GO Composites with High

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Applications of Polymer, Composite, and Coating Materials

Nano-biocatalysts of Cyt c@ZIF-8/GO composites with high recyclability via a de Novo Approach Qianqian Zhu, Wei Zhuang, Yong Chen, Zhanke Wang, Byron Villacorta Hernandez, Jinglan Wu, Pengpeng Yang, Dong Liu, Chenjie Zhu, Hanjie Ying, and Zhonghua Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00072 • Publication Date (Web): 24 Apr 2018 Downloaded from http://pubs.acs.org on April 24, 2018

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ACS Applied Materials & Interfaces

Nano-biocatalysts of Cyt c@ZIF-8/GO composites with high recyclability via a de Novo Approach Qianqian Zhu†, Wei Zhuang* †‡§⊥, Yong Chen†‡§, Zhanke Wang⊥, Byron Villacorta Hernandez⊥, Jinglan Wu†‡, Pengpeng Yang†‡, Dong Liu†‡, Chenjie Zhu†‡§, Hanjie Ying†‡§, Zhonghua Zhu *⊥

†

College of Biotechnology and Pharmaceutical Engineering, National Engineering

Technique Research Center for Biotechnology, Nanjing Tech University, No. 30 Puzhu South Road, Nanjing 211816, ‡

China

State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech

University, No. 5 Xinmofan Road, Nanjing 210009, China §

Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, No.

30 Puzhu South Road, Nanjing 211816 , China ⊥School

of Chemical Engineering, The University of Queensland, St Lucia,

Queensland 4072, Australia *To whom correspondence should be addressed. Contact information: Dr. Wei Zhuang Email: [email protected] Prof. Zhonghua Zhu Email: [email protected]

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ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract In order to improve the stability and recyclability of enzymes immobilized on metal organic framewoks (MOFs), graphene oxide (GO) with surface oxygen-rich functional groups was selected to form ZIF-8/GO nanocomposites with the zeolitic imidazolate framework (ZIF-8) for Cytochrome c (Cyt c) immobilization. It was found that the functional groups on GO surface were involved in the growth of ZIF-8 without affecting the crystal structure, but their particle size was reduced to about 200 nm. The storage stability and resistance to organic solvents of Cyt c were obviously improved after the immobilization on the ZIF-8/GO nanocomposite. On one hand, compared to Cyt c@ZIF-8 and Cyt c@GO with 30% and 60% protein leakage, the Cyt c@ZIF-8/GO displayed little protein leakage after 60 h of storage. On the other hand, the Cyt c@ZIF-8/GO retained a residual activity of approximately 100% after being stored in ethanol and acetone for 2 h, while the free enzyme, Cyt c@ZIF-8 and Cyt c@GO retained only about 10%, 50% and 40%, respectively. In addition, the Cyt c@ZIF-8/GO nanocomposites can be utilized up to 4 cycles with virtually no loss of activity, and may be further applied on H2O2 biosensing systems. The synergistic effect between MOFs and GO in ZIF-8/GO nanocomposites provides infinite possibilities as immobilized enzyme carriers. Key words: Cytochrome c; Graphene oxide; Metal organic frameworks; Zeolitic imidazolate framework; Enzyme immobilization


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Introduction Enzymes possess great potential in the production process of many chemicals, such as pharmaceutical synthesis, fuel production and food processing due to their high selectivity, catalytic efficiency and specificity under relatively mild conditions1-2. However, the industrial utilization of enzymes is obviously hampered because of their low operational stability, low tolerances to most organic solvents and difficulties in reuse

3-6

etc. Recently, enzyme immobilization is widely studied to address those

drawbacks 7-9. However, it is challenging that the carriers do not significantly damage the structure and activity of the enzymes

10-12

. Therefore, it is very important to find

an immobilization carrier and relative technique that bring the minimum loss of biocatalytic activity

