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. 2013 Oct 29:13:92.
doi: 10.1186/1472-6750-13-92.

Improving activity and enantioselectivity of lipase via immobilization on macroporous resin for resolution of racemic 1- phenylethanol in non-aqueous medium

Xiang Li, Shuangshuang Huang, Li Xu, Yunjun Yan  1
Affiliations

Improving activity and enantioselectivity of lipase via immobilization on macroporous resin for resolution of racemic 1- phenylethanol in non-aqueous medium

Xiang Li et al. BMC Biotechnol. .

Abstract

Background: Burkholderia cepacia lipase (BCL) has been proved to be capable of resolution reactions. However, its free form usually exhibits low stability, bad resistance and no reusability, which restrict its further industrial applications. Therefore, it is of great importance to improve the catalytic performance of free lipase in non-aqueous medium.

Results: In this work, macroporous resin NKA (MPR-NKA) was utilized as support for lipase immobilization. Racemic transesterification of 1-phenylethanol with vinyl acetate was chosen as model reaction. Compared with its free form, the enzyme activity and enantioselectivity (ees) of the immobilized lipase have been significantly enhanced. The immobilized BCL exhibited a satisfactory thermostability over a wide range of temperature (from 10 to 65°C) and an excellent catalytic efficiency. After being used for more than 30 successive batches, the immobilized lipase still kept most of its activity. In comparison with other immobilized lipases, the immobilized BCL also exhibits better catalytic efficiency, which indicates a significant potential in industrial applications.

Conclusion: The results of this study have proved that MPR-NKA was an excellent support for immobilization of lipase via the methods of N2 adsorption-desorption, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and Fourier transform-infrared spectroscopy (FT-IR). The improvement of enzyme activity and ees for the immobilized lipase was closely correlated with the alteration of its secondary structure. This information may contribute to a better understanding of the mechanism of immobilization and enzymatic biotransformation in non-aqueous medium.

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Figures

Figure 1
Figure 1
Effect of different kinds of MPRs on enzyme activity and immobilization efficiency.
Figure 2
Figure 2
Effect of substrate molar ratio on enzyme activity/ees of the free and immobilized BCL. Reaction condition: 0.1 g free and BCL was added to 5 mL n-heptane containing 1 mmol 1-phenylethanol, 1-9 mmol vinyl acetate. The reactions were performed at 35°C, 200 rpm for 1 h. The data were measured three times using the same sample and means. Vertical bars represent standard deviation.
Figure 3
Figure 3
Effect of water content on enzyme activity/ees of the free and immobilized BCL. Reaction condition: 0.1 g BCL was added to 5 mL n-heptane containing 1 mmol 1-phenylethanol, 4 mmol vinyl acetate. The reactions were performed at 35°C, 200 rpm for 1 h. The data were measured three times using the same sample and means. Vertical bars represent standard deviation.
Figure 4
Figure 4
Effect of temperature on enzyme activity/ ees of the free and immobilized BCL. Reaction condition: 0.1 g BCL was added to 5 mL n-heptane containing 1 mmol 1-phenylethanol, 4 mmol vinyl acetate. The reactions were performed at different temperatures, 200 rpm for 1 h. The data were measured three times using the same sample and means. Vertical bars represent standard deviation.
Figure 5
Figure 5
Effect of reaction time on conversion/ees of the free and immobilized BCL. Reaction condition: 0.1 g BCL was added to 5 mL n-heptane containing 1 mmol 1-phenylethanol, 4 mmol vinyl acetate. The reactions were performed at 35°C, 200 rpm for different reaction times. The data were measured three times using the same sample and means. Vertical bars represent standard deviation.
Figure 6
Figure 6
Reusability of the immobilized BCL. Reaction condition: 0.1 g BCL was added to 5 mL n-heptane containing 1 mmol 1-phenylethanol, 4 mmol vinyl acetate. The reactions were performed at 37°C, 200 rpm for 1 h. The data were measured three times using the same sample and means. Vertical bars represent standard deviation.
Figure 7
Figure 7
BJH pore size distributions.
Figure 8
Figure 8
SEM images of the pure MPR-NKA and MPR-NKA immobilized BCL. (a) Outer surface of MPR-NKA (Magnification: 100 000, Accelerating Voltage: 5.00 kV); (b) Internal surface of MPR-NKA (Magnification: 100 000, Accelerating Voltage: 5.00 kV); (c) MPR-NKA adsorbed with lipase (Magnification: 100 000, AcceleratingVoltage: 5.00 kV).
Figure 9
Figure 9
EDS spectra of MPR-NKA and MPR-NKA immobilized BCL. (a) EDS spectra of MPR-NKA; (b) EDS spectra of MPR-NKA adsorbed with lipase.
Figure 10
Figure 10
FT-IR spectra of (a: blue line) the pure MPR-NKA; (b: black line) MPR-NKA immobilized BCL and (c: red line) free BCL.
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