reaction efficiency were influenced by the molar ratio of enzymes, we herein ratio of GOx and Cyt c of 1:1 by optimization. Enzymatic activity increased with an increasing amount chose the mass ratio of GOx and Cyt c of 1:1 by optimization. Enzymatic activity increased with an of Cyt c, which might be due to the rapid consumption of the produced H2O2. Nevertheless, the excess increasing amount of Cyt c, which might be due to the rapid consumption of the produced H2O2. amount of Cyt c could reduce the enzymatic activity, and the reason might be the high concentration of Nevertheless, the excess amount of Cyt c could reduce the enzymatic activity, and the reason might c Cytbe thecould high suppressconcentration the activity of Cyt c of could the system. suppress the activity of the system. Under optimal conditions, the kinetic parameters of free GOx and Cyt c, and GOx-Cyt c@Cu(Im) Under optimal conditions, the kinetic parameters of free GOx and Cyt c, and GOx-Cyt c@Cu(Im)2 2 werewere determined determined to to evaluate evaluate the the enzymatic enzymatic performance performance of of the the multi-enzyme. multi-enzyme. As As shown shown in in Figure Figure5 a, c GOx-Cyt5a, GOx-Cyt@Cu(Im) c@Cu(Im)2 has2 anhas obvious an obvious activation activation stage stage in the in first the minutefirst minute of the of reaction, the reaction, whereas whereas the free GOx-Cytthe free GOx-Cytc showed c ashowed premature a premature inactivation. inactivation. The Kmvalue The K wasmvalue determined was determined to be 6.39 to mMbe 6.39 for mM GOx-Cyt for c@Cu(Im)GOx-Cyt 2c,@Cu(Im) indicating2, indicating that the mineralized that the mineralized multi-enzyme multi-enzyme of GOx-Cyt of GOx-Cytc@Cu(Im) c@Cu(Im)2 has a2 highhas aa highffinity foraffinity the substrate. for the substrate. This is due This toexcellent is due toexcellent adsorption adsorption of mesoporous of mesoporous structure structure [36], which [36], is which consistent is withconsistent the morphology with the morphology shown in theshown SEM in image the SEM (Figure image1). (Figure Moreover, 1). Moreover, the catalytic the catalytic e fficiency efficiency (kcat/Km ) 1 was(kcat/ determinedKm) was determined to be 413.2 to Mbe− 413.2s−1 forM−1 GOx-Cyts−1 for GOx-Cytc@Cu(Im) c@Cu(Im)2, which2, which was ~6-fold was ~6-fold higher higher than thatthan of freethat enzymes of free enzymes (Table3). (Table This corresponded 3). This corresponded to the chromogenic to the chromogenic reaction ofreaction GOx and of GOx Cyt cand(Figure Cyt 5c a, insert(Figure A) 5a, and insert GOx-Cyt A) andc@Cu(Im) GOx-Cyt2 (Figurec@Cu(Im)5a,2 insert(Figure B), 5a, which insertelucidates B), which thatelucidates GOx-Cyt that cGOx-Cyt@Cu(Im) 2 showsc@Cu(Im) a deeper2 shows color a deeper than that color of than free enzyme.that of free The enzyme. higher enzymaticThe higheractivity enzymatic of GOx-Cyt activity ofc@Cu(Im) GOx-Cyt2 is attributedc@Cu(Im)2 to is a attributed rapid activation to a rapid of H activation2O2 generated of H2O in2 thegenerated multi-enzyme in the multi-enzyme system by the system cooperation by the of thecooperation two enzymes of the (Figure two enzymes5b). (Figure 5b).

