Mussel-inspired surface modification of magnetic@graphite nanosheets composite for efficient Candida rugosa lipase immobilization

Abstract

Chen Hou and Hao Zhu contributed equally to this work.

This paper is dedicated to memory of pro. Yanfeng Li, who passed away recently.

Electronic supplementary material

The online version of this article (doi:10.1007/s10295-015-1602-0) contains supplementary material, which is available to authorized users.

Introduction

Immobilization of biocatalysts has been generally adapted for synthesis of high stereoselectivity and enantioselectivity products with the consideration of green, economic and sustainable chemistry. Enzymes are an important group of biocatalysts superior to chemical catalysts because of their high effectiveness, high specificity, and green reaction conditions [1]. Enzymes have found practical use in industrial processes and are extensively used in new fields, such as bioengineering [2], food industry [3], pharmaceuticals [4], fine chemical industry, biosensors, and biofuel cells [5]. However, the poor catalytic activity, stability and selectivity as well as recycling problems of free enzymes limited the applications in extensive fields [6]. In order to increase the performance of enzymes, many supporting materials have been developed including polysaccharide [7], inorganic materials [8, 9], organic polymeric carriers [10], and natural polymers [11], etc. Nevertheless, aiming at design facile, universal, economical, and environmental friendly supporting materials in balancing the key factors, which determine the efficiency of biocatalysts, including surface area, mass transfer resistance, and effective enzyme loading still remains challenging.

Graphite, with the excellent mechanical, thermal, optical, and electrical properties [12], simulated ever-increasing attention, and various kinds of graphite series based matrix and hybrids have been prepared for compound and immobilization [13–15]. Combining the merits of the unique magnetic properties of Fe₃O₄ nanoparticles and chemical stability, non-toxicity of graphite nanosheets, the magnetic nanoparticles doped graphene or graphite composites have been caused excited interests and used as carriers of drug delivery, purification and detection of biomolecules, immobilization of enzymes, and enrichment of proteins recently [16–19]. Chen and co-workers fabricated electrically and magnetically bi-functional graphene/γ-Fe₂O₃ hybrid aerogels for immobilizing β-glucuronidase [20]. Ma et al. [21] developed a cost-effective way to prepare Fe₃O₄ and graphite nanosheets composite to immobilize glucoamylase covalently by an inverse co-precipitation method. Taking advantage of the high loading capacity and abundant hydrophilic groups of graphene oxide (GO), and large surface areas and hydrophobicity of reduced graphene oxide (rGO), Chen et al. [22] prepared strong magnetic GO-Fe₃O₄ and rGO-Fe₃O₄ for the immobilization of protein and the enrichment of peptides, respectively. Li et al. [23] introduced an easy one-pot polyol approach to hybridize size- and density-tunable graphene-Fe₃O₄ composites for the immobilization of porcine pancreatic lipase. Despite these developments, there is still a need for some chemical modification schemes of magnetic graphene or graphite nanosheets that can derivatize a variety of materials in bio-applications.

Dopamine (DA) mimics the repetitive catechol-amine structure of 3,4-dihydroxyl-l-phenylalanine found in mussel’s foot protein, which has excellent adhesion property with virtually all organic and inorganic surfaces [24]. Polydopamine (PDA) has recently been introduced by Lee et al. who reported its versatility for various substrates and feasibility for facile functionalization with spontaneous oxidative polymerization of DA under mild conditions [25]. Since then, dopamine was utilized as a versatile and intriguing starting material for almost all kinds of solid surface modifications very extensively due to the green, simple, fast, and low-cost synthesis approach [26–28]. Especially, the coated polydopamine films also can be used for the step-functionalization with organic species for the creation of functional organic ad-layers [29, 30].

Although the utilization of magnetic graphite nanosheets and DA for immobilization has been reported several times, the composite that combines the excellent properties of both of them by facile adhesion way is still very innovative. Herein, inspired by the adhesion mechanism of mussels, we utilized DA as an attractive functional monomer for the modification of Fe₃O₄ decorated graphite nanosheets (Fe₃O₄@GNSs) for enzyme immobilization.

