Yang
Wang
,
Yun
Zhang
*,
Chen
Hou
,
Fu
He
and
Mingzhu
Liu
*
State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Institute of Biochemical Engineering and Environmental Technology, Lanzhou University, Lanzhou 730000, China. E-mail: zhangyun@lzu.edu.cn; mzliu@lzu.edu.cn; Fax: +86-931-8912113; Fax: +86-931-8912582
First published on 16th November 2015
Magnetic organic–inorganic hybrid microcapsules consisting of plant phenols, polyethylenimine (PEI) and FeIII ion complexes were prepared in a facile one-pot way. Porous CaCO3 microparticles were used as the hard template for the adsorption of negatively charged Fe3O4 nanoparticles, which acted as the source of magnetism for recycling. Moreover, the coated Fe3O4 nanoparticles also helped to improve the rigidness of the microcapsules away from rupturing during multiple reuses. Upon addition of tannic acid (TA), PEI and FeIII ions, the magnetic CaCO3 microparticles were coated with the adhesive complexes through chemical chelation and covalent bonding. Then the template was removed using EDTA to construct the target microcapsules. During the CaCO3 formation step, Candida rugosa lipase (CRL) was used as the biomolecule which was encapsulated in the CaCO3 microparticles. Characterizations demonstrated that the as-prepared magnetic microcapsules showed a robust structure, so that the enzyme inside could be protected physically. As a result, the magnetic hybrid microcapsules exhibited high efficiency in enzyme catalysis and stability against the environment, due to the high biocompatibility and robust structure.
Due to its unique structural properties, TA can make familiar interactions with a variety of materials to form metal–organic films via multiple reaction pathways, including electrostatic interactions, hydrogen bonding, hydrophobic interactions, and like many other polyphenols, metal chelation.9–11 Recently, Guo et al. reported the engineering of metal–phenolic networks to introduce a library of metal–phenolic motif materials, which provided an extensive field for the study and application of metal–organic films.8 As has been proved, the coordination between TA and FeIII ions is fast (in seconds), structurally rigid and highly biocompatible, forming a TA–FeIII film with desired properties, such as high biocompatibility, facile degradability, cyto-protectability and second-step functionality.2
Enzymes are superior over chemical catalysts because of their high effectiveness, high specificity, and green reaction conditions.12 Among the enzymes applied in biocatalysis, lipases [E.C. 3.1.1.3] have been widely studied due to their “interfacial activation” feature when catalyzing the hydrolysis of carboxylic acid esters to carboxylic acids and alcohols, and the reactions of chemo-, regio- and stereoselective esterification or trans-esterification under micro/non-aqueous conditions in an efficient and specific way.13,14 However, the industrial application of enzymes still has many challenges due to the low stability, high cost, and difficult recycling and regeneration. As a very promising strategy, the immobilization of enzymes shows lots of advantages involving enhancing the catalytic stability, feasibility for continuous operations, recycling the enzyme and a significant reduction of costs and so on.15 As a result, we decided to utilize natural phenols (TA) and inorganic materials (FeIII ions and magnetic Fe3O4 nanoparticles) to explore a high efficiency and time-saving method with a novel and much simpler route for the construction of enzyme reactors.
Herein, efforts were made to construct enzyme microcapsules using low-cost plant polyphenols (TA), polyethylenimine (PEI) and inorganic materials (FeIII ions and magnetic Fe3O4 nanoparticles) as precursors for the formation of Fe3O4/TA–FeIII–PEI hybrid microcapsules. More specifically, Fe3O4 nanoparticles were adsorbed on porous CaCO3 microparticles, then the organic–inorganic hybrid layer was formed in a one-pot step through chelation of TA and FeIII ions and the Schiff base reaction between TA and PEI. Lastly, CaCO3 templates were removed and the Fe3O4/TA–FeIII–PEI hybrid microcapsules were prepared. During the film formation process, covalent binding between PEI and TA could improve the toughness of the hybrid film more than sole chelation of TA and metal ions. The magnetic hybrid microcapsules could respond to external magnetic field stimuli, which is important for practical applications with enzyme recycling and reuse. Furthermore, the incorporated Fe3O4 nanoparticles also helped to retain an intact and rigid structure. In particular, compared to the wide application of polydopamine films, plant polyphenol-inspired coatings not only retain many of the advantages of polydopamine and deposit under similar conditions, but are also colorless and derived, in some cases, from reagents a hundredfold less costly than dopamine. Candida rugosa lipase (CRL) was immobilized in the microcapsules, accompanied with CaCO3 template formation, and the catalytic activity and stability were then investigated in detail.
