Supporting Information Design of Stable and Powerful ...
Supporting Information
Design of Stable and Powerful Nanobiocatalysts, Based on Enzyme Laccase Immobilized on Self- Assembled 3D Graphene/Polymer Composite Hydrogels
Nerea Ormategui†, Antonio Veloso†, Gracia Patricia Leal†, Susana RodriguezCouto‡§*, Radmila Tomovska† §*
†
POLYMAT and Departamento de Química Aplicada, Facultad de Ciencias Químicas, University of the Basque Country, UPV/EHU, Donostia-San Sebastián, Spain
‡
CEIT, Unit of Environmental Engineering, Paseo Manuel de Lardizábal, Donostia-San Sebastián, Spain §
IKERBASQUE, Basque Foundation for Science, Bilbao, Spain
Coresponding authors’ e-mail addresses: * [email protected] * [email protected]
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Experimental Section Materials All the chemicals for the experiments were used without further purification. Methyl methacrylate (MMA, Quimidroga), butylacrylate (BA, Quimidroga), 4-bromostyrene (StBr, Aldrich), 2-hydroxyethyl methacrylate (HEMA, Aldrich), potassium persulfate (KPS, Al-drich), 4,4-azobis(4-cyanovaleric acid) (V-501, Aldrich), ascorbic acid (AsA, Acros organics), GO plateletss (Graphenea, concentration 4 mg/mL, > 95% monolayer content), 2,2´-azino-di-[3-ethyl-benzo-thiazolin-sulphonate] (ABTS, Sigma), succinic acid (Sigma-Aldrich), Remazol Brilliant Blue R (1-amino-9,10-dihydro-9,10-dioxo-4[(3-{[2-(sulfooxy)ethyl]sulfonyl}phenyl)amino] 2-anthracenesulfonic acid (Sigma– Aldrich), sodium azide (N3Na, Alfa Aesar), bovine serum albumin (BSA, SigmaAldrich), Bradford protein assay reagent (Bio-Rad, USA).
Methods The nano-biocatalysts were prepared by immobilization of laccase enzyme on the 3D composite graphene/polymer hydrogels. Firstly, the hydrogels were prepared by selfassembling of GO platelets during their reduction in using the polymer latex as a matrix. The polymer latex was prepared by seeded semicontinuous emulsion polymerization of MMA and BA in relation 50/50 and 70/30 to each other and small amount of functional monomer (1 or 2% of HEMA or SBr, respectively), with aim to functionalize the polymer nanoparticles. The detailed procedures are as follows. Polymer latex synthesis Seed Latex preparation Semi-continuous seeded emulsion polymerization was used to synthesize poly(MMA/BA/SBr) at a ratio of 69/29/2 and poly(MMA/BA/HEMA) at a ratio of 49.5/49.5/1. First, 10 wt % solids content (s.c.) seed was prepared by batch emulsion polymerization in a 1 L-jacketed glass reactor equipped with an anchor type stirrer, a nitrogen inlet, a condenser and a thermocouple. The reaction mixture was degassed by nitrogen bubbling for 30 min, stirring constantly at 200 rpm, and then heated to 70 ºC. Once the desired reaction temperature was reached, in MMA/BA/SBr case the initiator potassium persulfate (KPS, Aldrich) (0.5 g dissolved in 20 g of water) was added in a single shot, while for MMA/BA/HEMA 4,4-azobis(4-cyanovaleric acid) (V-501, Aldrich) was used as a free radical initiator. After 3 h the reaction mixture was S-2
cooled and filtered through a muslin lining to remove the possible coagulum. The final solids content of the seed was 10 wt %. Seeded Emulsion Polymerization In a second step, an initial charge containing the seed latex and part of the water was added to the reactor and was degassed by nitrogen bubbling for 30 min stirring constantly at 200 rpm and let to reach 70 ºC. The preemulsion containing the rest of the water, the surfactant (SDS) and the monomers were feed by a feeding inlet. The initiator KPS dissolved in water was added in 1 shot when the first drop of the monomer mixture reached the reactor. The monomers and the emulsifier were fed along the reaction for 3 h at a feeding rate of 1.437 g/min in MMA/BA/SBr (69/29/2) latex production and for 4 h at a feeding rate of 2.604 g/min in MMA/BA/HEMA (49.5/49.5/1) latex production. In both cases the reaction was left to react for another 2 h before being cooled and filtered through a muslin lining to remove any small amounts of coagulum. The formulation used is presented in Table S1.