In this work, we prepared the non-toxic, high affinity and rapid magnetic response magnetic graphite nanosheets composite via a one-pot hydrothermal approach, and the polydopamine-coated Fe₃O₄@GNSs composite was then fabricated by in situ polymerization of dopamine. Then enzymes could be immobilized on polydopamine-coated Fe₃O₄@GNSs composite via Schiff base reaction. The Candida rugosa lipase (CRL) was used as the target enzyme because of the specific property and the increased activity when bound to an interface [31]. Thanks to the combination of high capacity, superior physical stability of GNSs and well biocompatibility, functional characteristics of PDA, the Fe₃O₄@GNSs-PDA composite shows the high performance as an enzyme immobilization supporting material. Moreover, the enzyme immobilized on Fe₃O₄@GNSs-PDA composite would promise to be a novel nano-biosensor and nano-reactor for the catalytic reaction.

Materials and methods

Materials

Natural flake graphite (NFG) 50 BS mesh, with the purity of 99 wt % was used as a precursor for the synthesis of GNSs supplied by ShanDong Qingdao Tianhe Graphite Company (China). Dopamine hydrochloride (DA), ethylene glycol (EG), sodium acetate (NaAc) and FeCl₃∙6H₂O were purchased from AiHua Fine Chemicals Co., Ltd. (China). Candida rugosa lipase (CRL, Type VII) and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. Other chemicals and reagents were of analytical grade, obtained from Tianjing Chemical Reagent Company (China).

Characterization

Fourier transform infrared (FTIR) spectra were obtained in transmission mode on a FT-IR spectrometer (American Nicolet Corp., Model 170-SX) using the KBr pellet technique. Raman spectra were carried out via a Reinishaw confocal spectrometer with 633 nm laser. Powder X-ray diffraction (XRD, Rigaku D/MAX-2400 X-ray diffractometer with Ni-filtered Cu Kα radiation (λ = 1.54056)) was used to investigate the crystal structure of the composites. All spectra were recorded under the same experimental conditions. The morphologies of the Fe₃O₄@GNSs and Fe₃O₄@GNSs-PDA were characterized by a field-emission scanning electron microscope (SEM, Hitachi S-4800, Japan), transmission electron microscope (TEM, FEI Tecnal G²F30) equipped with energy-dispersive X-ray spectroscopy (EDX, Oxford Instrument), high angle annular dark field (HAADF) and scanning transmission electron microscopy (STEM) to elucidate the dimensions and the structural details of the composites. The surface composition and oxidation state of the samples were performed by X-ray photoelectron spectroscopy (XPS, ESCALAB210). Magnetization measurements were characterized on a vibrating sample magnetometer (Lakeshore 7304, USA) at room temperature. The thermal stability of samples was studied with a thermogravimetry (TG) analyzer (STA449C, Netzsch, Germany) at heating rate of 10 °C min⁻¹ in a nitrogen atmosphere.

Preparation of GNSs

GNSs were prepared according to the previous work of our group [21]. Typically, the mixed acidic intercalating agent was collocated with 70 % concentrated sulfuric acid (45 g) and 68 % nitric acid (15 g). Then 20 g of NFG was added slowly to the mixed acid in a small portion, and 2.2 g KMnO₄ was dissolved into this mixture. The NFG was oxidized for 50 min under continuous stirring. Afterwards, the resulting solid mixture was washed with distilled water and a neutral elution solution through a centrifuge process was obtained; the precipitate was dried at 60 °C. The dried sample, i.e., graphite oxide (GO) was heat-treated abruptly at 600 °C for 15 s to obtain expanded GO. Then, dimethyl formamide (DMF) was used as dispersing agent to peel off the expanded GO. The prepared expanded GO was immersed in a dimethyl formamide (DMF) solution (2 mg/ml) in an ultrasonic bath and sonicated for 12 h, and lastly was filtered and dried to produce the graphite nanosheets (GNSs).

