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1 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, , Republic of Korea; b Department of Materials Science and Engineering, Korea University, Anam-Dong, Seongbuk-Gu, Seoul, , Republic of Korea These authors contributed equally to this work. *Corresponding authors. Mailing address: Department of Chemical Engineering, Konkuk University, Seoul , South Korea. jkrhee@konkuk.ac.kr; Fax: ; Tel: Department of Materials Science and Engineering, Korea University, Seoul , South Korea. yckang@korea.ac.kr; Fax: ; S-1
2 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 -M ± ± ± ±6.8 rgo-fe 3 O 4 -M ± ± ± ±5.2 rgo-fe 3 O 4 -M ± ± ± ±4.8 rgo NA c 80.7± ± ± ±4.0 Fe 3 O NA 49.0± ± ± ±4.2 a Immobilization yields. b Immobilization efficiency. c Not applicable. S-2
3 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 MSNPs wormhole framework NA f 2 MSNPs spherical NA MSNPs-Fe 3 O 4 spherical NA 4 magnetic-chitosan spherical magnetic polymers spherical NA 6 MSNPs tubular g 7 rgo-fe 3 O 4 -M1 spherical This study rgo sheet Fe 3 O 4 spherical 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
4 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 NK OK SK FeK Total S-4
5 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 ) r t 1/2 (h) S-5
6 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 ± ± ± ±4.9 guaiacol ± ± ± ±3.5 pyrogallol ± ± ± ±1.3 L-DOPA ± ± ± ±0.1 a Relative activity of free and immobilized laccase was considered as 100% for ABTS (1 mm). S-6
7 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
8 S-8
9 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
10 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
11 Figure S5. TG curves of graphene oxide (GO) and reduced graphene oxide (rgo) powders measured under an air atmosphere. S-11
12 Figure S6. Particle size distribution of the rgo-fe 3 O 4 -M1 composite particles measured by dynamic light scattering analysis. S-12
13 Figure S7. Hysteresis loop of the rgo-fe 3 O 4 -M1 composite particles. S-13
14 rgo-fe3o4-m1 Fe 2p 1/2 Fe 2p 3/2 Intensity (a. u.) Binding energy (ev) Figure S8. XPS Fe 2p spectrum of the rgo-fe 3 O 4 -M1 composite particles. S-14
15 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
16 Figure S10. Morphology and crystal structure of rgo powders prepared by spray pyrolysis: a) SEM image and b) XRD pattern. S-16
17 Figure S11. N 2 adsorption and desorption isotherms of the pure Fe 3 O 4 and rgo powders. S-17
18 Figure S12. Immobilization of on the particles at different ph values: a) laccase, and b) HRP. S-18
19 Figure S13. Immobilization efficiency of enzymes on the particles. Time profile: a) laccase and b) HRP. Loading: c) laccase and d) HRP. S-19
20 Figure S14. Zeta potential of synthesized rgo-fe 3 O 4 -M1 particles as function of ph values. S-20
21 Figure S15. CD analysis of free and immobilized laccase. S-21
22 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
23 Figure S17. Effect of substrate concentration on the activity of the free and immobilized laccase. S-23
24 Figure 18. Free and immobilized HRP: a) stability at 25 C, b) storage stability at 4 C, and c) reusability. S-24
25 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
26 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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