Table S1. Formulation for synthesis of MMA/BA/StBr (69/29/2) and MMA/BA/HEMA (49.5/49.5/1) latex by seeded semi-continuous emulsion polymerization. MMA/BA/StBr 69/29/2 Compound
Seed latex MMA BA StBr HEMA V-501 KPS SDS NaOH H2O
10 wt % s.c seed charge (g) _ 33.81 14.21 0.98 _ _ 0.5 0.5 _ 430
Initial charge (g) 100 _ _ _ _ _ _ _ _ 100
30 wt % s.c. latex stream (g) _ 94.7 39.8 2.7 _ _ 1.4+0.2* 1.4 _ 120
MMA/BA/HEMA 49.5/49.5/1 10 wt % 45 wt % Initial s.c seed s.c. latex charge (g) charge (g) stream (g) _ 100 _ 39.6 _ 154.36 39.6 _ 154.36 _ _ _ 0.8 _ 3.12 0.8 _ _ _ 1.87 _ 0.8 _ 3.15 1.9995 _ _ 719.4 75 310
* After 2 h another initiator KPS shot (0.2 g dissolved in 5 g of water) was added to the reaction mixture.
In MMA/BA/SBr (69/29/2) case the final solids content of the latex was 30 wt % while for MMA/BA/HEMA it was 37.5%. The reaction temperature and the feed flow rates were controlled by an automatic control system (Camile TG, Biotage).
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Latex Characterization Z-average particle diameters were determined by dynamic light scattering performed in a Malvern Zetasizer ZS using a scattering angle of 173º at 20 ºC. Samples were prepared by diluting a fraction of the latex with deionized water. Each measurement was done in triplicate, and the average of the three was taken. Average particle size for MMA/BA/SBr (69/29/2) latex was 213 nm and for MMA/BA/HEMA (49.5/49.5/1) latex was 278 nm. By means of a Soxhlet extraction using tetrahydrofuran (THF) as a solvent the gel part and the soluble part of the latex were separated. The gel content was determined gravimetrically and the average molecular weight (Mw) of the soluble part was measured via gel permeation chromatography (GPC-SEC, Waters) using polystyrene PS standards. The setting consisted of a pump, an autosampler (Waters 717 plus) a differential refractometer (Waters 2410), and 3 columns in series (Styragel HR2, HR4 and HR6; with a pore size from 102 to 106 Å). The analyses were performed at 35 ºC and THF was used as solvent at a flow rate of 1 mL/min. A series of PS standards in the range of 580 - 3,848,000 g/mol were used to prepare a universal calibration curve. No gel content was determined for MMA/BA/SBr (69/29/2), and the Mw was 411.097 g mol-1. For MMA/BA/HEMA (49.5/49.5/1) 15% of gel content was determined, and Mw was 513.903 g mol-1.
Synthesis of the rGO/polymer composite hydrogels 3D graphene/polymer composite hydrogels were formed by self-assembling of GO platelets (Graphenea, concentration 4 mg/mL, > 95% monolayer content) under heating in the presence of ascorbic acid (AsA, Acros organics) reducing agent and the polymer latex at different ratios. GO and latex in ratios 1/0.8; 1/1.8; and ½. 4 were stirred during 30 min with a magnetic stirrer, after which Ascorbic Acid (AsA) in various amounts (AsA/GO was 0.5; 1 and 2) was added and kept under 60 ºC during 2 h. Composite hydrogels were formed, however not all the amount of polymer presented in the latex was incorporated within it. In an attempt to increase the amount of polymer incorporated in the structure of the composite hydrogel, the procedure was modified and the contact between the GO and the polymer latex was increased to 3 h, before the addition of AsA. In order to remove residual reactants, the hydrogels were dialyzed for 1 week in ultrapure water controlling S-4
the conductivity until it remained constant. For that aim dialysis membrane Spectral/Por (Spectrumlabs) with MWCO: 12–14,000 Daltons was used. Xerogels were produced by freeze drying of hydrogels under vacuum in a lyophilizer (Telstar LyoQuest-85). Hydrogels characterization: Rheology Dynamic fre-quency experiments were performed on Tritec 2000 DMA (Triton Technology) using 10x10x2 mm samples between parallel steel plates at 25 ºC in compression mode with a 2 mm gap. Frequency experiments were performed in the range of 0.4 to 40 Hz. FTIR spectral analysis was carried out measuring the hydrogel directly using Alpha FTIR spectrophotometer Platinum ATR operated with OPUS software. SEM The surface morphology of the hydrogels was examined using scanning electron microscopes: Hitachi TM3030 tabletop sem at 15 kV accelerating voltage after coating the samples with a gold thin layer and FEI Quanta 250 FEG with low field detector (LFD) at 10 kV accelerating voltage under low or high vacuum mode with standard detectors. The samples were imaged after being breaked under liquid nitrogen. Laccase production and crude laccase preparation Laccase was produced by cultivation of the white-rot fungus Trametes pubescens MB 89 (Austrian Centre of Biological Resources and Applied Mycology, University of Natural Resources and Applied Life Sciences, Vienna, Austria), under semi-solid-state fermentation conditions using sunflower-seed shells as support-substrates as described by Rodríguez-Couto et al.1 Culture broth was collected at the maximum laccase activity, filtered, and centrifuged (8000xg, 15 min). No other ligninolytic activities were detected in it The resulting clear filtrate was concentrated using an ultrafiltration stirred cell apparatus (Amicon Corp., Lexington, MA, USA) with a YM10 membrane (molecular cut-off of 10kDa). The experiments were performed with this concentrated clear filtrate diluted in 25 mM succinic acid buffer (pH 4.5). Analytical determinations Laccase activity was spectrophotometrically determined as described by Niku-Paavola et al.2 with 2,2´-azino-di-[3-ethyl-benzo-thiazolinsulphonate] (ABTS) as a substrate. One activity unit (U) was defined as the amount of enzyme that oxidized 1 µmol of ABTS per min. The activities were expressed in U/L. Protein concentration was spectrophotometrically determined at 595 nm according to Bradford3 using the reagent commercialised by Bio-Rad (Richmond, USA). Bovine serum albumin (BSA) was used as a standard. S-5