13

. Many kinds of porous materials such as mesoporous silica,

mesoporous titania, organic microparticles, sol-gel matrices, and porous carbon are competitive candidates for enzyme immobilization due to their abundant of pore sizes, large specific surface areas and void volume, and thus have attracted much attention from the scientists and engineers during the past years 2, 14. As a kind of emerging porous materials, metal-organic frameworks (MOFs) derived from metal-containing nodes and organic ligands have been considered as promising candidates for gases separation, sensors fabrication and catalysts immobilization 15-17. Recently, they have emerged as supporting matrices on the enzyme immobilization due to their tunable pore size, very high surface area and large void volume, mild synthesis conditions and facile modification by other small molecules on both metal nodes and ligands

18-20

. NU-1003, a water-stable zirconium MOFs featuring the large



mesopores (4.6 nm), had been used to immobilize a kind of nerve agent hydrolyzing enzyme 21. Among different kinds of MOFs, ZIF-8 is widely known and is often used for the immobilization of enzymes because of their negligible cytotoxicity, superior chemical and thermal stability, and ease of synthesis in alcohol or water phase 22-24. To the best of our knowledge, methods used for enzyme immobilization on MOFs can be classified into three categories: (1) immobilization on the surface; (2) diffusion into the pores; and (3) in-situ encapsulation within the framework. The immobilization of enzymes on MOFs can be achieved by post-synthetic modification of MOFs. For example, after covalently bind with MIL53 (Al) that modified by amine group, glucose oxidase can retained the enzymatic activity and showed selectivity for glucose25. Enzyme could also be absorbed on the pore cages of MOFs. For instance, due to the strong interactions between MOFs and enzymes, the immobilization of one kind of enzyme microperoxidase-11 into the mesopores of MOFs was reported to prevent enzyme leaching

26

. In the third approach, the

catalase maintained its biocatalytic activities under unfolding conditions after being mounted into ZIF microcrystals via a de novo approach under a mild synthetic condition19. Although the utilization of MOFs is popular in the field of enzyme immobilization, their small channel may restrict the diffusion of substrates and products. Nanosized and well-dispersed MOFs are very useful for increasing diffusion performances 27. In order to achieve this requirement, graphene oxide (GO) with easy functionalization and strong hydrophilicity is selected as a suitable agent for MOFs.


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GO has a nano-meter scale thick layered structure and a nonstoichiometric chemical composition of carbon and oxygen. As normal, the basal plane of GO is decorated with oxygen-rich functional groups such as C-O-C (epoxy group) and OH (hydroxyl group), whereas the edge is mainly decorated with carboxylic groups24, 28-29. Hybrid composites from MOFs and graphene oxides enable the integration of the superior properties of these two fascinating materials, thus allowing the design of novel materials possessing properties not displayed by neither component alone 30-31. Recently, a few research groups have reported relative studies on the synthesis and utilization of graphene-based MOFs

24, 31-32

. Petit et al. have recently described that

GO–MOF composites can be used for the adsorption of nitric oxide and ammonia. Jahan et al. have reported conducting nanowires from MOF-5 and functionalized graphene where MOF-5 was inserted into the gap between the layers of graphene sheets

33-34

. Furthermore, Li and coworkers have reported the synthetic method of

MIL-101 and GO as well as its performance for the removal of azo dyes from water. Huang et al. have designed a layer by layer approach to synthesize ZIF-8@GO for hydrogen separation and purification24, 35. Also, the morphology controlled growth of MOF nanoparticles onto graphene sheets has also been explored recently30. Based on these facts, GO can be envisaged as a structure-directing agent and growth platform for MOF nanoparticles, whereby modulation may occurs due to the different functional groups on its surface

36

. In this context, preparing composites from ZIFs

and GO appeared to be very attractive18, 37.