The efficiency of immobilization was evaluated in terms of activity yield and immobilization yield as follows:
The relative activity (%) is the ratio between the activity of every 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 averages of triplicate experiments.
The effect of temperature on the catalytic activities of free and immobilized CRL were measured by the hydrolysis of olive oil in a water bath at 37 °C for 30 min, after they were first incubated in phosphate buffer (0.1 M, pH = 7.0) among the temperature range of 20–90 °C for 30 min. The relative activity was compared.
Thermal stabilities of the free and immobilized CRL were determined by measuring the activities after incubation in phosphate buffer (0.1 M, pH = 7.0) at 50 °C for 240 min with continuous stirring. A sample was removed after a 30 min interval and tested for enzymatic activity. The residual activity was calculated as above.
Fig. 2 demonstrates morphologies of the prepared microparticles and microcapsules. As can be seen from Fig. 2(a1), the as-prepared CaCO3 microparticles possessed a uniform, spherical shape with a diameter of about 3 μm. After adsorption of Fe3O4 nanoparticles, many tiny nanoparticles (with a diameter of about 10–15 nm) were assembled on CaCO3 microparticles to cover the original smooth surface (Fig. 2(a2)), indicating the successful preparation of Fe3O4–CaCO3 microparticles. Several batches of Fe3O4/TA–FeIII–PEI microcapsules were prepared to vary the concentration of TA, FeIII ions and PEI in the reaction mixtures as follows: TA 0.2 mg ml−1, FeIII ions 0.8 mg ml−1 and PEI 0.4 mg ml−1 (Fe3O4/TA0.2–FeIII0.8–PEI0.4), TA 0.2 mg ml−1, FeIII ions 0.8 mg ml−1 and PEI 0.8 mg ml−1 (Fe3O4/TA0.2–FeIII0.8–PEI0.8), and TA 0.4 mg ml−1, FeIII ions 1.6 mg ml−1 and PEI 1.6 mg ml−1 (Fe3O4/TA0.4–FeIII1.6–PEI1.6). For comparison, TA 0.2 mg ml−1 and FeIII ions 0.8 mg ml−1 repeated coating was performed 3 times (Fe3O4/(TA0.2–FeIII0.8)3). As can be seen from Fig. 2(b1), (c1), (d1) and (e1), after coating with the hybrid layer, the microparticles held a uniform surface and spherical structure, which were not affected by the interactions among TA, FeIII ions and PEI. After template removal, the hollowed microcapsules were formed and no obvious collapse appeared. The slight creases of the wall made it have a pisiform appearance (Fig. 2(d2) and (e2)). The wall of the hybrid microparticles became thicker and rougher with the increase of the coating concentration, and the spherical morphologies of the hybrid microparticles became distinct and intact (Fig. 2(b2), (c2), (d2) and (e2)). Besides, it can be clearly observed that Fe3O4 nanoparticles were wrapped in the hybrid layer after the CaCO3 microparticle dissolution (Fig. 2(f1) and (f2)). As for the microcapsule Fe3O4/(TA0.2–FeIII0.8)3, we also obtained the expected result (Fig. 2(b2)), though it would not be the optimized option for enzyme immobilization due to the weaker wall structure without PEI doping. Thus, it can be verified that the hollow and robust Fe3O4/TA–FeIII–PEI microcapsules were successfully achieved.
