Supporting Information Lipase immobilized on graphene oxide as ...
Supporting Information Lipase immobilized on graphene oxide as reusable biocatalyst Guanghui Zhanga, Jingwen Maa, Jun Wanga, Yang Lia, Guoliang Zhanga, Fengbao Zhanga, and Xiaobin Fan*a a
State Key Laboratory of Chemical Engineering, School of Chemical Engineering & Technology, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin University, Tianjin, China. E-mail: [email protected]; Fax: +86 22-27408778; Tel: +86 22-27408778
Characterization 1. The enzyme loading determination by using UV-vis spectroscopy through Bradford method
Figure S1. The absorption spectra of BSA after reaction with G-250. The concentrations of aqueous BSA solution in the UV-vis detection are 0, 20, 40, 60, 80 and 100 µg mL–1, respectively.
The enzyme loading on GO was determined by amount of residual lipase in the supernatant after centrifugation. Usually, the Bradford method is used to detect the protein amount by using UV-vis spectroscopy. After reaction between BSA solution (1 mL) and G-250 solution (5 mL) for 5 min, the peak at 595 nm can be utilized to obtain the concentration-absorption line. As shown in Table S1, the concentrations of aqueous BSA solution are 0, 20, 40, 60, 80 and 100 µg mL–1, respectively. As is showed in figure S1, the absorption value increases with the raise of BSA concentration. The concentration-absorption line at 595 nm is y=0.007x+0.776 1
(R2=0.9979). The concentration of free lipase can be also detected using this concentration-absorption line (Figure S2). Table S1 The absorption value at 595 nm using different BSA concentration Concentration (µg mL–1)
0
20
40
60
80
100
Absorption
0.770993 0.930827 1.057069 1.178113 1.326345 1.484499
line
Y=0.007X+0.7764 (R2=0.9979)
Figure S2. The protein concentration-absorption line of the Bradford method. In this UV-vis detection, BSA protein with a series of concentration (0, 20, 40, 60, 80 and 100 µg mL–1, respectively) was used for reaction with G-250.
2. The AFM characterization of free lipase To obtain a direct proof of the enzyme size, the AFM analysis of free lipase is given in the following image (Figure S3). The height profile of the free lipase clearly demonstrates that their thickness is in line with the size of the nanoparticles, proving the successful immobilization of enzyme molecules on the GO surface.
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Figure S3. The (a) AFM image of free lipase and (b) corresponding height profile.
3. The reusability of immobilized lipase through physical adsorption
Figure S4. The reusability of immobilize lipase through physical adsorption after 10 cycles. The catalytic reaction of the immobilized lipase was measured under pH 7.5 at 40 °C.
As a reference experiment, the reusability of GO-lipase through physical adsorption is provided in the supporting information (Figure S4). The immobilized lipase through covalent method exhibits a higher reusability after 10 cycles (about 69.9% of its initial activity) when compared with physical adsorption method (only 37.3% of its initial activity). For the physically adsorbed lipase, the repeated washings after the catalysis may disturb its interaction with the GO support and cause the enzyme bleeding gradually. The comparison of immobilized lipase demonstrates that the GO-lipase through covalent method shows great potential in practice biocatalysis system.
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State Key Laboratory of Chemical Engineering, School of Chemical Engineering & Technology, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin University, Tianjin, China. E-mail: [email protected]; Fax: +86 22-27408778; Tel: +86 22-27408778
Characterization 1. The enzyme loading determination by using UV-vis spectroscopy through Bradford method
Figure S1. The absorption spectra of BSA after reaction with G-250. The concentrations of aqueous BSA solution in the UV-vis detection are 0, 20, 40, 60, 80 and 100 µg mL–1, respectively.
The enzyme loading on GO was determined by amount of residual lipase in the supernatant after centrifugation. Usually, the Bradford method is used to detect the protein amount by using UV-vis spectroscopy. After reaction between BSA solution (1 mL) and G-250 solution (5 mL) for 5 min, the peak at 595 nm can be utilized to obtain the concentration-absorption line. As shown in Table S1, the concentrations of aqueous BSA solution are 0, 20, 40, 60, 80 and 100 µg mL–1, respectively. As is showed in figure S1, the absorption value increases with the raise of BSA concentration. The concentration-absorption line at 595 nm is y=0.007x+0.776 1
(R2=0.9979). The concentration of free lipase can be also detected using this concentration-absorption line (Figure S2). Table S1 The absorption value at 595 nm using different BSA concentration Concentration (µg mL–1)
0
20
40
60
80
100
Absorption
0.770993 0.930827 1.057069 1.178113 1.326345 1.484499
line
Y=0.007X+0.7764 (R2=0.9979)
Figure S2. The protein concentration-absorption line of the Bradford method. In this UV-vis detection, BSA protein with a series of concentration (0, 20, 40, 60, 80 and 100 µg mL–1, respectively) was used for reaction with G-250.
2. The AFM characterization of free lipase To obtain a direct proof of the enzyme size, the AFM analysis of free lipase is given in the following image (Figure S3). The height profile of the free lipase clearly demonstrates that their thickness is in line with the size of the nanoparticles, proving the successful immobilization of enzyme molecules on the GO surface.
2
Figure S3. The (a) AFM image of free lipase and (b) corresponding height profile.
3. The reusability of immobilized lipase through physical adsorption
Figure S4. The reusability of immobilize lipase through physical adsorption after 10 cycles. The catalytic reaction of the immobilized lipase was measured under pH 7.5 at 40 °C.
As a reference experiment, the reusability of GO-lipase through physical adsorption is provided in the supporting information (Figure S4). The immobilized lipase through covalent method exhibits a higher reusability after 10 cycles (about 69.9% of its initial activity) when compared with physical adsorption method (only 37.3% of its initial activity). For the physically adsorbed lipase, the repeated washings after the catalysis may disturb its interaction with the GO support and cause the enzyme bleeding gradually. The comparison of immobilized lipase demonstrates that the GO-lipase through covalent method shows great potential in practice biocatalysis system.
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