As mentioned above, although GO/MOFs nanocomposite attracts more and more attention, it seldom applies in the immobilization of enzymes. In order to characterize the performance of MOFs/GO nanocomposites for enzyme immobilization, Cytochrome c (Cyt c) which has molecular dimensions of ~2.6 nm × 3.2 nm × 3.3 nm was selected as a model enzyme. The protein consists of a covalently attached heme group, which is very good for the electron transport of reactions in mitochondria and apoptosis

38-40

. Therefore, Cyt c can be used as a bio-recognition element toward the

targets of biosensors with high sensitivity, good flexibility for rapid analysis, and relatively inexpensive instrumentation14, 41-42. In this work, we prepared Cyt c@ZIF-8/GO composites for H2O2 detection and developed a series of nanocomposites with different GO contents for enzyme encapsulation. Then, Cyt c was immobilized on the nanocomposites by a de Novo approach. FESEM, XPS, and FT-IR characterization were utilized to verify the binding efficiency of Cyt c on ZIF-8/GO nanocomposites. Also, we compared the differences of the enzymatic properties of Cyt c@ZIF-8/GO with free Cyt c, Cyt c@ZIF-8 and Cyt c@GO. The introduction of Cyt c on porous nanocomposites offers additional possibility for H2O2 biosensing systems.


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Materials and methods Reagents Zinc nitrate hexahydrate and 2-methylimidazole both with the purity higher than 99% were obtained from Aladdin Reagent (Shanghai, China). Graphite (100 meshes) was purchased from Qingdao Jinrilai Graphite Co. Ltd. (Qingdao, China). Cyt c, 2, 2'-amino-di (2-ethyl-benzothiazoline sulphonic acid-6) ammonium salt (ABTS), and polyvinylpyrrolidone (PVP) were obtained from Sigma-Aldrich (USA). All other chemicals were analytical grade and commercially available. 1. Preparation of ZIF-8/GO nanocomposites 1.1 Preparation of GO GO was firstly synthesized through the modified Hummers’ method29, 43-44. In detail, graphite (3 g) were added to a mixture of concentrated H2SO4 (12 mL), P2O5 (2.5 g), and K2S2O8 (2.5 g). The solution was then heated upto 80 °C and continuously stirred in an oil bath for 5 h. After reaction, the mixture was mixed with deionized water (500 mL), and the product was filtered and dried at room conditions. After 1 hour of exfoliation by sonication of the suspension of graphite oxide (0.1 mg/mL), the well dispersed GO was obtained by filtration and freeze vacuum drying. 1.2 Synthesis of ZIF-8 particles ZIF-8 particles were synthesized according to the previous reported work by Liu and co-workers 45. The methanol solution containing zinc nitrate hexahydrate (100 mL, 25



mM) and 2-methylimidazole (25 mM, 120 mL) was mixed and sonicated for 20 minutes. Then, the mixture was allowed standing for 24 h at room temperature. ZIF-8 was obtained by centrifugation (10,000 rpm/min), washed with methanol three times, and obtained after vacuum freeze drying. 1.3 Synthesis of ZIF-8/GO The synthesis of ZIF-8/GO is similar to the above procedure for the synthesis of ZIF-8 except that the methanol solution of zinc nitrate hexahydrate should be mixed with GO for 8 h. Based on the total mass of zinc nitrate hexahydrate and 2-methylimidazole, the addition ratio of GO was 0.25%, 0.5%, 1% and 2%, which weighed 2.5, 5, 10, and 20 mg of GO respectively. The synthesis method of ZIF-8/GO is the same as that described in section 1.1 The effect of growth time on the synthesis of ZIF-8/GO nanocomposites was studied by collecting ZIF-8/GO at the time of 45 min, 2 h, 4 h, 10 h, 22 h, 36 h, 48 h and 60 h. The growth process of ZIF-8/GO was observed by field emission scanning electron microscopy (FESEM). 2. Preparation and characterization of Cyt c@ZIF-8, Cyt c@GO and Cyt c@ZIF-8/GO 2.1 Synthesis of Cyt c@ZIF-8, Cyt c@GO and Cyt c@ZIF-8/GO A solution (100 µL) containing PVP (15 mg/mL) and Cyt c (50 mg/mL) was added to the mixed methanol solution containing 2-methylimidazole (25 mM, 120 mL) and zinc nitrate hexahydrate (25 mM, 100 mL). Similarly, the synthesis of Cyt c@GO was


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carried out by adding a mixed solution of PVP and Cyt c to the methanol solution of GO. Likewise, Cyt c@ZIF-8/GO was obtained by mixing the solution of PVP and Cyt c with the methanol solution of GO, zinc nitrate hexahydrate and 2-methylimidazole. All the enzymes nanocomposites were grown for 24 h at the room temperature and collected by centrifugation and freeze vacuum drying.