Preparation of Fe₃O₄@GNSs

An easy one-pot method was employed for preparation of Fe₃O₄@GNSs composite via a solvothermal method. In a typical procedure, GNSs (0.1 g) was added into ethylene glycol (30 ml) and ultrasounded under drastic stirring for 1 h. Then FeCl₃∙6H₂O (0.487 g) and sodium acetate (NaAc, 0.973 g) were dispersed into the aforementioned solution under continuous stirring so that Fe³⁺ chemically bonded onto expanded GO. After complete dissolution, the rich dark brown solution was transferred into a 50 mL Teflon reactor and heated at 200 °C for 8 h. After cooling down to room temperature (~20 °C), the solid product was gained by magnetic separation, and washed with deionized water and ethanol repeatedly several times. Finally the product was dried in vacuum at 40 °C for 8 h to obtain the uniform Fe₃O₄ nanoparticles embedded in GNSs.

Preparation of polydopamine modified Fe₃O₄@GNSs

The surface modification of Fe₃O₄@GNSs can be easily manipulated using dopamine with spontaneous oxidative polymerization under mild conditions. In a typical procedure, Fe₃O₄@GNSs (50 mg) was sonicated in a buffered solution (10 Mm Tris–HCl buffer, pH 8.5) for 30 min, and then a corresponding amount of dopamine (2 mg/ml) was added into the above suspension under vigorous stirring for a desired reaction time. The PDA-coated Fe₃O₄@GNSs was thoroughly rinsed with deionized water and ethanol repeatedly and then dried in a vacuum at room temperature for 8 h.

Immobilization of CRL

The CRL was immobilized onto Fe₃O₄@GNSs-PDA composite mainly via covalent binding [32]. Necessary quality of support was put into CRL solution (m/v, 1 %); then the lipase immobilization was carried out at 30 °C in a shaking-table with rotational speed at 120 rpm min⁻¹ for 5 h. After the reaction was completed, the immobilized CRL was obtained by magnetic separation and washed with phosphate buffer (0.1 M, pH = 7.0) several times to remove the unreacted CRL. Especially, the reaction solution and washing solution were collected to assay the amount of residual lipase. The ICRL was kept at 4 °C prior to use.

During the immobilization procedure, the amount of lipase added and reaction time on the activity of immobilized lipase were investigated. The relative activity was obtained after incubation under different amount of lipase added (100–500 mg/g support), immobilizing time (1–9 h), respectively.

Determination of immobilization efficiency and lipase activity

The immobilization efficiency was expressed by the amounts of enzyme bounded on supports of unite mass, and using BSA as the standard to determine the amount of enzyme by the Bradford method [33]. The enzymatic activities of free and immobilized lipase were measured by the titration of the fatty acid which comes from the hydrolysis of olive oil [34] and reverse titration was adopted. One unit of lipase activity (U) is defined as the amount of enzyme needed to hydrolyze olive oil liberating 1.0 μmol of fatty acid per min in the assay condition.

The relative activity (%) is the ratio between the activity of each sample and the maximum activity of the sample.

The residual activity (%) is the ratio between the activity of each sample and the initial activity of the sample.

All data used in these formulas are the average of triplicate of experiments.

Properties of Fe₃O₄@GNSs-PDA immobilized CRL

Effect of pH and temperature of free and immobilized lipase activities

A certain amount of free and immobilized CRL was incubated in 0.1 M 50 ml phosphate buffer under various pH (3.0–10.0) at 37 °C for 30 min with continuous stirring, respectively. Then the enzymatic activities were determined and the relative activities were calculated.

The effect of temperature on the activities of free and immobilized CRL was investigated in the temperature range 20–90 °C for 30 min. The relative activities were calculated.

Thermal stability of free and immobilized lipase activities

Thermal stabilities of the free CRL and immobilized CRL were determined by measuring the activities following incubation in 50 ml phosphate buffer (0.1 M, pH = 7.0) at 50 °C for 240 min with continuous stirring. A sample was removed at 30 min interval and assayed for enzymatic activity. Residual activity was calculated as mentioned above.

Effect of denaturant of free and immobilized lipase activities

A series of concentrations of urea-phosphate buffer solution (0.1 M, pH = 7.0) were added into the hydrolysis reaction of olive oil using free and immobilized lipase at 30 °C, respectively. Then the residual activity of free lipase and of the ICRL was calculated.

Reusability of ICRL

The reusability of the immobilized CRL was determined by hydrolysis of olive oil by the recovered lipase after magnetic separation and compared with that of the first running (activity defined as 100 %).