FigureFigure 5.5. ((aa)) KineticKinetic performanceperformance for GOx and and Cyt Cyt c,c, andand GOx-Cyt GOx-Cyt cc@Cu(Im)@Cu(Im)2, 2and, and the the chromogenic chromogenic reactionsreactions ofof GOx and and Cyt Cyt c,c, andand GOx-Cyt GOx-Cyt c@Cu(Im)c@Cu(Im)2 were2 were shown shown as insets as insets A and A B, and respectively; B, respectively; (b) (bSchematic) Schematic diagram diagram of GOx-Cyt of GOx-Cyt c@Cu(Im)c@Cu(Im)2 catalyzing2 catalyzing ABTS ABTS in the in presence the presence of glucose. of glucose.

Table 3. Kinetic parameters of GOx in free GOx and Cyt c, and GOx-Cyt c@Cu(Im)2 (mG:mC = 1). Table 3.Kinetic parameters of GOx in free GOx and Cyt c, and GOx-Cyt c@Cu(Im)2 (mG:mC = 1). 1 1 1 SampleSample kcat (skcat−1) (s− ) KKmm(mM) (mM) kcat/Kmk(Mcat/K−ms (M− )−1s−1) GOx-CytGOx-Cyt c c 0.09 0.09± 0.01 0.01 1.30 1.30 ±0.14 0.14 69.2 69.2 ± ± GOx-CytGOx-Cyt c@Cu(Im)c@Cu(Im)2 2.64 2.64± 0.24 0.24 6.39 6.39 ±0.57 0.57 413.2 413.2 2 ± ±

2.4.2.4. StabilityStability andand ReusabilityReusability Usually, free enzyme in aqueous solution can produce an unpleasant smell due to protein Usually, free enzyme in aqueous solution can produce an unpleasant smell due to protein putrefaction upon long-term storage. In this study, we found that the activity of both Cyt c@Cu(Im)2, putrefaction upon long-term storage. In this study, we found that the activity of both Cyt c@Cu(Im)2, and GOx-Cyt c@Cu(Im)2remained 100% after storage at 4 °C and at room temperature (RT) for a week and GOx-Cyt c@Cu(Im)2remained 100% after storage at 4 ◦C and at room temperature (RT) for a week (Figure6a), which indicates that bioinorganic material Cu(Im) 2 provides excellent protection for enzymes. The reusability of Cyt c@Cu(Im)2 was determined to further assess the immobilized enzyme performance. As shown in Figure6b, Cyt c@Cu(Im)2 still preserved more than 35% of the original activity after ﬁve cycles. The results showed that although the biocatalyst was less stable to recycle, they were more stable upon storage. Catalysts 2019, 9, x FOR PEER REVIEW 6 of 10 Catalysts 2019, 9, x FOR PEER REVIEW 6 of 10 (Figure 6a), which indicates that bioinorganic material Cu(Im)2 provides excellent protection for enzymes.(Figure The6a), reusabilitywhich indicates of Cyt cthat@Cu(Im) bioinorganic2 was determined material toCu(Im) further2 provides assess the excellent immobilized protection enzyme for performance.enzymes. The As reusability shown in ofFigure Cyt c @Cu(Im)6b, Cyt c2@Cu(Im) was determined2 still preserved to further more assess than the 35%immobilized of the original enzyme activityperformance. after five As cycles. shown The in resultsFigure showed6b, Cyt cthat@Cu(Im) although2 still the preserved biocatalyst more was than less 35% stable of tothe recycle, original Catalyststheyactivity were2019 aftermore, 9, 648 five stable cycles. upon The storage. results showed that although the biocatalyst was less stable to recycle,6 of 10 theyIn wereaddition, more stablewe investigated upon storage. the stability of free and immobilized enzyme against high temperatureIn addition, and organic we investigated solvents. The the resultsstability showed of free that and Cyt immobilized c@Cu(Im)2 preservedenzyme against the same high originaltemperatureIn addition, enzymatic and we activity investigatedorganic aftertreatment solvents. the stability The of results ofheating free andshowed at immobilized70 °C that or incubation Cyt enzyme c@Cu(Im) with against2 ethanolpreserved high temperature(Figure the S6).same and organic solvents. The results showed that Cyt c@Cu(Im) preserved the same original enzymatic Theseobservationsoriginal enzymatic indicated activity aftertreatmentthat inorganic ofmaterial heating Cu(Im) at 70 2°C2 can or incubationprotect the with encapsulated ethanol (Figure enzyme S6). activityfromTheseobservations degradation aftertreatment or inactivationindicated of heating that by at 70inorganicorganic◦C or solvents. incubation material Cu(Im) with ethanol2 can protect (Figure the S6). encapsulated Theseobservations enzyme indicatedfrom degradation that inorganic or inactivation material Cu(Im) by organic2 can protectsolvents. the encapsulated enzyme from degradation or inactivation by organic solvents.