Scheme 1. The illustration of the synthesis process of Cyt c@ZIF-8/GO nanocomposites. 2.2 Measurement of the protein concentration The protein concentration was detected using the Bradford method

46

and the load

amount of Cyt c was measured and calculated by the following formulation: Immobilized amount mg m: the initial amount of protein , mg C0: the concentration of protein after immobilization, mg/mL V0: the volume of methanol used to synthesize the immobilized enzyme, mL C: the concentration of protein in the eluent, mg/mL



V: the volume of methanol used to rinse the immobilized enzyme, mL 2.3 Assay of enzymatic activity The assay mixture was comprised of phosphate buffered saline (PBS, pH 7.0, 50 mM), ABTS (532 µM), H2O2 (2 mM), and Cyt c (8 µM) to a final volume of 1 mL. The reaction mixture was mixed and incubated at 25 ℃ for 1 min, and then the reaction solution was diluted 10 times with the buffer to detect an increase in absorbance at 415 nm. The immobilized enzyme should be filtered out before the reaction solution diluted. 2.4 Effect of protein addition on immobilized enzyme The effect of protein addition time on the immobilized enzyme was studied by adding protein at different times in the growth of the Cyt c@ZIF-8, Cyt c@GO and Cyt c@ZIF-8/GO, such as 0 h, 2 h, 5 h, 7 h, 9 h, 14 h, 20 h, and 24 h. The optimal time was obtained by comparing the enzyme activity. The effect of the adding amount of protein on the reaction activity of the immobilized enzymes nanocomposites was determined by immobilizing proteins of different concentrations, such as 2.50 mg, 3.75 mg, 5.00 mg, 6.25 mg and 7.50 mg, respectively. 2.5 Effects of H2O2 concentration on enzyme activity The concentration of H2O2 has an effect on enzymatic activity. In this study, four groups of experiments were performed, including free enzyme and immobilized enzymes at different concentrations of H2O2 for 1 min.


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2.6 Determination of kinetic parameters The kinetic parameters of Vmax and Km for immobilized and free enzymes were assayed in PBS buffer (pH 7.0, 50 mM) containing different concentrations of H2O2 (1-10 mM) for 1 min and were calculated and determined by the Lineweaver-Burk equation. 2.7 Storage stability of the enzyme nanocomposites Two groups of experiments for the stability measurement were carried out. One was determined by incubating Cyt c@ZIF-8, Cyt c@GO and Cyt c@ZIF-8/GO in PBS buffer (50 mM, pH 7.0) at 25 ℃ for 60 h, and by testing the concentration of protein at 10 h, 20 h, 30 h, 40 h, 50 h and 60 h. The other one was determined by incubating them at 4 ℃ for 11 days, and by measuring their residual enzymatic activity. 2.8 Tolerance of organic solvents 20 mg of Cyt c@ZIF-8, Cyt c@GO and Cyt c@ZIF-8/GO containing the same mass of protein were placed in 5 mL of ethanol and acetone for 2 h, and the immobilized enzymes were filtered off and rinsed with deionized water three times. The tolerance of the immobilized enzymes to the organic solvent was studied by comparing their enzymatic activity with that of the free enzyme after incubation in the organic solvent. 2.9 Operational stability Cyt c@ZIF-8, Cyt c@GO and Cyt c@ZIF-8/GO with the same quality of protein were used to study the reusability. After reaction, the immobilized enzymes were



