Activation of enzyme nanogel in organic solvents by PEG–substrate joint imprinting

Abstract

A substrate or polyethyleneglycol (PEG) imprinted lipase nanogel displayed increased apparent activity in organic solvents by 2.5–4.7 folds, compared to native lipase. It enabled a one-step synthesis of chloramphenicol palmitate with a yield of ∼99% and purity of ∼99%, indicating that the imprinted lipase nanogel is an appealing catalyst in organic media.

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The enzymatic synthesis of fine chemicals and therapeutic molecules in organic solvents has been extensively pursued to achieve a high chemo-, regio-, and stereo-selectivity for catalytic reactions utilizing hydrophobic substrates and/or inhibiting the adverse effects of water.¹ However, most enzymes exist in an aggregated and rigid form in organic solvents. This makes the active site of the enzyme less accessible to its substrate and, also hinders the conformational transition of the protein required for catalysis.¹ Thus, these reduce the apparent enzyme activity by one to several orders of magnitude, as compared to that in an aqueous solution.² The recent advancement in nanostructured enzyme catalysts³ resulted in the unlimited opportunities for modifying the nature-made enzymes with tailor-made chemical structures and functionalities to overcome the abovementioned problems. We have demonstrated that nanostructured materials, such as polymer nanogels,^4–6 inorganic nanoflowers,⁷ and polymer nanoconjugates^8,9 are effective in enhancing the performance of enzymes. Moreover, the apparent activity of the enzyme-polymer nanogel, which is prepared by aqueous in situ polymerization,⁵ in organic solvents can be elevated by substrate imprinting, a method known to enhance the substrate accessibility.¹⁰ Occasionally, we found that imprinting with PEG, which will be discussed in the text, did not increase the substrate accessibility, but led to a more significant increase in the apparent activity. Thus, we directed our efforts to explore the mechanism underpinning the enhanced apparent activity, using PEG and substrate.

Fig. 1 shows the preparation of the lipase-encapsulated polyacrylamide nanogel and the imprinting procedure. The transesterification reaction between chloramphenicol and vinyl palmitate to produce chloramphenicol palmitate was selected to examine the catalytic performance of the lipase nanogel with different imprinting treatments. Lipase from Thermomyces lanuginosus (lipozyme TL 100 L) was selected as the model enzyme and encapsulated into a nanogel using an aqueous two-step in situ polymerization (Fig. 1).⁵ The acryloylation of lipase with N-acryloxysuccinimide (NAS) was conducted at 30 °C in acetic buffer (pH 5.0, 50 mM) for 6 h, which generated vinyl groups on the surface of lipase for subsequent polymerization at room temperature using ammonium persulfate and N,N,N′,N′-tetramethylethylenediamine as initiators, and acrylamide as the monomer. The encapsulation yield of lipase in the nanogel was around 99%. The hydrolytic activity yield was 76% with reference to its native counterpart.

Fig. 1 Synthesis of imprinted lipase nanogels and application in the enzymatic synthesis of chloramphenicol palmitate in organic solvents.

Transesterification between p-nitrophenyl palmitate and n-butyl alcohol in n-heptane was selected as a standard assay to evaluate the activity of the native lipase and lipase nanogels with different treatments in organic media. The apparent activity of the lipase nanogel and the mixture of lipase with polyacrylamide was 219% and 171% compared to that of the native lipase, respectively. The increased apparent activity induced by polyacrylamide, as shown by both the mixture and the nanogel, is attributed to the improved dispersion of the lipase in organic solvents and consequently, the accessibility to substrates. This is more significant in the case of lipase nanogel, in which the enzyme molecules are more evenly encapsulated and are covalently bound with the polyacrylamide porous network.

Palmitic acid, and PEG with molecular weights of 200, 1000, 4000, and 12 000 were selected to prepare the imprinted lipase nanogel. During the experiment, palmitic acid or PEG was added to an acetic buffer (50 mM, pH 5.0) solution containing the pre-synthesized lipase nanogel (2 mg mL⁻¹ of protein) with the molar ratio of the imprinting molecule to lipase being 5 : 1, with vigorous stirring. Then, the mixture was lyophilized and subjected to extraction with toluene to remove palmitic acid or PEG, resulting in the imprinted lipase nanogel in powder form.

The transmission electron microscopy (TEM) images of the negatively stained non-imprinted lipase nanogel, lipase nanogel imprinted with palmitic acid and PEG are shown in Fig. 2a–c, all of which appear spherical in shape with diameters ranging from 20 to 30 nm. The scanning electron microscopy (SEM) images of the lyophilized non-imprinted, palmitic acid and PEG imprinted lipase nanogels in powder form are shown in Fig. 2d–f. Both the non-imprinted and the palmitic acid imprinted lipase nanogels show porous structure, which is advantageous for the substrate to access the enzyme encapsulated in nanogels. The mercury intrusion porosimetry method was used to analyze the specific surface area of different forms of lipase nanogels, and showed that the palmitic acid imprinted lipase nanogel has the largest total pore area (181.917 m² g⁻¹), whereas the non-imprinted lipase nanogel has a smaller one (88.127 m² g⁻¹). In contrast, from the SEM image, the PEG imprinted lipase nanogel did not appear a porous structure, with a total pore area of 63.327 m² g⁻¹.