To further observe the surface features of the microparticles and microcapsules, SEM images were conducted. As shown in Fig. 3, after adsorption of the negatively charged Fe3O4 nanoparticles, the surface of the CaCO3 microparticles became rough and coarse (Fig. 3(a) and (b)), which was consistent with the TEM images. It further indicated that the Fe3O4 nanoparticles were distributed uniformly on the surface of the CaCO3 microparticles. In the cases of the as-prepared hybrid microcapsules, all of them possessed a plump structure due to the inlaid Fe3O4 nanoparticles (Fig. 3(c)–(f)). Moreover, an absence of or decrease in the concentration of PEI would lead to an incompact wall structure (Fig. 3(c) and (d)). When the concentration of TA, FeIII ions and PEI doubled, the superfluous complex was adhered to the side of the microcapsules (Fig. 3(f)). Uniform and tidy microcapsules were obtained with the premium conditions for Fe3O4/TA0.2–FeIII0.8–PEI0.8 (Fig. 3(e)). In addition, compared with the PDA–Fe3O4 microcapsules we made,16 the newly prepared Fe3O4/TA–FeIII–PEI microcapsules had a more micromesh and homogeneous wall structure, attributed to the rapid coating process.
FTIR was conducted to examine the functional groups of the prepared magnetic hybrid materials. As shown in Fig. 4(a), the characteristic peak at 580 cm−1 of Fe3O4–CaCO3 can be attributed to the lattice absorption of the Fe3O4 nanoparticles, the absorption bands appearing at 1491/1434 cm−1, 1087 cm−1, and 876 cm−1 can be assigned to the vibrations of the carbonate group in CaCO3. In the spectrum of Fe3O4/(TA0.2–FeIII0.8)3, the decreased intensity of the C–OH stretching peak of TA at around 1250 cm−1 shows evidence that the phenolic groups are coordinated with FeIII ions.8 The same situation is also observed in the Fe3O4/TA0.2–FeIII0.8–PEI0.8 microcapsule. The absorption peak at 1622 cm−1 is attributed to the o-benzoquinone derivative arising from the oxidation of TA22 which evidenced the rationality of the step reaction with PEI. After reaction with PEI, the old peak of Fe3O4/(TA0.2–FeIII0.8)3 at 1622 cm−1 disappeared and the new peak of Fe3O4/TA0.2–FeIII0.8–PEI0.8 at 1601 cm−1 represented the aromatic CN, which successfully confirmed the reaction between TA and PEI.
To further confirm the reaction phenomenon of the compounds, images of the different reacting mixtures are displayed (Fig. 4(b)). Under neutral conditions, the mixture of FeIII ions and TA solution transformed into a dark blue solution due to the formation of tris-pyrogallato iron complexes. After PEI was added into the above mixture, it turned into a prunosus sticky liquid immediately. The mixture of TA and PEI became milky white hydrogels immediately as they were mixed. Thus the reactions among FeIII ions, TA and PEI were proven to be fast, visually.
XPS was performed to identify the presence of metal ions and PEI in the microcapsule shells (Fig. 5). As displayed in Fig. 5, C1s, O1s, N1s, and Fe2p peaks were detected in the survey spectra, and this is in agreement with the hybrid wall compositions. From the O1s photoelectron spectrum (Fig. 5(b)), the peaks at ∼531.29, ∼530.07, ∼529.18, and ∼528.28 eV can be assigned to the CO, C–O, Fe–OH, and Fe–O species, respectively. C
O corresponded to the o-benzoquinone derived from TA and Fe–O/Fe–OH arose from the coordination between TA and FeIII ions. From the N1s spectra (Fig. 5(c)), the peaks appearing at ∼399.81, ∼397.94, and ∼397.37 eV can be attributed to –NH2, –NH–, and –N
, respectively, which suggested successful chemical crosslinking between TA and PEI.
