MOFzyme: Enzyme Mimics of Fe/Fe-MIL-101

In this work, metal-organic frameworks (MOFs) Fe-MIL-101 was synthesized by hydrothermal method, and Fe/Fe-MIL-101 with different loadings was prepared. The crystal structure of the Fe/Fe-MIL-101 sample was characte-rized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and specific surface area measurement (BET). Fe/Fe-MIL-101 was found to posses an intrinsic enzyme mimicking activity similar to that found in natural horseradish peroxidase (HRP). The Michaelis constant (K m ) of 5% Fe/Fe-MIL-101 with ABTS as the substrate is about 10-fold smaller than Fe-MIL-101 and about 3-fold smaller than HRP, and about 108 times less than that of CuO NPs (K m = 10.28 mM), indicating a much higher affinity for ABTS than HRP and most of the peroxidase mimetics.


Introduction
Natural enzymes have attracted much attention because of their high catalytic efficiency and strong response characteristics. However, natural enzymes have many problems such as inactivation and denaturation, difficulty in purification, high cost and difficulty in storage and easily affected by environmental conditions including pH, temperature and inhibitors [1] [2]. These defects largely limit the application of enzyme. Therefore, the preparation and application of artificial mimic enzymes is a current research topic in recent years. In the past few decades, many mimic enzymes have been discovered [3] [4], for example, cytochrome P450 mimetics [5], serine proteases mimetics [6], dioxygenase mimetics [7], phosphodiesterase mimetics [8] and so on. Among these, a large number of peroxidase mimics, such as hemin [9] [10], hemeatin [11], hemoglobin [12], cyclodextrin [13], and porphyrin [14] [15], etc, have been used for H 2 O 2 and ascorbic acid detection [13] [14]. However, these peroxide mimic enzymes have lower catalytic activity and poorer selectivity than natural enzymes. Therefore, it is necessary to work harder to design a mimetic enzyme with high catalytic activity.
Metal-organic frameworks (MOFs) are topologically formed by organic ligands and inorganic metal clusters, a type of zeolite-like crystalline porous materials, have been recently researched as new functional materials. Compared with traditional zeolite, activated carbon and other materials, it has the advantages of high specific surface area, adjustable pore structure and easy modification. It has extensively application prospects in the fields of gas storage [16], separation [17] and catalysis [18], and has attracted much attention in science and biological systems. In view of the above advantages of MOFs, we consider it to be a material suitable for simulating enzymes. Indeed, Fe-MIL-101 [19], Fe-MIL-88NH 2 [20], MIL-53(Fe) [21], MIL-100(Fe) and MIL-68(Fe) [22] were found to have the activity of peroxide mimicking enzyme. Cu-MOF [23] was synthesized for catalyzing hydrolysis of bovine serum albumin and casein by solvothermal method. Despite the many exciting and convincing developments recently, we believe that the MOF-based catalytic field is still in the development and immature stage. In this work, we make use of the novel properties of 5% Fe/Fe-MIL-101 as peroxidase mimetics to catalyze oxidation of the ABTS by H 2 O 2 .

Synthesis of Fe/Fe-MIL-101
The Fe-MIL-101 is synthesized by hydrothermal method using p-dibenzoic acid as ligand iron as metal active center [24]. FeCl 3 ·6H 2 O (0.675 g) was added slowly into DMF (15 mL) solution, followed by adding H 2 BDC (0.206 g). The mixture was under continuous mechanical stirring for 10 min at room temperature, and then transferred into a Teon-lined stainless steel autoclave and heated at 110˚C for 20 h. The resulting brown solid was separated from the reaction medium, and purified by using the hot ethanol (70˚C), the purification process was carried out several times, followed by drying in an oven (70˚C, 30 min). The particles were separated by centrifuging and washed with DMF and ethanol to remove any unreacted raw materials.

Preparation of 1% Fe/Fe-MIL-101
FeCl 3 ·6H 2 O (0.0195 g) and Fe-MIL-101 (0.4 g) were dissolved in ethanol, stirred at room temperature for 1 h, centrifuged and washed 3 times in ethanol and dried under vacuum at 80˚C to obtain the product 1. NaBH 4 (2 mL, 1.875 mg/mL) solution was added dropwise to the solution of product 1 (product 1:150 mg, ethanol: 10 mL, dichloromethane: 50 mL), stirred at 0˚C under nitrogen for 30 min and then stirred at room temperature for 30 min. The precipitate was collected by centrifuging to obtain the product 1% Fe/Fe-MIL-101.