Figure 6. (a) Stability for Cyt c@Cu(Im)2and GOx-Cyt c@Cu(Im)2against long-term placing; (b) Figure 6. (a) Stability for Cyt c@Cu(Im) and GOx-Cyt c@Cu(Im) against long-term placing; ReusabilityFigure 6. of(a Cyt) Stability c@Cu(Im) for2. Cyt c@Cu(Im)22and GOx-Cyt c@Cu(Im)2against2 long-term placing; (b) (b)Reusability Reusability of of Cyt Cyt c@Cu(Im)c@Cu(Im)2.2 . 2.5. Glucose Detection 2.5. Glucose Detection The catalytic performance of the GOx-Cyt c@Cu(Im)2 was evaluated by the detection of glucose The catalytic performance of the GOx-Cyt c@Cu(Im)2 was evaluated by the detection of glucose The catalytic performance of the GOx-Cyt c@Cu(Im)2 was evaluated by the detection of glucose2 in KPi buffer buﬀer (40 mM, pH pH 7.4). 7.4). As As shown shown in in Figure Figure 7a,7a, an an excellent excellent linearity linearity (y ( =y 0.0082= 0.0082x +x 0.0371,+ 0.0371, R in KPi buffer (40 mM, pH 7.4). As shown in Figure 7a, an excellent linearity (y = 0.0082x + 0.0371, R2 R= 20.9921)= 0.9921) between between the theabsorbance absorbance and andthe concentration the concentration of glucose of glucose in the in range the range of 2.5 of to 2.5 150 to µM 150 wasµM = 0.9921) between the absorbance and the concentration of glucose in the range of 2.5 to 150 µM was wasobtained. obtained. The limit-of-detection The limit-of-detection (LOD) (LOD) of glucose of glucose was calculated was calculated to be to0.7 be µM, 0.7 whichµM, which was lower was lower than obtained. The limit-of-detection (LOD) of glucose was calculated to be 0.7 µM, which was lower than thanmost mostof the ofreported the reported colorimetric colorimetric glucose glucose sensors sensors[37,38]. The [37,38 reason]. The for reason high activity for high and activity sensitivity and most of the reported colorimetric glucose sensors [37,38]. The reason for high activity and sensitivity sensitivitytoward detection toward of detection glucose ofwas glucose the more was theeffic moreient activation eﬃcient activation of the intermediate of the intermediate productproduct (H2O2). Moreover,toward detection we studied of theglucose selectivity was the of GOx-Cytmore effic c@Cu(Im)ient activation2 toward of glucosethe intermediate against various product analogs, (H2O 2). (H2O2). Moreover, we studied the selectivity of GOx-Cyt c@Cu(Im)2 toward glucose against various Moreover, we studied the selectivity of GOx-Cyt c@Cu(Im)2 toward glucose against various analogs, analogs,including including fructose, fructose,mannose, mannose, xylose, xylose,lactose, lactose, galactose, galactose, sucrose sucrose and andgalactose. galactose. Although Although the including fructose, mannose, xylose, lactose, galactose, sucrose and galactose. Although the theconcentration concentration of the of theinterfering interfering compounds compounds (menti (mentionedoned above) above) was was 10-times 10-times higher higher than than that of concentration of the interfering compounds (mentioned above) was 10-times higher than that of glucose, a negligible enzymatic activity toward these competing compounds was found (Figure 77b),b), glucose, a negligible enzymatic activity toward these competing compounds was found (Figure 7b), suggesting the high selectivity towardtoward glucose.glucose. suggesting the high selectivity toward glucose.

