Efficient biocatalyst by encapsulating lipase into nanoporous gold
© Du et al.; licensee Springer. 2013
Received: 13 March 2013
Accepted: 8 April 2013
Published: 19 April 2013
Lipases are one of the most important biocatalysts for biotechnological applications. Immobilization is an efficient method to increase the stability and reusability of lipases. In this study, nanoporous gold (NPG), a new kind of nanoporous material with tunable porosity and excellent biocompatibility, was employed as an effective support for lipase immobilization. The pore size of NPG and adsorption time played key roles in the construction of lipase-NPG biocomposites. The morphology and composition of NPG before and after lipase loading are verified using a scanning electron microscope, equipped with an energy-dispersive X-ray spectrometer. The resulting lipase-NPG biocomposites exhibited excellent catalytic activity and remarkable reusability. The catalytic activity of the lipase-NPG biocomposite with a pore size of 35 nm had no decrease after ten recycles. Besides, the lipase-NPG biocomposite exhibited high catalytic activity in a broader pH range and higher temperature than that of free lipase. In addition, the leaching of lipase from NPG could be prevented by matching the protein’s diameter and pore size. Thus, the encapsulation of enzymes within NPG is quite useful for establishing new functions and will have wide applications for different chemical processes.
KeywordsLipase Immobilization Nanoporous gold Catalysis Reusability
Immobilization of enzymes on insoluble supports is a significant process due to its promising potential in improving enzyme thermal or pH stability, easing product purification, and facilitating enzyme recycling [1, 2]. Therefore, immobilized enzymes have a broader range of applications such as bioconversion, bioremediation, biodetection, and biosensing [3–8].
Among the various supports used for enzyme immobilization, nanoporous gold (NPG) has attracted much attention recently [9–12]. NPG, fabricated by a simple dealloying method, possesses the following unique properties: (1) it is a bulky material with microstructure, which means it can be easily employed and recovered; (2) it has an open three-dimensional structure while possessing a comparably high surface area, which favors strong adsorption and can afford high enzyme loading; (3) the pore size is tunable in a wide range from a few nanometers to many microns, which fits for a wide range of enzyme molecules with specific molecular weight and function; (4) it well reserves a biocompatible, clean, and active surface, which efficiently alleviates enzyme denaturation. Coupled with a rich surface chemistry for further functionalization and excellent conductivity, NPG has great potential for applications in heterogeneous catalysis, electrocatalysis, fuel cell technologies, and biomolecular sensing in comparison with other mesoporous materials [10–13].
In our previous work, enzyme-NPG biocomposites were successfully constructed by assembling various enzymes (such as lipase, catalase, and horseradish peroxidase) onto NPG . Among these enzymes, lipase has gained particular interest as one of the most frequently used biocatalysts in the hydrolysis and the synthesis of esters from glycerol and long-chain fatty acids . In addition, lipase is commercially important and has many applications in food industry and clinical analysis . Especially, lipases are important drug targets or marker enzymes in the medical field. Recently, the development of lipase sensors has been strongly focused on biosensors for the detection of triglycerides and cholesterol . Therefore, further studies were carried out on the catalytic performance of the lipase-NPG biocomposite in this study. It is revealed that the pore size of NPG and adsorption time play significant roles in enzyme loading, leaching, activity, and reusability. The finding should be useful for the creation of biocatalysts and biosensors.
4-Nitrophenyl palmitate, p-nitrophenol, pyrogallol, and lipase (Aldrich 534641 from Pseudomonas cepacia) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
NPG was made by chemically dealloying AgAu alloy foils (Ag78Au22 at.%, 25 μm in thickness, purchased from Changshu Noble Metal Company, China) in concentrated HNO3 (approximately 67%). NPG with a pore size of 35 nm was obtained by chemically dealloying AgAu alloy foils in concentrated HNO3 (approximately 67%) at 30°C for 2 h. The preparation of NPG with a pore size of 100 nm was that AgAu alloy foils was chemically dealloyed in concentrated HNO3 (approximately 67%) at 30°C for 2 h and then annealed at 250°C for 10 min. After rinsing in distilled water, the samples were dried and kept in a desiccator for further use. The morphology of the samples was observed with a JSM-6700 F field emission scanning electron microscope (SEM; JEOL Ltd., Tokyo, Japan), equipped with an Oxford INCA x-sight energy-dispersive X-ray spectrometer (EDS; Oxford Instruments, Abingdon, Oxfordshire, UK) for compositional analysis.