Laccase immobilization procedure Crude laccase was immobilized onto the graphene-polymer based supports synthesized above. For this, 0.09±0.03 g of xerogel supports or 5.43±0.38 g of hydrogel supports, according to the experiment, were immersed in 15 mL of crude laccase solution (activity: 9113 U/L; specific activity: 10.6 U/mg) for 72 hours at room temperature. Afterwards, the supernatant was removed and the supports were washed several times with 25 mM succinate buffer (pH 4.5) to remove the unbounded laccase and protein and kept at 4ºC until further use. Bound laccase and bound protein were determined as a difference between the initial and residual laccase and protein concentrations, respectively. Immobilized and free laccase stability against sodium azide. The hydrogel-immobilized laccase and the free laccase were exposed to the well-known laccase inhibitor compound sodium azide (N3Na). For this both laccases were incubated with 0.1 mM N3Na in 25 mM succinic buffer at pH 4.5 for 10 and 20 min. The residual laccase activity was determined using the standard ABTS assay. Laccase activity in the absence of the inhibitor was taken as 100%. Dye discoloration experiments The recalcitrant anthraquinonic dye Remazol Brilliant Blue R (1-amino-9,10-dihydro9,10-dioxo-4-[(3-{[2-(sulfooxy)ethyl]sulfonyl}phenyl)amino]
2-anthracenesulfonic
acid; CI name Reactive Blue 19; CI number 61200; Mw 626.54 g/mol; molecular formula: C22H16N2Na2O11S3), purchased from Sigma–Aldrich (St. Louis, MO, USA), was selected to perform the present study as a model of synthetic dye. A stock solution of RBBR dye (1% w/v in water) was prepared and stored in the dark at room temperature. Dye discoloration was carried out by directly adding aliquots of this RBBR stock solution to get the desired concentration into 250-mL Erlenmeyer flasks containing the immobilized laccase (5.43±0.38 g) and 25 mL of 25 mM succinic acid buffer (pH 4.5). Dye discoloration was performed in three successive batches. In the first batch a dye concentration of 100 mg/L was tested and in the other two the dye concentration was increased up to 200 mg/L. Laccase immobilized on HG3Br was also subjected to a fourth batch at a high RBBR concentration of 400 mg/L (final concentration in the flasks). After each batch the supports with the immobilized laccase were removed from the reaction mixture by filtration, washed 3 times with buffer (25 S-6
mM succinic acid, pH 4.5) and transferred into 25 mL of fresh dye solution for the next batch. Samples were taken at the beginning of each dye addition and at determined intervals, centrifuged (8000xg, 5 min) and the residual RBBR concentration was spectrophotometrically (Helios alfa, Thermo Fisher Scientific Inc.) measured from 450500 to 700 nm and calculated by measuring the area under the plot. This approach takes into account the conversion of the dye molecules to other compounds absorbing at different wavelengths and then, the ratio of the area under the visible spectrum is always equal or lower than the ratio of the absorbencies at the peak. Dye discoloration was expressed in terms of percentage. Control tests containing no laccase were also performed in parallel. Adsorption of the dye to the supports was determined by immersing them in methanol for 3 days at room temperature. The amount of the dye bound to the supports was calculated from the absorption of the supernatants. Analysis of RBBR degradation metabolites Mass Spectrometry MALDI-TOF-MS measurements were performed on a Bruker Autoflex Speed system (Bruker, Germany) instrument equipped with a 355 nm NdYAG laser. All spectra were acquired in the positive-ion reflectron mode. Different MALDI matrices were used α-cyano-4hydroxycinnamic
acid
(CHCA),
trans-2-[3-(4-tert-Butylphenyl)-2-methyl-2-
propenylidene]malononitrile (DTCB), 2-mercaptobenzothiazole (MBT). All the matrices were dissolved in THF at a concentration of 10 g/L. Potassium trifluoroacetate (KTFA), sodium iodide (NaI) and sodium trifluoroacetate (NaTFA) were added as cationic ionization agents (approximately 10 g/L dissolved in THF). The matrix, salt and polymer solutions were premixed in the ratio 10:1:10 (matrix: salt: sample). Approximately 0.5 µL of the obtained mixture were hand spotted on the ground steel target plate. For each spectrum 5000 laser shots were accumulated operating at 1 kHz. The spectra were externally calibrated using a mixture of polyethylene glycol standard of Mw 600 Da (PEG, Varian).
