A new lipase–inorganic hybrid nanoflower with enhanced enzyme activity

Abstract

A new hybrid nanoflower was synthesized using the organic component of Burkholderia cepacia lipase and inorganic component of calcium phosphate. Under the optimum conditions, the nanobiocatalyst exhibited an activity of 849.8 U, 308% folds of the free one, with relatively good stability, highlighting its potential for industrialization.

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In the past decades, numerous immobilization strategies have been utilized to improve the lipase properties.^1,2 Immobilization supports, as one of the most important factors in immobilization procedures, have attracted considerable attention. During the selection of supports, the material composition, size, shape, mechanical strength, hydrophilicity, functional group species and some other properties should be fully considered according to the lipase type and applications.³ Solid supports provide predominant advantages in separation and reuse, lipase stability, and material modification. To date, various solid supports have been reported for immobilization, such as pore glass beads, membranes, macroporous resin, graphene, nanotubes, and polymer-/silica-based monolithic materials. Nanobiocatalysis, which involves enzymes immobilized on nanostructured materials, are attracting increasing attention due to its large surface–volume and great potential in applications. Nanoparticles with different materials and morphologies have been widely reported as immobilization supports for different applications.⁴ Kavosi et al. reported that gold nanoparticles encapsulated in a polyamidoamine dendrimer composed of a type of immunosensor⁵ was used to detect the PSA concentration, which is promising in the clinical screening of cancer biomarkers. Ranjbakhsh et al.⁶ immobilized porcine pancreas lipase on silica-coated modified magnetite nanoparticles and improved the lipase kinetic parameters and stability. Cellulase from Trichoderma reesei was immobilized on an activated magnetic nanoparticle, resulting in enhanced enzymatic saccharification of the pretreated hemp biomass.⁷ With the increase in the number of reports focusing on enzyme immobilization, nanoparticles and nanobiocatalysts have great potential for industrial applications.

Lipase (EC3.1.1.3) has been widely used in food, biodiesel preparation, chiral pharmacy and many other biosynthetic industries.^8,9 Many studies have been carried out to improve the catalytic properties of lipases; however, some limitations still block their applications in industry, such as poor stability, narrow pH range adaptability, and sensitivity to harsh environments.¹⁰ It is also difficult to separate free lipase from a reaction mixture, which can be overcome by immobilization. Moreover, immobilization has been demonstrated to be one of the most useful strategies to improve the catalytic properties of free enzyme. In this study, Burkholderia cepacia lipase (BCL) was chosen as the immobilization object because of its versatile catalysis in biofuel synthesis, biorefinery and a variety of reactions in aqueous and non-aqueous phases. In our previous study, BCL have been employed in biofuel synthesis and in its immobilized form they exhibited very high catalytic activity. Li et al.¹¹ examined the chiral resolution properties of BCL and the form immobilized on multi-walled carbon nanotubes has extremely high activity, showing great promise in the biopharmaceutical industry.

In 2012, Ge et al.¹² first reported nanoflowers made of organic–inorganic components using BSA–Cu₃(PO₄)₂·3H₂O; subsequently, some enzyme–inorganic hybrids have been reported.^13,14 However, there are few reports focusing on lipases and their nanoflower hybrids,^15,16 and the activities are rather low. Therefore, in this study, we synthesized a new nanoflower hybrid from Burkholderia cepacia lipase (BCL) and calcium phosphate (Ca₃(PO₄)₂). It has an interesting flower-like structure on the nanoscale. Its properties were examined and discussed, and the results showed that the obtained nanobiocatalyst exhibited a higher enzyme activity and an enhanced operational stability in the resolution reaction.

The nanoflowers were synthesized in a typical experiments: 100 μL CaCl₂ (200 mM) was added to 5 mL of a phosphate buffered saline (PBS) solution (20 mM) containing 24 mg mL⁻¹ BCL at pH 6.7, and the mixture was kept at room temperature (∼25 °C) for 24 h. In all the following experiments, the conditions were kept the same as the typical experiment, unless stated otherwise. The mixture was centrifuged at 4 °C and 12 000 rpm for 10 min to remove the supernatant. The protein content of the supernatant was measured using a Bradford protein assay¹⁷ to calculate the immobilization efficiency. The immobilized lipase (lipase–inorganic hybrid nanoflowers) was then dried in a thermostatic vacuum drier for subsequent use. SEM was utilized to display the morphologies of nanoflowers. As shown in Fig. 1, the morphology turns to be flower-like nanoparticles from the sheets stacking ones when the lipase protein was added. The petals were about 2–3 μm wide.

Fig. 1 SEM images of the synthetic products without lipase (a) and with lipase (b). (Conditions: 24 mg mL⁻¹ lipase loading, 20 mM PBS).

With increasing PBS concentration (from 5 mM to 30 mM), the petals of hybrids gradually increased in size. At a lower PBS concentration, the petals were incomplete and there were no obviously flower-like nanoparticles. A distinct and stable structure was formed when the PBS concentration was at 20 mM. Beyond 20 mM, the petals were embedded with redundant calcium phosphate and the flower-like structure did not exist anymore (Fig. 2).

Fig. 2 SEM images of BCL–Ca₃(PO₄)₂ hybrids in different PBS concentrations ((a to f) from 5 mM to 30 mM); the lipase loading was 24 mg mL⁻¹.