Fig. 2 TEM images of (a) non-imprinted, (b) palmitic acid imprinted, (c) PEG imprinted lipase nanogel. SEM images of the powder of (d) non-imprinted, (e) palmitic acid imprinted, (f) PEG imprinted lipase nanogel.

The abovementioned lipase nanogels were subjected to the transesterification reaction between p-nitrophenyl palmitate and ethanol in n-heptane, using native lipase as the reference. It is shown in Table 1 that the relative activity of non-imprinted lipase nanogel and the palmitic acid imprinted one was 189% and 293%, respectively, compared to the native lipase imprinted with palmitic acid showed activity similar to the native one. To investigate the mechanism of the increased activity of the palmitic acid imprinted lipase nanogel, we compared the adsorption of palmitic acid by these imprinted lipase nanogels to that by non-imprinted lipase nanogel. FITC-labeled palmitic acid with a concentration of 0.05 mg mL⁻¹ in acetonitrile was added to 0.1 mg of imprinted (or non-imprinted) lipase nanogels at 25 °C, followed by an immediate analysis of the fluorescence intensity within the lipase nanogels under a laser scanning confocal microscope (LSCM). The increase in the fluorescence intensity within the lipase nanogel in a selected region of interest reflects the adsorption of the substrate. Fig. 3a–c present the fluorescence images of FITC-labeled palmitic acid adsorbed by the palmitic acid imprinted lipase nanogel, showing a remarkable increase in the fluorescence intensity after 0 s, 250 s, and 500 s incubation. As shown in Fig. 3d, the palmitic acid imprinted lipase nanogel showed a higher adsorption rate and capacity than the non-imprinted lipase nanogel. From the abovementioned facts it is concluded that the substrate imprinting favors the substrate uptake and thus results in a higher apparent activity.¹⁰

Table 1 Relative activities of different preparations of the lipase catalysts compared with that of native lipase

Imprinting molecule	Apparent activity (%)
	Native lipase	Lipase nanogel
Non-imprinted	100.00	189.05
Palmitic acid	89.00	292.66
PEG (Mw: 200)	220.58	281.69
PEG (Mw: 1000)	252.22	313.15
PEG (Mw: 4000)	247.78	255.31
PEG (Mw: 12 000)	169.54	223.12
PEG (Mw: 1000) + palmitic acid	240.01	476.30

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Fig. 3 LSCM images of palmitic acid-imprinted lipase nanogel incubated with FITC-labeled palmitic acid in acetonitrile for (a) 0 s, (b) 250 s, (c) 500 s. (Red circles indicate the selected region for analysis). (d) Increase in relative fluorescence intensity within different lipase nanogels (all data were obtained from three parallel experiments).

For the PEG imprinted lipase nanogel having a non-porous structure (Fig. 2f), the mass transfer hindrance is expected to be significant and thus is disadvantageous for the catalysis. Surprisingly, the PEG imprinted lipase nanogel gave a relative activity of 223–313% compared to native lipase (Table 1), while the PEG imprinting increased the apparent activity of the native lipase by 169–252%. To elucidate the mechanism underpinning the enhanced activity, we examined the adsorption of the substrate by the PEG imprinted lipase nanogel using the LCSM method under the abovementioned conditions. As shown in Fig. 3d, the PEG imprinted lipase nanogel showed considerably slow adsorption of palmitic acid compared to that for both the non-imprinted and the palmitic acid imprinted lipase nanogel. This indicates the transport of the substrate in the PEG-imprinted lipase nanogel is hindered, as expected from the non-porous structure illustrated by SEM (Fig. 2f). Considering the improved apparent activity achieved at a hindered mass transport of the substrate, it is concluded that the increased apparent activity of PEG-imprinted lipase nanogel is attributed to the activation effect of PEG. Otero, et al.¹¹ has reported that PEG induces a conformational change, which uncovers the ‘lid’ covering the active site of lipase. It is possible that in our study the activated conformation of lipase attributed to PEG imprinting was retained in organic solvents, giving an elevated catalytic activity.