Phytotoxicity studies The toxicity of the original and the decolorized dye was assessed by measuring the phytotoxicity effect of water solutions (1:3) on seeds germination of radish (Raphanus sativa) according to Zucconi et al.4 Four replicates of 10 seeds were used for each test. After 5 days of incubation in the dark, the seed germination percentage and root length of seeds immersed in the dye solutions as well as in deionised water (control) were S-7
determined. The germination index (GI) was calculated as follows: GI = GP x La/Lc, where GP is the number of germinated seeds expressed as a percent-age of control values, La is the average value of root length in the dye solutions and Lc is the average value of the root length in the control. Determination of RBBR biotransformation products To identify the metabolites of RBBR degradation by the immobilized laccase, the original dye solution and the laccase-treated dye solution were analyzed by MALDITOF MS. In Table S2 the all the identified peaks (m/z values) together with the assigned structures are presented. Table S2. Identified species by MALDI-TOF MS
Structure
Addu ct H
Detected m/z (Da)
Theoret. m/z (Da)
103.694
103.954
Na
125.803
125.936
NaO3SH
K
142.814
142.918
-
NaO4SH
Na
142.814
142.938
-
C8H8O3S
H
184.804
185.027
[1]*
-
-
190.748
-
[5] **
C8H11NO3S
H Na 2M+ Na
200.787 201.993 224.992 425.111
201.045 202.053 224.035 425.081
C8H10NO3S
H
201.993
202.053
H
208.161
207.008
Molecular formula
O S
ONa
NaO3S
Ref.
-
O
ESI MS detected but not identified
[1]*
[1]*
[1]
S-8
C8H7NaO3S
ESI MS detected but not identified
O
NH2
Na
230.942
228.991
K
244.966
244.964
[5] **
-
-
260.046
-
[5] **
C8H10NNaO6S2
Na
326.820
325.974
[1]
C14H8NaO6S
-
326.993
325.974
[1]*
C14H7NNaO5S
Na
346.928
346.983
[1]*
C14H6NaO6S
-
346.928
347.967
[1]*
C22H17N2NaO7S
H
509.303
509.045
-
2M+ Na
701.428
700.999
-
O S
ONa
O
O
O
O S O
O
ONa
O
2
C14H8N2NaO5S
* Proposed species, but not identified by LC-MS in reference [1] ** Detected mass values by ESI-MS, but not assigned in reference [5]
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REFERENCES [1] Rodríguez-Couto, S.; Osma, J.F.; Toca-Herrera, J.L., Removal of Synthetic Dyes by an Eco-Friendly Strategy, Eng. Life Sci. 2009, 9 (2) 116–123 [2] Niku-Paavola, M.L.; Raaska, L.; Itavaara, M., Detection of White-rot Fungi by a Non-Toxic Stain, Mycol. Res. 1990, 94, 27-31. [3] Bradford, M.M., A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding, Anal. Biochem. 1976, 72, 248-254 [4] Zucconi, F.; Monaco, A.; Forte, M.; De-Bertoldi, M., Phytotoxins during the stabilization of organic matter. Gasser, J.K.R., Commission European Communities (Eds.), Composting of Agricultural and Other Wastes, Elsevier Applied Science Publishers, London 1985, pp. 73–86, [5]
Sathishkumar, P., PhD thesis, Periyar University, Salem, India, 2010
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Design of Stable and Powerful Nanobiocatalysts, Based on Enzyme Laccase Immobilized on Self- Assembled 3D Graphene/Polymer Composite Hydrogels
Nerea Ormategui†, Antonio Veloso†, Gracia Patricia Leal†, Susana RodriguezCouto‡§*, Radmila Tomovska† §*
†
POLYMAT and Departamento de Química Aplicada, Facultad de Ciencias Químicas, University of the Basque Country, UPV/EHU, Donostia-San Sebastián, Spain
‡
CEIT, Unit of Environmental Engineering, Paseo Manuel de Lardizábal, Donostia-San Sebastián, Spain §
IKERBASQUE, Basque Foundation for Science, Bilbao, Spain