During the immobilization procedure, the effects of the lipase loading and PBS concentration on the immobilization efficiency and resolution ability were examined. The optimum immobilization conditions determined were as follows: lipase loading of 24 mg mL⁻¹ and PBS concentration of 20 mM (Fig. 3). The enzyme activity and enantioselectivity (ee value) were evaluated using the reaction of the enzymatic kinetic resolution of (R,S)-1-phenylethanol. As many reports on the enantioselective transesterification of 1-phenylethanol with vinyl acetate are available in the literature,^18,19 it can be regarded as a model reaction. Under the optimum immobilization conditions, the highest immobilization efficiency and ee value were 51.2% and 91.1%, respectively, and the enzyme activity was 849.8 U, which was 308% of the free lipase. Moreover, upon measuring stability and reusability of the hybrid nanoflower to enantioselective resolution, it was observed that it could maintain more than 75% of its original activity after 15 days storage and about 80% after reuse of five times, indicating that the hybrid biocatalyst possesses an approving stability. However, the hydrolysis and esterification capacity of the hybrid, which were also measured, were observed to decrease to some extent (data not shown). The detailed reason requires further investigation.²⁰

Fig. 3 Effects of the lipase loading (a) and PBS concentration (b) on the immobilization efficiency and ee value. Storage stability (c) and reusability (d) of the immobilized lipase at a lipase loading of 24 mg mL⁻¹ and 20 mM PBS concentration, indicating the hybrid nanobiocatalyst holds an approving stability. The other conditions were maintained the same as the typical experiment.

As shown in Fig. 4, FT-IR spectroscopy provided direct proof for the formation of lipase–inorganic hybrid nanoparticles. A strong characteristic peak of P–O vibrations and stretches (spectra a and c) was observed at 1037 cm⁻¹. The relatively small band at 530–670 cm⁻¹ was probably the bending vibrations of bridging phosphorous, such as O=P–O, which contributes to the phosphate groups. These two peaks proved the existence of Ca₃(PO₄)₂. Spectrum b exhibits the characteristic peak of the free BCL protein. There are three wide bands at 1650–1680 cm⁻¹, 1350–1460 cm⁻¹ and 1020–1220 cm⁻¹, which are the characteristics for the peptide bond and its stretches of the protein, such as –NH₂ and C=O. The wide and strong band at 2800–3000 cm⁻¹ was attributed to –CH₂ and –CH₃. After immobilization, the characteristic peaks of BCL at 1650–1680 cm⁻¹ and 2800–3000 cm⁻¹ were maintained, while the bands at 1350–1460 cm⁻¹ and 1020–1220 cm⁻¹ were weakened. This indicates that the protein secondary structures had changed during the gradual formation of Ca₃(PO₄)₂, which probably leads to the variation of the catalytic property.²¹ As reported by Barbe et al.,²² there is ‘lid’ structure on the BCL active sites.

Fig. 4 FT-IR spectra of Ca₃(PO₄)₂·nH₂O (a); BCL (b); and the BCL–inorganic hybrid (c).

The different extent of opening in the ‘lid’ structure results in different enzyme activity.²³ The formation process of Ca₃(PO₄)₂ was invisible and complicated, which possibly opened the ‘lid’ to some extent, leading to an increase in the transesterification activity of the lipase.

Energy dispersive spectrometer (EDS) analysis was utilized to characterize lipase–calcium phosphate nanoflower (BCL–Ca₃(PO₄)₂·nH₂O) composition. As shown in Fig. 5, the peak of carbon (C), nitrogen (N) and sulfur (S) was attributed to lipase protein, and the existence of the sodium (Na) peak was due to the sample preparation buffer. The result further confirms the successful preparation of lipase–calcium phosphate hybrid.

Fig. 5 EDS spectra of the BCL–inorganic hybrid nanobiocatalysts.

In conclusion, this study reported a novel and facile method of synthesizing a new lipase–inorganic hybrid nanoflower. The immobilized lipase appeared to be a flower-like porous nanostructure with a high surface-to-volume ratio. SEM analysis could directly observe the morphologies of the hybrid, and the flower-like structure was obtained under the optimum immobilization conditions of a lipase loading of 24 mg mL⁻¹ and PBS concentration of 20 mM.

The resolution activity of the immobilization lipase was 849.8 U, which was 308% higher than the free lipase. The immobilized lipase exhibited a high stability and retained more than 75% of its original activity after 15 days of storage and about 80% after reuse for five times. In addition, FT-IR spectroscopy provided evidence of the structural change in the lipase protein, which probably results in the variation of the BCL properties. Considering the flower-like hybrid nanostructure with wide applications, this new lipase–inorganic hybrid nanobiocatalyst may be promising in many industrial applications, such as biofuels, biopharmaceuticals, and biosensors.

Acknowledgements

This study was financially supported by the National Natural Science Foundation of China (No. 31170078 and J1103514), the National High Technology Research and Development Program of China (No. 2011AA02A204 and 2013AA065805), the National Natural Science Foundation of Hubei Province (No. 2015CFA085), and the Fundamental Research Funds for HUST (No. 2014NY007, 2014QN119 and 2012SHYJ004). The authors are grateful to Analytical and Testing Centre of HUST for their valuable assistances in SEM, EDS and FT-IR measurement.

Notes and references

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