Figure 7. ((a)) Linear Linear correlation correlation for for glucose glucose (2.5–150 (2.5–150 µM)µM) detection; ( b) Selectivityfor Selectivityfor glucose glucose (100 (100 µM)µ M) detectionFigure 7. withwith (a) interferingLinear interfering correlation compounds compounds for glucose (1.0 (1.0 mM (2.5–150 mM oﬀructose, offructose, µM) mannose, detection; mannose, xylose, (b) Selectivityfor xylose, lactose, lactose, galactose, glucose galactose, sucrose, (100 µM) andsucrose,detection galactose). and withgalactose). interfering compounds (1.0 mM offructose, mannose, xylose, lactose, galactose, sucrose, and galactose). 3. Materials and Methods

3.1. Materials and Instruments Cytochrome c from equine heart (oxidation state, petal-like crystal) and glucoseoxidase (50 ku, Roche subpackage) were purchased from Regal (Shanghai, China). Rhodamine B isothiocyanate Catalysts 2019, 9, 648 7 of 10

(RhB) was from Hefei Ruibio (Hefei, China), 2-methylimidazole (Im) was obtained from Adamas-beta (Shanghai, China). D-glucose was from Kermerl Corporation (Tianjin, China). Fluoresceinisothiocyanate (FITC), ABTS [2,20-Azinobis (3-ethylbenzothiazoline-6-sulfonic Acid Ammonium Salt], xylose, fructose, galactose, lactose, mannose and sucrose were purchased from Aladdin (Shanghai, China). All other reagents were of analytical grade, which include CuSO 5H O, KH PO , KOH and H O . 4· 2 2 4 2 2 SEM images were obtained on FEI HELIOS NanoLab 600i SEM (Hillsboro, OR, USA). UV-Vis spectral measurements were recorded on a Hewlett-Packard 8453 diode array spectrometer. XRD patternsof various samples were obtained from Bruker D8 Advance (Billerica, MA, USA), AXS and TGA experiments were performed by using STA2500, NETZSCH-Gerätebau GmbH. High-resolution microscope imageswere obtained by using a high-resolution microscope (Delta Vision Elite, GE, Boston, MA, USA).

3.2. Preparation of Enzyme@ Cu(Im)2

Cyt c@Cu(Im)2 was prepared by mixing Cyt c (0.4 mg/mL, 2 mL),Im (160 mM, 1 mL) with CuSO4 solution (40 mM, 1 mL) and incubating for 8 h. GOx-Cyt c@Cu(Im)2 was obtained by a similar process except for replacing Cyt c (0.4 mg/mL, 2 mL) with 1 mL 0.4 mg/mL Cyt c and 1 mL 0.4 mg/mL GOx. The encapsulated enzymes were centrifuged (4000 rpm, 1 min) and washed with double distilled water three times.

3.3. Fluorescent Labeling of Enzymes Fluorescent labeling of enzymes was carried out by dripping a small amount of FITC and RhBisothiocyanate into Cyt c and GOx solutions, respectively, and then incubating them for 15h in the refrigerator of 4 ◦C. In order to remove uncoupled ﬂuorescent substances, the mixtures were centrifuged (4000 rpm, 15 min) with Amicon® Ultra-4 10 K centrifugal ﬁlter devices (Sigma-Aldrich, Saint Louis, MO, USA) several times. Finally, the mineralization process is similar to that of Cyt c@Cu(Im)2.