Coresponding authors’ e-mail addresses: * [email protected] * [email protected]
S-1
Experimental Section Materials All the chemicals for the experiments were used without further purification. Methyl methacrylate (MMA, Quimidroga), butylacrylate (BA, Quimidroga), 4-bromostyrene (StBr, Aldrich), 2-hydroxyethyl methacrylate (HEMA, Aldrich), potassium persulfate (KPS, Al-drich), 4,4-azobis(4-cyanovaleric acid) (V-501, Aldrich), ascorbic acid (AsA, Acros organics), GO plateletss (Graphenea, concentration 4 mg/mL, > 95% monolayer content), 2,2´-azino-di-[3-ethyl-benzo-thiazolin-sulphonate] (ABTS, Sigma), succinic acid (Sigma-Aldrich), Remazol Brilliant Blue R (1-amino-9,10-dihydro-9,10-dioxo-4[(3-{[2-(sulfooxy)ethyl]sulfonyl}phenyl)amino] 2-anthracenesulfonic acid (Sigma– Aldrich), sodium azide (N3Na, Alfa Aesar), bovine serum albumin (BSA, SigmaAldrich), Bradford protein assay reagent (Bio-Rad, USA).
Methods The nano-biocatalysts were prepared by immobilization of laccase enzyme on the 3D composite graphene/polymer hydrogels. Firstly, the hydrogels were prepared by selfassembling of GO platelets during their reduction in using the polymer latex as a matrix. The polymer latex was prepared by seeded semicontinuous emulsion polymerization of MMA and BA in relation 50/50 and 70/30 to each other and small amount of functional monomer (1 or 2% of HEMA or SBr, respectively), with aim to functionalize the polymer nanoparticles. The detailed procedures are as follows. Polymer latex synthesis Seed Latex preparation Semi-continuous seeded emulsion polymerization was used to synthesize poly(MMA/BA/SBr) at a ratio of 69/29/2 and poly(MMA/BA/HEMA) at a ratio of 49.5/49.5/1. First, 10 wt % solids content (s.c.) seed was prepared by batch emulsion polymerization in a 1 L-jacketed glass reactor equipped with an anchor type stirrer, a nitrogen inlet, a condenser and a thermocouple. The reaction mixture was degassed by nitrogen bubbling for 30 min, stirring constantly at 200 rpm, and then heated to 70 ºC. Once the desired reaction temperature was reached, in MMA/BA/SBr case the initiator potassium persulfate (KPS, Aldrich) (0.5 g dissolved in 20 g of water) was added in a single shot, while for MMA/BA/HEMA 4,4-azobis(4-cyanovaleric acid) (V-501, Aldrich) was used as a free radical initiator. After 3 h the reaction mixture was S-2
cooled and filtered through a muslin lining to remove the possible coagulum. The final solids content of the seed was 10 wt %. Seeded Emulsion Polymerization In a second step, an initial charge containing the seed latex and part of the water was added to the reactor and was degassed by nitrogen bubbling for 30 min stirring constantly at 200 rpm and let to reach 70 ºC. The preemulsion containing the rest of the water, the surfactant (SDS) and the monomers were feed by a feeding inlet. The initiator KPS dissolved in water was added in 1 shot when the first drop of the monomer mixture reached the reactor. The monomers and the emulsifier were fed along the reaction for 3 h at a feeding rate of 1.437 g/min in MMA/BA/SBr (69/29/2) latex production and for 4 h at a feeding rate of 2.604 g/min in MMA/BA/HEMA (49.5/49.5/1) latex production. In both cases the reaction was left to react for another 2 h before being cooled and filtered through a muslin lining to remove any small amounts of coagulum. The formulation used is presented in Table S1.