Results

Preparation and characterization of Fe₃O₄@GNSs-PDA

The size and morp hologies of Fe₃O₄@GNSs and Fe₃O₄@GNSs-PDA composites were characterized by SEM and TEM observations. Figure 1 shows SEM images of Fe₃O₄@GNSs and Fe₃O₄@GNSs-PDA composites. As can be seen from Fig. 1a, b, spherical Fe₃O₄ nanoparticles with a diameter about 300–350 nm deposited on the surface or in the interlayer of GNSs uniformly, which owns to the controllable one-pot hydrothermal process. After the in situ polymerization reaction, PDA film was coated on Fe₃O₄@GNSs so that the surface of Fe₃O₄@GNSs became coarse (Fig. 1c, d) and the diameter of Fe₃O₄@GNSs-PDA composite increased by ~30 nm (Fig. 1d). TEM images present a consistent result compared with the SEM images. It is seen from Fig. 2A a, b, that Fe₃O₄ nanoparticles are an assembly of many small Fe₃O₄ nanocrystals [38] and well dispersed on GNSs, plenty of wrinkles of GNSs can be observed obviously due to the thin structure of the sheet. After the formation of Fe₃O₄@GNSs-PDA composite, a distinct gray thin PDA layer was wrapped on the Fe₃O₄ core (dark) and the surface of GNSs (gray) made it rough and thicker (Fig. 2A c, d). Energy-dispersive X-ray spectroscopy elemental mapping was used to understand the distribution of elements in the composites. From the elemental distribution of iron, oxygen and nitrogen in Fig. 2B, we can recognize that Fe, O and N are distributed uniformly throughout the specific area in Fe₃O₄@GNSs-PDA. Thus, we can speculate that the Fe₃O₄@GNSs was synthesized by the one-pot hydrothermal method and the PDA coating layer was successfully wrapped on the surface of Fe₃O₄@GNSs so that the enzyme would be immobilized on it.

Fig. 1

SEM images of Fe₃O₄@GNSs (a and b) and Fe₃O₄@GNSs-PDA(c and d)

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Fig. 2

A TEM images of Fe₃O₄@GNSs (a and b), and Fe₃O₄@GNSs-PDA (c and d), B STEM image and corresponding iron, oxygen, and nitrogen elemental mapping of Fe₃O₄@GNSs-PDA composite

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FT-IR was employed to examine the surface composition of the synthesized GNSs, Fe₃O₄@GNSs and Fe₃O₄@GNSs-PDA composites. In the spectrum of bare GNSs (Fig. 3a), multiple peaks were observed in the range of 900 to 1,500 cm⁻¹, which can be assigned to the functional groups, including C–O (ν C–O at 1,060 cm⁻¹), C–O-C (ν C–O-C at 1,247 cm⁻¹), C = O in carboxylic acid and carbonyl moieties (ν C = O at 1,764 cm⁻¹). The intense band at 1,594 cm⁻¹ attributed to the O–H bending vibration, epoxide groups and skeletal ring vibrations [39]. After decorated with Fe₃O₄, the strong peak at 579 cm⁻¹ is related to the vibration of the Fe–O function group and peaks at 1,634 and 3,435 cm⁻¹ are corresponding to the surface-adsorbed water and hydroxyl groups during the hydrothermal process. New peaks at 1,239, 1,293, 1,500 cm⁻¹ and stronger peaks at 1,634 cm⁻¹ in the spectrum of Fe₃O₄@GNSs-PDA derive from the aromatic rings in the PDA polymer. In addition, it could be noted that the peak at 3,435 cm⁻¹ of Fe₃O₄@GNSs-PDA is broader than that of Fe₃O₄@GNSs, which resulted from the overlapping of hydroxyls, water adsorbed in PDA polymer and amines of PDA [40]. The FT-IR analysis suggested that PDA adhered onto Fe₃O₄@GNSs surface successfully.