filtered out and their enzymatic activity was tested. Then, the immobilized enzymes were washed by PBS buffer (pH 7.0, 50 mM) and started for the next round of usage. 2.10 Characterization Field emission scanning electron microscopy (FESEM) was performed with an acceleration voltage of 20 KV using an instrument of Hitachi S-4800 (Hitachi, Japan). Transmission electron microscopy (TEM) images were determined on a Tecnai-G20 (FEI, USA) transmission electron microscope. X-ray photoelectron spectroscopy (XPS) analysis was conducted on an Axis-Nova instrument (Kratos Analytical, Manchester, UK) for the surface elemental and energy analysis. Fourier transform infrared spectroscopy (FT-IR) was conducted by scanning the spectrum range from 400 to 4000 cm-1. X-ray diffractograms were obtained with a Bruker D8 (Bruker, Germany) diffraction meter to detect the crystal structure of the nanocomposites.


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Results and discussion

Fig. 1 FESEM images of the ZIF-8/GO nanocomposites with different amount of GO: (a, b) 0.5%; (c, d) 1.0%; (e, f) 1.5%; (g, h) 2.0%. ZIF-8/GO nanocomposites were successfully synthesized by rinsing GO with zinc nitrate solution, followed by adding the solution of 2-Methylimidazole. Field emission scanning electron microscopy (FESEM) images were determined to investigate the



morphology changes of the nanocomposites. As shown in Fig. 1a, b, after adding 0.5% weight percentage of GO into the precursor solution of ZIF-8, many ZIF-8 crystals are grown on the GO layers due to the capillary force and the covalent bond between them, thus forming dense ZIF-8 layers (Fig. 1b)24, 30. Because the amount of GO is very low, there are many ZIF-8 particles dispersed in the solution.

Fig. 2 XRD patterns of the nanocomposites: (a) GO; (b) 2% GO-ZIF-8; (c) 1% GO-ZIF-8; (d) 0.5% GO-ZIF-8; (e) 0.25% GO-ZIF-8; (f) ZIF-8; (g) the simulated XRD structure of ZIF-8. Moreover, the ZIF-8 on GO is not a single layer, many ZIF-8 particles are adsorbed on GO30. As shown in Fig. 1, the number of ZIF-8 particles supported on GO sheet increases, so the dispersion in solution clearly decreases according to the increasing in the GO content. As shown in Fig. 2, the synthesized ZIF-8 (Fig. 2f) exactly matched the diffraction peak of the simulated XRD structure of ZIF-8 (Fig. 2g), indicating that the synthesized sample is ZIF-8 crystal. Also, the formation of the ZIF-8/GO nanocomposites with high crystallinity was proved by the X-ray diffraction (XRD)


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characterization, which indicated that all peaks of the spectrum match well with those of ZIF-8 standard sample 31.

Fig. 3 TEM images of ZIF-8 (a, b) and ZIF-8/GO nanocomposites (c, d).

Fig. 4 FESEM images of ZIF-8 (a, b) and GO (c, d). It is confirmed that ZIF-8 nanoparticles on the composites have the similar morphology as that of the pure ZIF-8 (Fig. 3a, b) by the transmission electron microscopy (TEM) images. However，their particle size is about 200 nm, which is



smaller than that of the pure ZIF-8 particles about 500 nm, shown in Fig. 4a, b and c. The results are very similar to other previous published results 45. The size of ZIF-8 particles decreases with the amount of GO added30. As shown in Fig. 4d, compared to pure GO sample in which the layers compact to each other, the GO layers in ZIF-8/GO seem very well dispersed

31

. This result illustrates that the interlayers of

zinc ions between the GO layers form before the ZIF-8 particles start growing. As revealed in Fig. 1 h, the ZIF-8 nanoparticles have an average size about 200 nm and displays the similar morphology and surface structure as those of the pure ZIF-8 particles (Fig. 4b). Also, the similar existing peaks in XRD pattern of Cyt c@ZIF-8/GO indicates that the incorporation of enzyme does not affect much of the crystal structure of ZIF-8.