3.4. Peroxidase Activity Assay

Peroxidase activity assays for Cyt c@Cu(Im)2 was added into KPi (pH 7.4, 40 mM) containing 1 mM ABTS, then adding H2O2, and the ﬁnal volume was 1 mL. The dynamic reaction was determined by UV-vis and the absorbance at 420 nm was recorded for 3 min. The enzyme activity assay of free Cyt c was the same as that of Cyt c@Cu(Im)2 under the same conditions. Peroxidase activity assays for GOx-Cyt c@Cu(Im)2 was added into 1 mL of KPi (pH 7.4, 40 mM) containing glucose with diﬀerent concentrations (0–10 mM), 1 mM ABTS. The dynamic reaction was determined by UV-vis and the absorbance at 420 nm was recorded for 3 min. The limit-of-detection (LOD) was calculated by using a formula of LOD = 3S/N, whereS and N represent the standard deviation of the regression line for three independent measurements, and the Slope, respectively. The enzyme activity assay of free GOx and Cyt c was the same as that of GOx-Cyt c@Cu(Im)2 under the same conditions.

3.5. Enzyme Stability Test

The storage stability of Cyt c@Cu(Im)2 and GOx-Cyt c@Cu(Im)2 continued to hatch at 4 ◦C and room temperature for a week. Cyt c@Cu(Im)2 was reused at 25 ◦C for 5 min in 100 mM KPi (pH 7.4) to evaluate the reusability.

4. Conclusions In summary, we have constructed a highly active enzyme-inorganic composite material by biomimetic mineralization. The prepared Cyt c@Cu(Im)2 exhibits excellent catalytic eﬃciency,long-term storage stability, and reusability, as compared to the free enzyme, which demonstrates that the Catalysts 2019, 9, 648 8 of 10 mineralization could well maintain and ﬁx the conformation of protein. Moreover, we have introduced GOx into Cyt c@Cu(Im)2 to develop multi-enzyme system of GOx-Cyt c@Cu(Im)2. As designed, this multi-enzyme exhibits an improved enzymatic activity. The cascade reaction of GOx-Cyt c@Cu(Im)2 was used to detect low concentrations (2.5 to 150 µM) of glucose with excellent selectivity (LOD of 0.7 µM). Therefore, the multi-enzyme system constructed in this study may confer potential applications in biocatalysis and biosensors, etc.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4344/9/8/648/s1. Figure S1: The preparation process of Cyt c@Cu(Im)2, and GOx-Cyt c@Cu(Im)2, Figure S2: UV-Vis spectra of free Cyt c before encapsulation (red) and the supernatant of Cyt c after encasing in Cu(Im)2 (black), Figure S3: SEM images of the GOx@Cu(Im)2 and GOx-Cyt c@Cu(Im)2, Figure S4: X-ray diffraction (XRD) patterns of the Cu(Im)2, Cyt c@Cu(Im)2, GOx@Cu(Im)2, and GOx-Cyt c@Cu(Im)2, Figure S5: The effect of pH on activity of (a) Cyt c and Cyt c@Cu(Im)2, (b) GOx-Cyt c, and GOx-Cyt c@Cu(Im)2, Figure S6: Relative activities of Cyt c@Cu(Im)2 after water bath at 70 ◦C and incubation in ethanol for 0.5 h. Author Contributions: X.-J.W. and Y.-W.L. designed the project. X.-Q.G., C.-W.W. J.-K.X., and S.-Q.G. performed the experiments. X.-Q.G., C.-W.W., J.-K.X., X.-J.W. and Y.-W.L. analyzed the data. X.-Q.G., X.-J.W. and Y.-W.L. organized the experimental results and wrote the manuscript. Funding: This work was supported by the National Natural Science Foundation of China (21807058), Special Scientific Research Funds for Central Non-profit Institutes, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (20603022016011), Financial Fund of the Ministry of Agriculture and Rural Affairs, P. R. of China (NFZX2018), the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology (Qingdao) (2018SDKJ0304-4-2), Hunan Provincial Natural Science Foundation (2019JJ50492, 2019JJ40259), Hunan Provincial Education Foundation (18C0420), and the Doctoral scientific research foundation of the University of South China (2014XQD32, 2017XQD10). Conflicts of Interest: The authors declare no conflict of interest.

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