Fig. 3

a FT-IR spectra of GNSs, Fe₃O₄@GNSs and Fe₃O₄@GNSs-PDA, b Raman spectra of GNSs, Fe₃O₄@GNSs, and Fe₃O₄@GNSs-PDA

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In Raman spectrum (Fig. 3b), GNSs shows D-band peak at 1,332 cm⁻¹ and G-band peak at 1,576 cm⁻¹, respectively [41]. The G-band is due to the first-order scattering of E_2g mode and is associated with the in-plane vibration of sp² carbon domains in a 2D hexagonal lattice, while the D-band arises from the κ-point phonons of A_1g mode and is related to the vibration of sp³ carbon domains in plane terminations of disordered graphite [42]. Compared with pristine GNSs, the D- and G-bands of Fe₃O₄@GNSs become lower and broader, suggesting a higher level of disorder of the graphite layers during the functionalization process [43]. The G-bands of Fe₃O₄@GNSs, occur at 1,581 cm⁻¹, which is up shifted by 5 cm⁻¹ compared to that of GNSs clearly demonstrates that there is a strong interaction between the Fe₃O₄ and GNSs. In the Raman spectra of Fe₃O₄@GNSs-PDA, the slightly split peak at 1,320 cm⁻¹ with the sharp peak at 1,581 cm⁻¹ can be characterized as the stretching and deformation of aromatic rings of PDA [44] which confirms the existence of PDA in Fe₃O₄@GNSs-PDA composite. What is more, it can be seen from Fig. 3b that these two characteristic peaks became sharper and more obvious than that of Fe₃O₄@GNSs at the same position, which resulted from the overlap of the two peaks between PDA and the typical D- and G-bands of Fe₃O₄@GNSs. The similarity in FT-IR and Raman spectroscopy further supported the affinity between Fe₃O₄@GNSs and PDA film.

X-ray photoelectron spectroscopy (XPS) analysis was applied to investigate the chemical elements on surface of the materials. The wide-scan XPS spectra for Fe₃O₄ nanoparticles, Fe₃O₄@GNSs and Fe₃O₄@GNSs-PDA composites are shown in Fig. 4. As can be seen from Fig. 4a, except for the characteristic peaks of C1s and O1s, the spectrum of Fe2p appeared in the spectrum of Fe₃O₄@GNSs, and the high intensity C1s came from GNSs suggested the Fe₃O₄@GNSs was effectively obtained. After the Fe₃O₄@GNSs composite was wrapped by PDA film, the Fe₃O₄ peak from the underlying composite was screened (Fig. 4b), indicating that the surface was coated with a film that was thicker than the analysis depth of XPS (~10 nm). Meanwhile, a new peak of N1s in Fig. 4b appeared, and the nitrogen-to-carbon signal ratio (N/C) of 0.126 was similar to that of the theoretical value for PDA (N/C = 0.125), implying the formation of a polydopamine layer.

Fig. 4

a XPS spectra of Fe₃O₄@GNSs, b XPS spectra of Fe₃O₄@GNSs-PDA

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XRD was used to confirm the phase and structure of the prepared samples. Figure 5 shows that both Fe₃O₄@GNSs (Fig. 5a) and Fe₃O₄@GNSs-PDA (Fig. 5b) possess diffraction patterns similar to those of Fe₃O₄ (JCPDS no. 19–629): the 111, 220, 311, 400, 422, 511, 440 planes of Fe₃O₄ were observed at 2θ = 18.25, 30.19, 35.39, 43.04, 53.44, 56.80, 62.46°, respectively. Meanwhile, the sharp diffraction peak at 2θ = 26.50° in Fe₃O₄@GNSs and Fe₃O₄@GNSs-PDA can be attributed to the graphite-like 002 reflection. This suggests that the crystal structure of the magnetite component was preserved during the whole synthesis process.