Fig. 5 FT-IR spectra of the nanocomposites: (a) GO; (b) GO-ZIF-8; (c) ZIF-8; (d) Cyt c; (e) Cyt c@GO; (f) Cyt c@ZIF-8, and (g) Cyt c@ZIF-8/GO


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As shown in Fig. 5, the FT-IR spectra of GO, ZIF-8 and its composites have some differences. The spectrum of GO/ZIF-8 is a little similar to that of ZIF-8, with the exception of the peaks at 1729 cm-1 belong to the stretching vibration of C=O bond due to the presence of GO. Also, the peaks at 1265 cm-1 are attributed to -C-O-C bond that are associated with the surface structure of GO. Furthermore, the peaks at 1580cm-1, 1145cm-1 and 990cm-1 corresponding to the C=N bond and the C-N bond, respectively, due to the existence of 2-methylimidazole. It is expected that zinc clusters might anchor on GO surfaces, because epoxy and carboxylic groups are present on its surface

33

. After the modification of GO with

ZIF-8, the infrared range absorption peaks of the functional groups mainly correspond to ZIF-8 due to the small amount of GO. These data further prove that the structure of ZIF-8 in the composites does not change significantly. The peaks at about 1647 cm-1 correspond to the stretching vibration of C=O that belongs to the amide bond of Cyt c 45

. After the modification of Cyt c, above peaks appear in the spectrum, which further

prove that the enzymes were immobilized successfully. XPS can be used to determine the different elements and structure information on the surface of materials. To further analyse the process of ZIF-8/GO nanocomposite synthesis, XPS characterization was conducted to confirm their elemental composition. It is clear that elements of C, O, N and Zn appear in the prepared composites (Fig. 6 A).



(B)

(A) Zn2p

(b)

O1s N1s

C1s

(c)

C 1s

C=O

(a)

Counts/a.u.

Counts/a.u.

C-O C-C

(a)

(b)

(c)

1200

1000

800

600

400

200

0

292

(C)

290

288

286

284

282

280

Binding Energy/eV

Binding Energy/eV

O 1s

(D)

(a) (b)

N 1s

(a)

Counts/a.u.

Counts/a.u.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(b)

(c) (c)

540

538

536

534

532

530

528

526

410 408 406 404 402 400 398 396 394 392

Binding Energy/eV

Binding Energy/eV

Fig. 6 X-ray photoelectron spectra (XPS) of the nanocomposites: (A) Survey spectra, (B) C1s spectrum, (C) O1s spectrum, and (D) N1s spectrum of the samples (a) GO; (b) ZIF-8; (c) ZIF-8/ GO. As shown in Fig. 6 B, there are three peaks at 284.6, 286.3 and 288.6 eV, which are corresponding to the C-H or -C-C, C-O or C-N, and O=C-O moieties in the nanocomposites, respectively. Besides, the peak at 532.3 eV is attributed to the C=O bonds in the nanocomposites [39] (Fig. 6 C). The N 1s spectra of the samples have one peak, but can be divided into two components located at 401.7 eV and 399.2, respectively, which should be assigned to protonated amine (amine cation) and amine nitrogen. This phenomenon may come from the strong acid-base interaction between the carboxyl groups and amine sites (Fig. 6D). The ZIF-8 covalently bound to the GO layer, which can be proved by XPS characterization.


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Fig. 7 FESEM images of ZIF-8/GO nanocomposites for different synthesis times: (a) 45 min; (b) 2 h; (c) 5 h; (d) 7 h; (e) 9 h; (f) 24 h. The synthesis time is very important to determine the morphology and structure of the ZIF-8/GO nanocomposites. For a very short synthesis time (45 min), the coverage of ZIF-8 crystals with small size on the GO nanosheets is low (Fig. 7a). So, the GO nanosheets show high flexibility and are easily aggregated. For longer synthesis times, after 9 h for example, the GO surface is fully covered by ZIF-8 nanoparticles, which is uniformly covered on the GO surface(Fig. 7b, c, d, e, f)18.