Peroxidase-Like Activity of 5% Fe/Fe-MIL-101
The effect of pH, temperature, H 2 O 2 concentration and ABTS concentration on the peroxidase-like activity of 5% Fe/Fe-MIL-101 was performed in a reaction volume of 3 mL of buffer solution. Buffers used in this experiment were acetate buffer (pH 4.0-6.0) and borate buffer (pH 8.0-10.0). The steady state kinetic assays of 5% Fe/Fe-MIL-101 were carried out by changing the concentration of ABTS at a fixed concentration of H 2 O 2 or vice versa at 50˚C. After 5 minutes of reaction, the absorbance of the reaction solution was measured at a wavelength of 420 nm using a Shi-madzu UV-2450 spectrophotometer. The kinetic parameters were calculated based on the equation: where ν is the initial velocity, V max is the maximal velocity, [S] is the concentration of the substrate, and K m is the Michaelis constant.

The Characterization of Fe/Fe-MIL-101
The X-ray diffraction (XRD) pattern of the as-synthesized Fe-MIL-101 is shown in Figure 1(a). The diffraction peaks all corresponded to the products synthesized in the literature [24] and generally consistent with the pattern calculated from the crystallographic data in this reference. Moreover the diffraction peaks position of obtained Fe/Fe-MIL-101 by different iron loadings are the same as Fe-MIL-101, but the intensity of the diffraction peaks are weaker than

Peroxidase-Like Activity of 1% Fe/Fe-MIL-101
In order to study the peroxidase-like activity of 1% Fe/Fe-MIL-101, the conventional catalytic oxidation of the peroxidase substrate ABTS in the presence of    selecting the substrates ABTS and H 2 O 2 as a model reaction system. The peroxidase-like activity of 5% Fe/Fe-MIL-101 was measured at different pH (3.0-9.0) and various temperature (30˚C -50˚C) (Figure 4(a) and Figure 4(b)). As can be seen from Figure 5, the pH 4.0 and 45˚C were the optimal reaction condition, which are very similar to the values for HRP [25].

Kinetic Analysis
We used steady-state kinetics to further study the peroxidase-like catalytic mechanism and kinetic parameters of 5% Fe/Fe-MIL-101. The kinetic data was collected by changing the concentration of one substrate at a fixed concentration of the other substrate. Within the concentration range of TMB and H 2 O 2 used, Journal of Biosciences and Medicines  typical Michaelis-Menten curves were observed ( Figure 5). A Lineweaver-Burk plot can be obtained with a nearly linear relationship ( Figure 5(b) and Figure  5(d)), from which important kinetic parameters can be derived ( Table 2). The kinetic parameters, such as the Michaelis-Menten constant (K m ) and maximum initial velocity (V max ) were from a Lineweaver-Burk plot. The Michaelis constant, K m , the smaller the K m value, the stronger the affinity of the enzyme to the substrate, and the higher the catalytic activity of the enzyme. As shown in Table 2 [26] Fe-MIL-101 H2O2 3.7 2.498 × 10 −11 [26] CuO NPs ABTS 10.28 [27] CuO NPs H2O2 120.3 [27] HRP ABTS 0.319 [25] HRP H2O2 5.44 [25] lower than HRP (0.319 mM) , and this value is about 108 times less than that of CuO NPs (K m = 10.28 mM) and about 10-fold lower than Fe-MIL-101 (0.916 mM), indicating a much higher affinity for H 2 O 2 than HRP and most of the peroxidase mimetics at pH 4.0.

Conclusion
The present study demonstrates that the 5% Fe/Fe-MIL-101 exhibited excellent peroxidase-like activity, catalyzing the oxidation of ABTS in the presence of H 2 O 2 . The Michaelis constant (K m ) of 5% Fe/Fe-MIL-101 with ABTS as the substrate is about 10-fold smaller than Fe-MIL-101 and about 3-fold smaller than HRP, and about 108 times less than that of CuO NPs (K m = 10.28 mM), indicating a much higher affinity for ABTS than HRP and most of the peroxidase mimetics. The above findings will open such catalytic systems for a variety of potential applications in biological systems in the future because of their ease of preparation, high activity and stability.