Fig. 5

XRD patterns of Fe₃O₄@GNSs (a), Fe₃O₄@GNSs-PDA (b), and GNSs (c)

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For quantifying the amount of PDA in the Fe₃O₄@GNSs-PDA composite, thermogravimetric analysis (TGA) was carried out in nitrogen atmosphere at a rate of 10 °C min⁻¹. Figure 6 displayed the TGA curves of the Fe₃O₄@GNSs and Fe₃O₄@GNSs-PDA samples along with bare Fe₃O₄. As can be seen from Fig. 6, the Fe₃O₄@GNSs showed a mild weight loss (1.45 wt %) below 200 °C which can be assigned to the evaporation of absorbed water and solvent. The significant decomposition occurred between 200 and 400 °C (3.83 wt %) mainly due to the pyrolysis of stable oxygen-functioned groups. Compared with the Fe₃O₄@GNSs composite, bare Fe₃O₄ lost weight about 5.61 % at the temperature range 200–400 °C, which is indicative of the remarkable thermo-stability of Fe₃O₄@GNSs. The TG curve of Fe₃O₄@GNSs-PDA kept same trend between 35 and 400 °C, while a large mass loss (18.33 %) is observed from 513 to 589 °C which is presumed to result from the decomposition of PDA. The final weight loss of Fe₃O₄@GNSs-PDA is 35.10 %, indicating that PDA comprises about a half mass of the Fe₃O₄@GNSs-PDA.

Fig. 6

TG curves of Fe₃O₄, Fe₃O₄@GNSs and Fe₃O₄@GNSs-PDA

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In the use of PDA polymer coated Fe₃O₄@GNSs for lipase immobilization, it is important that the Fe₃O₄@GNSs-PDA should possess sufficient magnetic properties. The magnetic properties of bulk Fe₃O₄ nanoparticles, Fe₃O₄@GNSs and Fe₃O₄@GNSs-PDA composites were studied using a vibrating sample magnetometer at room temperature. From Fig. 7a we can see that the as-prepared magnetic materials have a fairly strong magnetization and the saturation magnetization (MS) values are about 84.57 emu g⁻¹ for Fe₃O₄, 53.09 emu g⁻¹ for Fe₃O₄@GNSs and 37.28 emu g⁻¹ for Fe₃O₄@GNSs-PDA, respectively. The decrease in magnetic saturation of Fe₃O₄@GNSs-PDA in comparison with Fe₃O₄@GNSs may be attributed to the increased mass of modified polydopamine shell on the surface of Fe₃O₄@GNSs. The Fe₃O₄@GNSs-PDA composite also shows fast response to the applied magnetic field (2000 Oe), and after dispersing the Fe₃O₄@GNSs-PDA in water by shaking, vortexing or sonication, it can be easily collected within several minutes by placing a magnet on the side of the vessel (Fig. 7b). Though the saturation magnetizations of Fe₃O₄@GNSs-PDA is lower than bulk Fe₃O₄ and Fe₃O₄@GNSs, it is strong enough for effective magnetic separation.

Fig. 7

a Magnetic hysteresis loops of Fe₃O₄, Fe₃O₄@GNSs and Fe₃O₄@GNSs-PDA, b Schematic of the simple magnetic separation of Fe₃O₄@GNSs-PDA

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Activity of Fe₃O₄@GNSs-PDA immobilized CRL

Optimum conditions of ICRL

For the preparation of Fe₃O₄@GNSs-PDA immobilized CRL, the activity of lipase amount added is shown in Fig. 8a. From Fig. 8a we can see, with the lipase amount increased the activity of ICRL rose gradually and decreased after the lipase amount added was up to 350 mg g⁻¹. The excellent ability of lipase loading was much higher than our previous works [23, 47] which correlated to the high capacity of GNSs and high activity of PDA polymer both for chemistry and physics. The relative activity declined when the lipase amount added was up to 400 mg g⁻¹ because of the over cross-linking of CRL during the immobilizing process. Relevant research confirmed that the improved activity of the immobilized lipase was involved with the alteration of its secondary structure, especially to the increase of α-helix content and the decrease of β-sheet content [48], which demonstrates that the Fe₃O₄@GNSs-PDA have well biocompatibility and affinity with CRL to enhance its activity.

Fig. 8

a The effect of the amount of CRL added, b The effect of the immobilization time of CRL. The enzyme immobilization was conducted in phosphate buffer solution (0.1 M, pH = 7.0) at 37 °C

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Figure 8b shows the result of the immobilizing time of CRL. It can be seen that the optimum immobilizing time is 5 h, after 6 h the relative activity of ICRL decreased sharply. The main reason is that an appropriate reaction time contributed to couple the lipase on Fe₃O₄@GNSs-PDA, while the excessive coupling time may lead to the inactivation of CRL.