(a)

(b)

100

Z-Cyt c

ZG-Cyt c

100

Relative Activity/%

Relative activity/%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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90 80 70

ZG-Cyt c Z-Cyt c G-Cyt c

60 50 0

2

3

4

5

6

7

-1

Concentration of Cyt C/gL

8

80

60

0 0

2

5

7

9

14

20

24

Time/h

Fig. 8 (a) Effect of amount of protein added on the activity of the immobilized enzymes; (b) Effect of protein addition time on immobilized enzymes activities. As shown in Fig. 8a, b, both the amount of protein added and the protein addition time have effects on the reactivity of the enzymes. With the increase of protein content, the relative activities of immobilized enzymes increase, and reached the highest value when the protein content was 5 mg in the 1 mL reaction system. However, when the amount of protein exceeds 5 mg, excessive protein might stack on the surfaces of the carriers, not only the inner ones, but also the outer, which affects the effective contact of the substrate with the active sites of the enzyme, leading, in turn, to reduce the enzymatic activity. In contrast, protein addition time is also essential to the enzymatic activity, as shown in Fig. 8b. The optimal protein addition time is 7 h, corresponding to the highest enzymatic activity. The reason for this is that the amount of adsorption and the mass transfer have the best match at this time.


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Fig. 9 (a) Effect of H2O2 concentration on the reaction rate of free enzyme and immobilized enzymes; (b) Lineweaver-Burk curves of free enzyme and three immobilized enzymes. Table 1 Kinetic parameters of the enzymes.

Samples

Free

Z-Cyt c

G-Cyt c

ZG-Cyt c

R2

0.993

0.997

0.998

0.997

Vmax

254

242

231

393

Km

3.58

5.90

9.94

0.85

It is well-known from the definition of enzymatic activity that the enzyme can only maximize the catalytic efficiency at the optimum substrate concentration. As shown in Fig. 9a, with the increase of the concentration of H2O2, the reaction rate of free enzyme and immobilized enzymes show a "bell structure" with the increase of substrate concentration. The optimal substrate concentration of free enzyme is 0.8 mM while the optimum substrate concentration of immobilized enzymes is increased to 1 mM. This difference might be due to the fact that the distribution of the protease



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in the immobilized enzymes is not concentrated and requires a higher concentration of substrate for effective contact. The Michaelis-Menten constant Km can reflect the degree of affinity of the enzyme with the substrate. Fig. 9b shows the Lineweaver-Burk curves of the free and immobilized Cyt c enzymes. The dynamic parameters are displayed in Table 1. As shown in Table 1, the Cyt c@ZIF-8/GO exhibits a Km value of 0.85 mM and a Vmax value of 392.78 mM/min, whereas the Km and Vmax of free Cyt c, Cyt c@ZIF-8 and Cyt c@GO are 3.58 mM, 254.91 mM/min, 5.90 mM, 294.18 mM/min and 9.94 mM, 230.64 mM/min, respectively. The decrease of Km of the nanobicatalyst Cyt c@ZIF-8/GO indicates a strong affinity for H2O2, which might be for the reason that the embedding of Cyt c in methanol and the microenvironment created by the ZIF-8/GO carrier. Previous studies showed that methanol could change the conformational structure of Cyt c to expose its heme group, which means that more enzymatic active sites could be bound to the substrate

47

. Also, some studies

suggested that carriers could significantly affect enzymatic activity by adjusting the hydrophobicity and dispersibility to create the suitable microenvironments for enzymatic catalysis 45, 48. It is very important to study the stability of immobilized enzymes for the industrial applications. As shown in Fig. 10a, the activity values of Cyt c@ZIF-8 and Cyt c@GO decreased by 35% and 55%, respectively; while the activity of Cyt c@ZIF-8/GO remained almost unchanged after storage in buffer for 11 days. Fig. 10d also prove that the Cyt c@ZIF-8/GO composites have a good storage stability. The


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protein leakage rate of Cyt c@ZIF-8 and Cyt c@GO are 30% and 60%, respectively, but the protein leakage rate of Cyt c@ZIF-8/GO is negligible. The tolerance of the free Cyt c to the organic solvents is the worst of the studied set and almost becomes completely inactivated because the organic solvent deprives the "essential water" of the surface of the enzyme molecule and it may also cause changes in the conformation of the protein.