The above results encouraged us to probe a new imprinting strategy, which is using PEG and substrate simultaneously, to achieve a facilitated substrate uptake and an activated enzyme. This idea was validated by an improved apparent activity, up to 476% compared to that of native lipase (Table 1), in the transesterification reaction between p-nitrophenyl palmitate and ethanol. The substrate adsorption behavior of the palmitic acid–PEG imprinted lipase nanogel was also examined under the same conditions. The increase in the fluorescence intensity within the palmitic acid–PEG imprinted lipase nanogel in a selected region of interest is shown in Fig. 3d, which indicates that the adsorption rate by the PEG and palmitic acid jointly imprinted lipase nanogel was lower than that of the palmitic acid imprinted one, but higher than the PEG imprinted one. The specific area analysis showed that the PEG and palmitic acid jointly imprinted lipase nanogel has a total pore area of 109.381 m² g⁻¹. The complementary input of activity and structure assay led us to the conclusion that the increased activity shown by the palmitic acid–PEG jointly imprinted lipase nanogel can be attributed to the activation effect by PEG imprinting and increased accessibility of the substrate by palmitic acid imprinting. Although the molecular imprinting^12,13 and PEG treatment¹¹ of native enzymes have been well studied to increase enzymatic activity in organic media, in this study, we for the first time demonstrated that the substrate and PEG joint imprinting can significantly increase the apparent activity of lipase nanogel, which is a robust nanostructured enzyme catalyst with promising applications in industrial biocatalysis.

The chemical synthesis of chloramphenicol palmitate, a broad-spectrum bacteriostatic antimicrobial already used in clinics¹⁴ requires selective modification with the protection and deprotection steps. Lipase, which exhibits regioselectivity between the two hydroxyl groups,^4,15,16 may enable a one-step synthesis of the target molecule. Using native lipase as the control, the lipase catalysts prepared with the above mentioned imprinting procedures were subjected to the synthesis of chloramphenicol palmitate in acetonitrile at 20 °C. Enzyme samples applied in the transesterification reaction including free lipase, lipase nanogel and imprinted lipase nanogel were all obtained in powder form by lyophilisation, with the same water content of ∼5% w/w as measured by the Karl-Fischer titration. As shown in Fig. 4, the native lipase gave a yield below 40% in 24 h, which was mainly because of the lipase deactivation caused by the extraction of the ‘essential water’ by acetonitrile. The non-imprinted lipase nanogel, which inhibited the extraction of essential water, elevated the yield to ∼99% in 24 h.⁴ The palmitic acid imprinted lipase nanogel took only 12 h to reach a ∼99% yield because of the facilitated uptake of the substrate and the preservation of the essential water of the enzyme by the polyacrylamide network. This conclusion was supported by measuring the water content in reaction media of the catalysis by using different enzyme samples. For native lipase, the water content in the acetonitrile solution was measured as 0.01% w/w, while for lipase nanogel (non-imprinted and imprinted) it was 0.002% w/w which demonstrates the capability of preservation of water by polyacrylamide shells. The PEG (M_w: 1000) imprinted lipase nanogel displayed similar performance compared to that of the substrate imprinted one, which again confirmed the activation effect of PEG. The lipase nanogel jointly imprinted with PEG and palmitic acid reached a ∼99% yield within only 6 h, because of both the activation effect by PEG imprinting and the facilitated uptake of substrate by palmitic acid imprinting. Interpreted from the initial reaction rate, the catalytic activity of the non-imprinted, palmitic acid imprinted, PEG imprinted and palmitic acid–PEG imprinted lipase nanogel was increased by 4.0, 6.8, 8.2, 15.5 fold, respectively, compared to the native lipase. Moreover the two by-products, (1R,2R)-2-[(dichloroacetyl)amino]-1-(4-nitrophenyl)-3-(palmitoyloxy)propylpalmitate [(2′R,3′R)-chloramphenicol 1′,3′-dipalmitate and (1R,2R)-2-[(dichloroacetyl)amino]-3-hydroxy-1-(4-nitrophenyl)propyl decanoate (2′R,3′R)-chloramphenicol 1′-palmitate, which are the 1′,3′-di-substituted product and the 1′-mono-substituted product were not detected by high performance liquid chromatography (HPLC),¹⁷ suggesting that the imprinted lipase nanogel gave the expected regioselectivity.

Fig. 4 Yields of chloramphenicol palmitate using different lipase catalysts.

In summary, we described a PEG and substrate joint imprinting method, which activates the encapsulated lipase and facilitates the uptake of the substrate, leading to an enhanced apparent catalytic activity in organic media. The enzyme-catalyzed transesterification reaction between chloramphenicol and vinyl palmitate demonstrated the high performance of the PEG and substrate jointly imprinted lipase nanogel and its advantages in terms of enhanced stability, improved apparent activity, in addition to the high selectivity, making the imprinted enzyme nanogels appealing for industrial biocatalysis.

Acknowledgements

This work was supported by the National High Technology Research and Development Program (“863” Program) of China under the grant number of 2014AA020507, the National Natural Science Foundation of China under the grant number of 21036003 and 21206082, Tsinghua University Initiative Scientific Research Program under the grant number of 20131089191.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra06660j

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