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Fig. 5 XPS of Fe3O4/TA0.2–FeIII0.8–PEI0.8 microcapsules: (a) survey spectrum, (b) O1s core-level spectrum, and (c) N1s core-level spectrum. |
The hysteresis loops of the prepared magnetic nanoparticles are shown in Fig. 6. From Fig. 6 we can see that the saturation magnetization (MS) values are about 66.67 emu g−1 for the CA–Fe3O4 nanoparticles, and 34.69 emu g−1 for the Fe3O4/TA0.2–FeIII0.8–PEI0.8 microcapsules, respectively. As a result, the microcapsules used for the CRL immobilization could be separated quickly and easily from the reaction medium with an external field. Compared to the magnetic PDA microcapsules we previously made,16 the as-prepared microcapsules possessed a significantly higher saturation magnetization, and they would obtain an improved efficiency for the immobilized enzyme recycling and reuse. Furthermore, there is no hysteresis in the magnetization, with both remanence and coercivity being zero, indicating that the as-prepared Fe3O4/TA0.2–FeIII0.8–PEI0.8 microcapsules are superparamagnetic at room temperature.23
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Fig. 6 Magnetic hysteresis loops of CA–Fe3O4 nanoparticles, and Fe3O4/TA0.2–FeIII0.8–PEI0.8 microcapsules. |
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Fig. 7 Effect of enzyme amount and encapsulation efficiency of Fe3O4/TA0.2–FeIII0.8–PEI0.8 microcapsules. |
K m (mg ml−1) | V max (U mg−1) | |
---|---|---|
Free CRL | 0.43 | 6.05 |
CRL–Fe3O4/TA0.2–FeIII0.8–PEI0.8 | 0.54 | 5.10 |
The pH stabilities of CRL–Fe3O4/TA0.2–FeIII0.8–PEI0.8 microcapsules and free CRL are compared in Fig. 8(a). The CRL–Fe3O4/TA0.2–FeIII0.8–PEI0.8 kept >71% of its initial activity at pH 5.0–8.0, and showed a decline below pH 4.0 and above 9.0. In comparison, the free CRL retained 29% of the relative activity at pH 3.0 and 42% of the relative activity at pH 9.0. In addition, CRL–Fe3O4/TA0.2–FeIII0.8–PEI0.8 showed a broader pH scope. As a result, the Fe3O4/TA0.2–FeIII0.8–PEI0.8 used for CRL immobilization exhibited a markedly improved adaptability in a wide pH range, which can greatly expand the applications of lipase in chemical and biocatalytic industries. This phenomenon can be explained by the buffering effect of the hybrid layer of the microcapsules. The abundant –OH/–O− pairs on TA and the –NH2/–NH3+ pairs on PEI could tune the local pH value under basic or acidic conditions. Therefore, the CRL near the hybrid walls would stay in the buffer region, against environmental mutation, to maintain the activity of the CRL due to the positive influence of the TA and PEI ingredients.
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Fig. 8 Effect of (a) pH value, (b) temperature, (c) stability, and (d) number of reuses of free and immobilized CRL. |
When the hydrolysis for olive oil emulsion was operated at a series of temperature ranges, the immobilized CRL showed enhanced relative activities compared to the free CRL (Fig. 8(b)). Compared with free lipase, the immobilized CRL kept its relative activity up to 80% in the temperature range of 20–60 °C and exhibited more than 60% of its relative activity at 90 °C, revealing a more superb heat endurance than that of the free lipase. It seemed that the interaction between the positively charged PEI and negatively charged CRL molecules would prevent enzymes from denaturing at high temperatures.24
Strong thermal stability is one of the critical factors in industrial applications. Fig. 8(c) shows the residual activity of the free and immobilized lipase at 50 °C for the hydrolysis reaction of olive oil. From Fig. 8(c) we can see that after being incubated for 150 min, free CRL lost its activity while immobilized CRL retained its residual activity as high as 58%, until the incubation time reached 240 min. This phenomenon probably resulted from the excellent thermal stability, good mechanical hardness and high biocompatibility of the prepared organic–inorganic hybrid microcapsules, which protected the CRL from unfolding and conformational transitions.
A high reusability of lipase is critical for potential applications in industry. As presented in Fig. 8(d), the immobilized enzyme kept a high activity at 75% after reuse 12 times, due to the sturdy stability of the hybrid microcapsules which effectively ameliorated the denaturation and leakage of enzyme under multiple reaction circles. Moreover, the loss of microcapsules during each recycle cannot be ignored. As a result, the layer assembled by Fe3O4 and TA–FeIII–PEI had a high biocompatibility and strong mechanical properties, which could effectively mitigate the deactivation, leaching and embedding of the encapsulated enzymes.
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