(a)

110 As prepared

(b)

11 day

120 As prepared

Ethanol

Aceton

100

90

Relative Activity(%)

Relative Activity(%)

100

80 70 60 50

80 60 40 20 0

40 tc Z-Cy

t c -0.25% G-Cy B

% B-0.5

Free G-Cyt c Z-Cyt c B-0.25% B-0.5%

B-2%

B-1%

(c)

B-1%

B-2%

(d)

60

100 G-Cyt c Z-Cyt c GZ-Cyt c(0.25) GZ-Cyt c(0.50) GZ-Cyt c(1.0) GZ-Cyt c(2.0)

80 60 40 20

Z-Cyt c

Release percent/%

RelativeActivity(%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


50

G-Cyt c

40

GZ-Cyt c(0.25) GZ-Cyt c(0.50) GZ-Cyt c(1.0) GZ-Cyt c(2.0)

30 20 10 0

0 1

2

3

4

5

6

7

8

0

10

20

Reaction Batches

30

40

50

60

Time/h

Fig. 10 (a) Storage stability of immobilized enzymes; (b) Tolerance of organic solvents for free and immobilized enzymes; (c) The reusability curves of immobilized enzymes; (d) Enzyme shedding curves of immobilized enzymes The enzyme activities of Cyt c@ZIF-8 and Cyt c@GO are reduced to about 50% after treatment with organic solvent. However, the tolerance of Cyt c@ZIF-8/GO to



organic solvent is obviously improved, which may be due to

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the prominent

protective effect of the nanocomposite and the proper interaction between the enzyme and the carrier that improve the structure of the enzyme molecule (Fig. 10b)49. As shown in Fig. 10c, the relative activity values of the immobilized enzymes decrease after repeated use, which are caused by enzyme shedding or active site clogging. It is worth noting that Cyt c@ZIF-8 and Cyt c@GO are used up to 2 batches to retain 80% of the original enzymatic activity while the Cyt c@ZIF-8/GO is used up to 4 batches, and the reusability was increased by 100%. Also, as shown in Fig. 11, the ease of recycling of ZG-Cyt c is also very important to the catalysis performance of reusability.


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Fig. 11 Digital photos after mixing the different nanocomposites for 60 minutes: (a) G-Cyt c; (b) Z-Cyt c; (c) ZG-Cyt c.



Conclusions In summary, ZIF-8/GO nanocomposites with 200 nm particle size were successfully synthesized by adjusting the added amount of GO and the growth time of GO, and then the nanocomposites were successfully used for Cyt c immobilization. The results showed that the Cyt c@ZIF-8/GO had improved storage stability, tolerance to ethanol and acetone as well as operational stability. After incubation in organic solvent for 2 h, the Cyt c@ZIF-8/GO have the residual activity about 100%, while the free Cyt c, Cyt c@ZIF-8 and Cyt c@GO retained only about 10%, 50% and 40%, respectively. It was also noticed that the Km of Cyt c@ZIF-8/GO was much lower than that of free Cyt c, Cyt c@ZIF-8 and Cyt c@GO, which also indicates that the Cyt c@ZIF-8/GO had a high affinity for H2O2. Thus, the ZIF-8/GO nanocomposites and Cyt c@ZIF-8/GO may have a bright future for applications in biotechnology and biomedicine. Acknowledgements This work was supported by grants from the Program for Changjiang Scholars and Innovative Research Team in University (IRT_14R28), the Major Research Plan of the National Natural Science Foundation of China (21390204), the National Natural Science Foundation of China (Grant No. : 21636003, 21506090), the Technology Support Program of Jiangsu (Grant No. BE2014715), the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (SICAB), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References


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Graphical Abstract


GO Composites with High

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