As a result, the optimum conditions of immobilizing lipase were obtained: amount of lipase was 350 mg g⁻¹ support, reaction time was 5 h at pH value of the reaction system was 7.0. The bounded protein of the immobilized lipase under this condition was 337.96 mg g⁻¹ support and the activity yield of the ICRL was 65.43 % (Table 1). Therefore, the results obtained in our work seemed to be quite promising.

Discussion

Figure 9 displays the reaction mechanism of polydopamine modified Fe₃O₄@GNSs in this work. The Fe₃O₄ doped graphite nanosheets were firstly prepared by a one-pot hydrothermal method, the process of which is a versatile chemical approach for the synthesis of nano-materials with well-defined shapes and controlled sizes. Compared with the similar Fe₃O₄ decorated graphene, graphene oxide and reduced graphene oxide synthesis [35–37], graphite nanosheets obtained from the expanded graphite is more economical-effective, manufacture easily and environmentally friendly. Then, the adhesive-polymer (PDA) was deposited on the surface of Fe₃O₄@GNSs uniformly via the dip-coating way (tris-buffer solution, pH 8.5). The familiar adhesion between PDA platform and Fe₃O₄@GNSs composite is fast, green and process controllable, and the versatile chemical properties as a binding regent and the excellent biocompatibility of PDA film played an important role in CRL immobilization.

Fig. 9

Schematic illustration of the synthesis process used to produce Fe₃O₄@GNSs-PDA and CRL immobilized on Fe₃O₄@GNSs-PDA

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In this work, the self-polymerization of DA formed a thin, surface-adherent PDA film onto Fe₃O₄@GNSs under a controllable condition. Its bioactive surface can graft macromolecules directly via Schiff base formation between animo group of macromolecules and quinone group of PDA [32] along with some physical effects by the noncovalent functionalities such as amino and hydroxyl groups as well as π–π bonds [29]. Compared with many related works which usually use some synthetic reagents (such as glutaraldehyde, epichlorohydri, formaldehyde, carbodiimide (EDC), and hexamethy-lene-1,6-diamino-carboxysulphonate (HDACS)) as the cross-linkers between supporting materials and enzymes [45, 46], we provided a fast, effective, and most importantly, non-toxic process for lipase immobilization. Additionally, the chemical stability, high affinity of GNSs also contributed to the effective lipase immobilization. Fe₃O₄ nanoparticles embedded in GNSs not only acted as an easy separation tool in Fe₃O₄@GNSs-PDA composites, but also mitigated the restacking and aggregation of GNSs during the one-pot process. The principle of CRL immobilized on Fe₃O₄@GNSs-PDA composite is illustrated in Fig. 9.

Optimal conditions of enzymatic activity

The effect of pH value on the activity of ICRL is given in Fig. S1A. The optimal pH of both free CRL and ICRL is 7.0. In addition, compared to the pH endurance of free CRL, ICRL showed enhanced stability especially in the alkaline zone. It can be explained that by covalent attachment, the configuration of lipase was fixed on the surface of Fe₃O₄@GNSs-PDA so the tolerability of CRL to pH in surroundings increased [49]. The abundant–OH/-O⁻ pairs and the–NH₂/-NH₃⁺ pairs on PDA made it a zwitter-ion under different pH range, which could tune the local pH value. Thus the immobilized CRL would stay in the buffer region against the environmental mutation. This indicates that the lipase immobilized on Fe₃O₄@GNSs-PDA possesses markedly improved adaptability in a wide pH range, which can greatly expand the applications of lipase in chemical and biocatalytic industry.

The operating temperature range is another important factor in limiting the practical applications of lipase. Figure S1B shows the effect of temperature of the solutions on the hydrolysis activity of free and immobilized lipase for olive oil. The optimum reaction temperature of both free and immobilized lipase is 30 °C, while the activity of free lipase decreased sharply with the increase of the temperature. Compared with free lipase, the ICRL kept its relative activity up to 80 % in the temperature range of 20–60 °C and exhibited the high relative activity of 42 % at 90 °C, revealed much superb heat endurance than that of the free lipase. The increase of the optimum reaction temperature for the immobilized CRL may be due to the covalent combination between the Fe₃O₄@GNSs-PDA and lipase as well as the change of the conformational integrity in lipase structure. Moreover, the protective effect provided by the flexible structure of the prepared Fe₃O₄@GNSs-PDA also conducted a tough performance with enzymes from denaturation at high temperature.