Table S1. Formulation for synthesis of MMA/BA/StBr (69/29/2) and MMA/BA/HEMA (49.5/49.5/1) latex by seeded semi-continuous emulsion polymerization. MMA/BA/StBr 69/29/2 Compound
Seed latex MMA BA StBr HEMA V-501 KPS SDS NaOH H2O
10 wt % s.c seed charge (g) _ 33.81 14.21 0.98 _ _ 0.5 0.5 _ 430
Initial charge (g) 100 _ _ _ _ _ _ _ _ 100
30 wt % s.c. latex stream (g) _ 94.7 39.8 2.7 _ _ 1.4+0.2* 1.4 _ 120
MMA/BA/HEMA 49.5/49.5/1 10 wt % 45 wt % Initial s.c seed s.c. latex charge (g) charge (g) stream (g) _ 100 _ 39.6 _ 154.36 39.6 _ 154.36 _ _ _ 0.8 _ 3.12 0.8 _ _ _ 1.87 _ 0.8 _ 3.15 1.9995 _ _ 719.4 75 310
* After 2 h another initiator KPS shot (0.2 g dissolved in 5 g of water) was added to the reaction mixture.
In MMA/BA/SBr (69/29/2) case the final solids content of the latex was 30 wt % while for MMA/BA/HEMA it was 37.5%. The reaction temperature and the feed flow rates were controlled by an automatic control system (Camile TG, Biotage).
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Latex Characterization Z-average particle diameters were determined by dynamic light scattering performed in a Malvern Zetasizer ZS using a scattering angle of 173º at 20 ºC. Samples were prepared by diluting a fraction of the latex with deionized water. Each measurement was done in triplicate, and the average of the three was taken. Average particle size for MMA/BA/SBr (69/29/2) latex was 213 nm and for MMA/BA/HEMA (49.5/49.5/1) latex was 278 nm. By means of a Soxhlet extraction using tetrahydrofuran (THF) as a solvent the gel part and the soluble part of the latex were separated. The gel content was determined gravimetrically and the average molecular weight (Mw) of the soluble part was measured via gel permeation chromatography (GPC-SEC, Waters) using polystyrene PS standards. The setting consisted of a pump, an autosampler (Waters 717 plus) a differential refractometer (Waters 2410), and 3 columns in series (Styragel HR2, HR4 and HR6; with a pore size from 102 to 106 Å). The analyses were performed at 35 ºC and THF was used as solvent at a flow rate of 1 mL/min. A series of PS standards in the range of 580 - 3,848,000 g/mol were used to prepare a universal calibration curve. No gel content was determined for MMA/BA/SBr (69/29/2), and the Mw was 411.097 g mol-1. For MMA/BA/HEMA (49.5/49.5/1) 15% of gel content was determined, and Mw was 513.903 g mol-1.
Synthesis of the rGO/polymer composite hydrogels 3D graphene/polymer composite hydrogels were formed by self-assembling of GO platelets (Graphenea, concentration 4 mg/mL, > 95% monolayer content) under heating in the presence of ascorbic acid (AsA, Acros organics) reducing agent and the polymer latex at different ratios. GO and latex in ratios 1/0.8; 1/1.8; and ½. 4 were stirred during 30 min with a magnetic stirrer, after which Ascorbic Acid (AsA) in various amounts (AsA/GO was 0.5; 1 and 2) was added and kept under 60 ºC during 2 h. Composite hydrogels were formed, however not all the amount of polymer presented in the latex was incorporated within it. In an attempt to increase the amount of polymer incorporated in the structure of the composite hydrogel, the procedure was modified and the contact between the GO and the polymer latex was increased to 3 h, before the addition of AsA. In order to remove residual reactants, the hydrogels were dialyzed for 1 week in ultrapure water controlling S-4
the conductivity until it remained constant. For that aim dialysis membrane Spectral/Por (Spectrumlabs) with MWCO: 12–14,000 Daltons was used. Xerogels were produced by freeze drying of hydrogels under vacuum in a lyophilizer (Telstar LyoQuest-85). Hydrogels characterization: Rheology Dynamic fre-quency experiments were performed on Tritec 2000 DMA (Triton Technology) using 10x10x2 mm samples between parallel steel plates at 25 ºC in compression mode with a 2 mm gap. Frequency experiments were performed in the range of 0.4 to 40 Hz. FTIR spectral analysis was carried out measuring the hydrogel directly using Alpha FTIR spectrophotometer Platinum ATR operated with OPUS software. SEM The surface morphology of the hydrogels was examined using scanning electron microscopes: Hitachi TM3030 tabletop sem at 15 kV accelerating voltage after coating the samples with a gold thin layer and FEI Quanta 250 FEG with low field detector (LFD) at 10 kV accelerating voltage under low or high vacuum mode with standard detectors. The samples were imaged after being breaked under liquid nitrogen. Laccase production and crude laccase preparation Laccase was produced by cultivation of the white-rot fungus Trametes pubescens MB 89 (Austrian Centre of Biological Resources and Applied Mycology, University of Natural Resources and Applied Life Sciences, Vienna, Austria), under semi-solid-state fermentation conditions using sunflower-seed shells as support-substrates as described by Rodríguez-Couto et al.1 Culture broth was collected at the maximum laccase activity, filtered, and centrifuged (8000xg, 15 min). No other ligninolytic activities were detected in it The resulting clear filtrate was concentrated using an ultrafiltration stirred cell apparatus (Amicon Corp., Lexington, MA, USA) with a YM10 membrane (molecular cut-off of 10kDa). The experiments were performed with this concentrated clear filtrate diluted in 25 mM succinic acid buffer (pH 4.5). Analytical determinations Laccase activity was spectrophotometrically determined as described by Niku-Paavola et al.2 with 2,2´-azino-di-[3-ethyl-benzo-thiazolinsulphonate] (ABTS) as a substrate. One activity unit (U) was defined as the amount of enzyme that oxidized 1 µmol of ABTS per min. The activities were expressed in U/L. Protein concentration was spectrophotometrically determined at 595 nm according to Bradford3 using the reagent commercialised by Bio-Rad (Richmond, USA). Bovine serum albumin (BSA) was used as a standard. S-5
