Supporting Information Eco-friendly Composite of Fe 3 O 4 -Reduced Grapene Oxide Particles for Efficient Enzyme Immobilization Sanjay K. S. Patel a,, Seung Ho Choi b,, Yun Chan Kang b,*, Jung-Kul Lee a,* Addresses: a Department of Chemical Engineering, Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul, 143-701, Republic of Korea; b Department of Materials Science and Engineering, Korea University, Anam-Dong, Seongbuk-Gu, Seoul, 136-713, Republic of Korea These authors contributed equally to this work. *Corresponding authors. Mailing address: Department of Chemical Engineering, Konkuk University, Seoul 143-701, South Korea.E-mail: jkrhee@konkuk.ac.kr; Fax: +82-2-458-3504; Tel: +82-2-450-3505 Department of Materials Science and Engineering, Korea University, Seoul 136-713, South Korea.E-mail: yckang@korea.ac.kr; Fax: +82-2-928-3584; +82-2-3290-3282. S-1
Table S1. Immobilization of enzymes on the particles particles particle size (µm) BET surface area (m 2 /g) pore size (nm) laccase HRP IY a (%) IE b (%) IY (%) IE (%) rgo-fe 3 O 4 -M1 1-3 30 4.8 91.1±6.0 112±10 86.4±6.5 89.8±6.8 rgo-fe 3 O 4 -M2 1-3 136 3.6 86.5±6.2 84.3±7.1 77.8±7.3 68.5±5.2 rgo-fe 3 O 4 -M3 1-3 177 4.6 87.8±5.7 73.6±6.7 70.1±7.0 55.1±4.8 rgo 1-2 145 NA c 80.7±7.0 70.4±6.8 76.6±7.2 39.3±4.0 Fe 3 O 4 0.5-1 2 NA 49.0±5.0 45.3±4.4 41.8±4.3 43.5±4.2 a Immobilization yields. b Immobilization efficiency. c Not applicable. S-2
Table S2. Immobilization of Trametes versicolor laccase through adsorption on magnetic composite particles particles a structure immobilized properties reusability e reference IY% b loading c IE% d carbon based Magnetic hierarchical 19.6 492 91.0 50.0 1 MSNPs wormhole framework 72.6 72.6 81.5 NA f 2 MSNPs spherical NA 80.0 55.2 88.0 3 MSNPs-Fe 3 O 4 spherical 91.0 82.0 79.4 NA 4 magnetic-chitosan spherical 81.0 16.2 82.6 87.0 5 magnetic polymers spherical 17.0 17.0 20.0 NA 6 MSNPs tubular 42.7 34.8 66.2 8.0 g 7 rgo-fe 3 O 4 -M1 spherical 91.1 418 112 92.6 This study rgo sheet 80.7 258 70.4 73.9 Fe 3 O 4 spherical 49.0 123 45.3 21.3 a MSNPs: Magnetic SiO 2 nanoparticles. b Immobilization yields. c mg of enzyme/g of support. d Immobilization efficiency. e Residual activity after 10 cycles. f Not applicable. g After 4 cycles. S-3
Table S3. Energy dispersive spectroscopy analysis of immobilized laccase on rgo-fe 3 O 4 - M1 particles Elements rgo-fe 3 O 4 -M1 particles composition (%) Before immobilization After immobilization CK 39.27 47.54 NK 1.86 3.12 OK 30.10 34.95 SK 1.65 1.96 FeK 27.12 12.43 Total 100 100 S-4
Table S4. Determination of the denaturation constant (k d ) and half-life (t 1/2 )values for the free and immobilized laccase at 25 C laccase parameter free immobilized rgo-fe 3 O 4 -M1 rgo Fe 3 O 4 k d (h -1 ) 0.041 0.0027 0.0078 0.0085 r 2 0.97 0.97 0.98 0. 99 t 1/2 (h) 16.9 257 88.8 81.5 S-5
Table S5. Oxidation of the phenolic compounds by free and immobilized laccase phenolic compound absorbance molar extinction coefficient (ε max /M/cm) free relative activity (%) a immobilized rgo-fe 3 O 4 -M1 rgo Fe 3 O 4 2,6-DMP 470 35645 46.9±4.3 54.0±4.8 48.9±4.6 53.8±4.9 guaiacol 436 6400 35.4±3.2 41.5±4.6 34.9±3.5 37.6±3.5 pyrogallol 450 4400 12.4±1.0 16.6±1.3 12.8±1.2 14.2±1.3 L-DOPA 460 38000 1.3±0.1 2.5±0.2 1.4±0.1 1.5±0.1 a Relative activity of free and immobilized laccase was considered as 100% for ABTS (1 mm). S-6
Figure S1. Schematic diagram of one-pot and continuous spray pyrolysis process applied in the preparation of the rgo-fe 3 O 4 composite particle. S-7
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Figure S3. SAED patterns of (a) rgo-fe 3 O 4 -M1, (b) rgo-fe 3 O 4 -M2, and (c) rgo-fe 3 O 4 -M3 composite particles. S-9
Figure S4. Elemental mapping images of the rgo-fe 3 O 4 -M1 composite particle: a) TEM image, b) iron, c) oxygen, d) carbon, e) sulfur, and f) nitrogen components. S-10
Figure S5. TG curves of graphene oxide (GO) and reduced graphene oxide (rgo) powders measured under an air atmosphere. S-11
Figure S6. Particle size distribution of the rgo-fe 3 O 4 -M1 composite particles measured by dynamic light scattering analysis. S-12
Figure S7. Hysteresis loop of the rgo-fe 3 O 4 -M1 composite particles. S-13
rgo-fe3o4-m1 Fe 2p 1/2 Fe 2p 3/2 Intensity (a. u.) 740 735 730 725 720 715 710 705 Binding energy (ev) Figure S8. XPS Fe 2p spectrum of the rgo-fe 3 O 4 -M1 composite particles. S-14
Figure S9. Morphology and crystal structure of pure Fe 3 O 4 powders prepared by spray pyrolysis: a) SEM image and b) XRD pattern. S-15
Figure S10. Morphology and crystal structure of rgo powders prepared by spray pyrolysis: a) SEM image and b) XRD pattern. S-16
Figure S11. N 2 adsorption and desorption isotherms of the pure Fe 3 O 4 and rgo powders. S-17
Figure S12. Immobilization of on the particles at different ph values: a) laccase, and b) HRP. S-18
Figure S13. Immobilization efficiency of enzymes on the particles. Time profile: a) laccase and b) HRP. Loading: c) laccase and d) HRP. S-19
Figure S14. Zeta potential of synthesized rgo-fe 3 O 4 -M1 particles as function of ph values. S-20
Figure S15. CD analysis of free and immobilized laccase. S-21
Figure S16. Analysis of immobilized laccase on rgo-fe 3 O 4 -M1: a) FTIR, b-c) CLSM in green and in bright channels, and d) TG curves. S-22
Figure S17. Effect of substrate concentration on the activity of the free and immobilized laccase. S-23
Figure 18. Free and immobilized HRP: a) stability at 25 C, b) storage stability at 4 C, and c) reusability. S-24
Figure S19. Magnetic separation of immobilized laccase on rgo-fe 3 O 4 -M1: a) in absence and b) in the presence of a magnet. S-25
Figure S20. Determination of EC 50 values: a) commercial Fe 3 O 4 and b) synthesized rgo-fe 3 O 4 - M1 particles towards V. fischeri. S-26
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