The strong thermal stability is one of the critical factors in the industrial applications. Figure S2A shows the residual activity of free and immobilized lipase at 50 °C on the hydrolysis reaction of olive oil. Both the free CRL and ICRL exhibited the similar trend: the residual activity declined along with the reaction time prolonged. While the ICRL declined less and more slowly compared with the free form. After 150 min, free CRL lost the activity while ICRL kept it at 30 % and did not lose activity when the reaction time was at 240 min. These results probably resulted from the excellent thermal tolerance, good mechanical hardness and high specific surface area of the prepared supporting material, which protected the CRL from unfolding and prevented conformational transitions.

A denaturant such as urea can cause the protein to unfold, which may highly restrict the industrial applications of lipase. In order to investigate the influence of denaturant on the inactivation of lipase, the effect of urea with different concentrations on the hydrolysis activity of free and immobilized lipase was determined at 30 °C in phosphate buffer (0.1 M, pH = 7.0). From Fig. S2B we can see the activity of CRL shows a progressive loss with the concentration increase of urea. The CRL showed a reasonable decrease and significantly prevented from denaturation after immobilization on Fe₃O₄@GNSs-PDA composite. The ICRL kept the excellent residual activity of 87 % in 3 M urea and still held it at about 75 % in 6 M urea. However, free lipase possessed only 21 % activity in 3 M urea and deactivated totally when the concentration of urea was up to 6 M, respectively. One of the possible reasons is the lipase was attached on Fe₃O₄@GNSs-PDA mainly via covalent bonding, and it is possibly conducted a higher activation energy for the molecules to recognize and prevented the ternary structure of lipase from denaturation [50].

In order to achieve the reuse of lipase and then for the potential application in industry, it is important to investigate the well reusability of lipase. The variation of the activity of the ICRL after multiple-reuse was shown in Fig. 10. It can be observed from Fig. 10 that the ICRL kept the high activity at 80 % after 12 recycles. The decline in activity was considered as the denaturation and leakage of protein after some reaction circles. Thus, it can be concluded that the Fe₃O₄@GNSs-PDA could significantly increase the operational stability as well as confer additional stability against denaturation and provide a protected microenvironment that could enhance the catalytic properties of the biocatalyst.

Fig. 10

Number of reuses of the immobilized CRL. The recovered CRL was washed with phosphate buffer solution (0.1 M, pH = 7.0) thoroughly and reused for the next cycle. The enzyme activity was compared with the first running (activity defined as 100 %)

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Conclusion

In this work, mussel-inspired magnetic graphite composite was successfully synthesized via the simple dip-coating way of dopamine. Then the as-prepared composite was used to immobilize CRL covalently under mild conditions and avoided any other toxic functional chemicals. The novel Fe₃O₄@GNSs-PDA composite was proved to possess high magnetization, a uniform well-defined structure and high affinity towards lipase immobilization. By means of measuring various properties of ICRL, the Fe₃O₄@GNSs-PDA composite was demonstrated to have high capacity and well biocompatibility for lipase immobilization, and the Fe₃O₄@GNSs-PDA immobilized CRL showed excellent abilities in environmental stability and catalytic activities. Significantly, the ICRL retained 80 % activity after 12 times of recycling for batch hydrolysis of olive oil emulsion. Overall, the successful application in enzyme immobilization using the adhesive magnetic graphite composite confirmed the suitability for various biochemical applications.

Acknowledgments

The authors thank the financial supports from the National Natural Science Foundation of China (No. 21374045), the scientific research ability training of under-graduate students majoring in chemistry by the two patters based on the tutorial system and top students (J1103307) and the Opening Foundation of State Key Laboratory of Applied Organic Chemistry (SKLAOC-2009-35).

Conflict of interest

The authors declare no competing financial interest.

References