Laccase immobilization procedure Crude laccase was immobilized onto the graphene-polymer based supports synthesized above. For this, 0.09±0.03 g of xerogel supports or 5.43±0.38 g of hydrogel supports, according to the experiment, were immersed in 15 mL of crude laccase solution (activity: 9113 U/L; specific activity: 10.6 U/mg) for 72 hours at room temperature. Afterwards, the supernatant was removed and the supports were washed several times with 25 mM succinate buffer (pH 4.5) to remove the unbounded laccase and protein and kept at 4ºC until further use. Bound laccase and bound protein were determined as a difference between the initial and residual laccase and protein concentrations, respectively. Immobilized and free laccase stability against sodium azide. The hydrogel-immobilized laccase and the free laccase were exposed to the well-known laccase inhibitor compound sodium azide (N3Na). For this both laccases were incubated with 0.1 mM N3Na in 25 mM succinic buffer at pH 4.5 for 10 and 20 min. The residual laccase activity was determined using the standard ABTS assay. Laccase activity in the absence of the inhibitor was taken as 100%. Dye discoloration experiments The recalcitrant anthraquinonic dye Remazol Brilliant Blue R (1-amino-9,10-dihydro9,10-dioxo-4-[(3-{[2-(sulfooxy)ethyl]sulfonyl}phenyl)amino]
2-anthracenesulfonic
acid; CI name Reactive Blue 19; CI number 61200; Mw 626.54 g/mol; molecular formula: C22H16N2Na2O11S3), purchased from Sigma–Aldrich (St. Louis, MO, USA), was selected to perform the present study as a model of synthetic dye. A stock solution of RBBR dye (1% w/v in water) was prepared and stored in the dark at room temperature. Dye discoloration was carried out by directly adding aliquots of this RBBR stock solution to get the desired concentration into 250-mL Erlenmeyer flasks containing the immobilized laccase (5.43±0.38 g) and 25 mL of 25 mM succinic acid buffer (pH 4.5). Dye discoloration was performed in three successive batches. In the first batch a dye concentration of 100 mg/L was tested and in the other two the dye concentration was increased up to 200 mg/L. Laccase immobilized on HG3Br was also subjected to a fourth batch at a high RBBR concentration of 400 mg/L (final concentration in the flasks). After each batch the supports with the immobilized laccase were removed from the reaction mixture by filtration, washed 3 times with buffer (25 S-6
mM succinic acid, pH 4.5) and transferred into 25 mL of fresh dye solution for the next batch. Samples were taken at the beginning of each dye addition and at determined intervals, centrifuged (8000xg, 5 min) and the residual RBBR concentration was spectrophotometrically (Helios alfa, Thermo Fisher Scientific Inc.) measured from 450500 to 700 nm and calculated by measuring the area under the plot. This approach takes into account the conversion of the dye molecules to other compounds absorbing at different wavelengths and then, the ratio of the area under the visible spectrum is always equal or lower than the ratio of the absorbencies at the peak. Dye discoloration was expressed in terms of percentage. Control tests containing no laccase were also performed in parallel. Adsorption of the dye to the supports was determined by immersing them in methanol for 3 days at room temperature. The amount of the dye bound to the supports was calculated from the absorption of the supernatants. Analysis of RBBR degradation metabolites Mass Spectrometry MALDI-TOF-MS measurements were performed on a Bruker Autoflex Speed system (Bruker, Germany) instrument equipped with a 355 nm NdYAG laser. All spectra were acquired in the positive-ion reflectron mode. Different MALDI matrices were used α-cyano-4hydroxycinnamic
acid
(CHCA),
trans-2-[3-(4-tert-Butylphenyl)-2-methyl-2-
propenylidene]malononitrile (DTCB), 2-mercaptobenzothiazole (MBT). All the matrices were dissolved in THF at a concentration of 10 g/L. Potassium trifluoroacetate (KTFA), sodium iodide (NaI) and sodium trifluoroacetate (NaTFA) were added as cationic ionization agents (approximately 10 g/L dissolved in THF). The matrix, salt and polymer solutions were premixed in the ratio 10:1:10 (matrix: salt: sample). Approximately 0.5 µL of the obtained mixture were hand spotted on the ground steel target plate. For each spectrum 5000 laser shots were accumulated operating at 1 kHz. The spectra were externally calibrated using a mixture of polyethylene glycol standard of Mw 600 Da (PEG, Varian).