The immobilized lipase was prepared as previously described . For enzyme immobilization, 1 ml of lipase solution (1.0 mg ml−1 of lipase in 50 mM, pH 8.0 Tris–HCl buffer) was mixed with 18 mg of NPG. Then, the mixture was incubated at 4°C without shaking for a certain period of time. After incubation, the supernatant was removed by centrifugation (5,000×g for 5 min), and the resulting lipase-NPG biocomposite was washed five times with Tris–HCl buffer (50 mM, pH 8.0) to remove the weakly adsorbed enzyme. The amount of immobilized enzyme was determined by Bradford protein assays .
For leaching test, the lipase-NPG biocomposite was incubated in Tris–HCl buffer (50 mM, pH 8.0) for 0.5 and 5 h at 40°C, respectively. Then, the Tris–HCl buffer was removed. The catalytic activity of the lipase-NPG biocomposite was determined.
The catalytic activities of free lipase and the lipase-NPG biocomposite were determined by measuring the initial hydrolysis rate of 4-nitrophenyl palmitate (pNPP) by lipase at 40°C, using a spectrophotometer (2100), following the increase of p-nitrophenol (pNP) concentration at 410 nm . One unit (U) of catalytic activity is defined as the amount of lipase which catalyzes the production of 1 μg p-nitrophenol under the experimental conditions. For reusability test, the lipase-NPG biocomposite was washed with Tris–HCl buffer (50 mM, pH 8.0) for three times after catalytic activity determination in each cycle, and then used in the next cycle.
Results and discussion
Characterization of lipase-NPG biocomposites
Catalytic activity of lipase-NPG biocomposites
The catalytic activity of free lipase during adsorption processes
Adsorption time (h)
Catalytic activity (U μg−1 protein)
55.7 ± 1.7
54.3 ± 2.7
54.8 ± 3.1
57.6 ± 0.9
Reusability of lipase-NPG biocomposites
Effect of buffer pH and temperature on lipase-NPG biocomposite
The effects of reaction temperature on the catalytic activity of free lipase and the lipase-NPG biocomposite with a pore size of 35 nm were also investigated by varying temperatures from 30°C to 80°C. Figure 4B shows that the maximum catalytic activity of the lipase-NPG biocomposite was observed at 60°C, whereas free lipase exhibited the highest activity at 50°C. Additionally, higher catalytic activity was maintained by the lipase-NPG biocomposite within the temperature range investigated. The improvement in the denaturation resistance of the lipase-NPG biocomposite was probably a consequence of increasing conformational stability by being adsorbed within nanoscale pore channels .
In conclusion, NPG with a three-dimensional spongy morphology was demonstrated to be a suitable support for lipase immobilization. The pore size of NPG and adsorption time played key roles in achieving high stability and reusability. The resulting lipase-NPG biocomposites with a pore size of 35 nm exhibited excellent catalytic activity and stability compared with the native lipase at different pH and temperatures. The leaching of lipase from NPG could be prevented by matching the protein’s diameter and pore size. These results suggest that NPG with unique structure properties has great potential for applications in biomolecule separation systems, biocatalysis, electrocatalysis, and biosensors.
This work was supported by grants from the National Natural Science Foundation of China (21177074), New Teacher Foundation of Ministry of Education of China (20090131120005), and Excellent Middle-Aged and Youth Scientist Award Foundation of Shandong Province (BS2010SW016).
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