Phytotoxicity studies The toxicity of the original and the decolorized dye was assessed by measuring the phytotoxicity effect of water solutions (1:3) on seeds germination of radish (Raphanus sativa) according to Zucconi et al.4 Four replicates of 10 seeds were used for each test. After 5 days of incubation in the dark, the seed germination percentage and root length of seeds immersed in the dye solutions as well as in deionised water (control) were S-7
determined. The germination index (GI) was calculated as follows: GI = GP x La/Lc, where GP is the number of germinated seeds expressed as a percent-age of control values, La is the average value of root length in the dye solutions and Lc is the average value of the root length in the control. Determination of RBBR biotransformation products To identify the metabolites of RBBR degradation by the immobilized laccase, the original dye solution and the laccase-treated dye solution were analyzed by MALDITOF MS. In Table S2 the all the identified peaks (m/z values) together with the assigned structures are presented. Table S2. Identified species by MALDI-TOF MS
Structure
Addu ct H
Detected m/z (Da)
Theoret. m/z (Da)
103.694
103.954
Na
125.803
125.936
NaO3SH
K
142.814
142.918
-
NaO4SH
Na
142.814
142.938
-
C8H8O3S
H
184.804
185.027
[1]*
-
-
190.748
-
[5] **
C8H11NO3S
H Na 2M+ Na
200.787 201.993 224.992 425.111
201.045 202.053 224.035 425.081
C8H10NO3S
H
201.993
202.053
H
208.161
207.008
Molecular formula
O S
ONa
NaO3S
Ref.
-
O
ESI MS detected but not identified
[1]*
[1]*
[1]
S-8
C8H7NaO3S
ESI MS detected but not identified
O
NH2
Na
230.942
228.991
K
244.966
244.964
[5] **
-
-
260.046
-
[5] **
C8H10NNaO6S2
Na
326.820
325.974
[1]
C14H8NaO6S
-
326.993
325.974
[1]*
C14H7NNaO5S
Na
346.928
346.983
[1]*
C14H6NaO6S
-
346.928
347.967
[1]*
C22H17N2NaO7S
H
509.303
509.045
-
2M+ Na
701.428
700.999
-
O S
ONa
O
O
O
O S O
O
ONa
O
2
C14H8N2NaO5S
* Proposed species, but not identified by LC-MS in reference [1] ** Detected mass values by ESI-MS, but not assigned in reference [5]
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REFERENCES [1] Rodríguez-Couto, S.; Osma, J.F.; Toca-Herrera, J.L., Removal of Synthetic Dyes by an Eco-Friendly Strategy, Eng. Life Sci. 2009, 9 (2) 116–123 [2] Niku-Paavola, M.L.; Raaska, L.; Itavaara, M., Detection of White-rot Fungi by a Non-Toxic Stain, Mycol. Res. 1990, 94, 27-31. [3] Bradford, M.M., A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding, Anal. Biochem. 1976, 72, 248-254 [4] Zucconi, F.; Monaco, A.; Forte, M.; De-Bertoldi, M., Phytotoxins during the stabilization of organic matter. Gasser, J.K.R., Commission European Communities (Eds.), Composting of Agricultural and Other Wastes, Elsevier Applied Science Publishers, London 1985, pp. 73–86, [5]
Sathishkumar, P., PhD thesis, Periyar University, Salem, India, 2010
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