Supplementary Data
Comparison of lignin peroxidase and horseradish peroxidase for catalyzing the removal of nonylphenol from water
Shipeng Dong, Liang Mao*, Siqiang Luo, Lei Zhou, Yiping Feng, Shixiang Gao*
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210046, P. R. China
FivePages
Three Figures
Sec. I. Stereo view of the catalytic cavity contained in lignin peroxidase (LiP) andhorseradish peroxidase (HRP)
FIGURE SM-1. Stereo view of the catalytic cavity contained in LiP (left) andHRP (right).These enzyme structures were downloaded fromRCSB protein data bank ( and viewed in HyperChem Molecular Modeling System.
Sec. II. Additional description of certain experimental procedures
Assessment of Michaelis-Menten reaction kinetics
The initial reaction rates of NP at various initial concentrations were assessed, and the data were used to construct Michaelis-Menten curves and for associated kinetic analysis.
The reactions were carried out inglass test tubes as batch reactors under room temperature.Each reactor contained a 2-mL reaction medium preparedin 10mMbuffer solution. The reaction solution initiallycontained predeterminedamounts of nonylphenol(NP) and enzyme.Hydrogenperoxide was added to each reactor as the last componentto initiate the reaction. Following the initiation of the reaction by the addition of H2O2, the reactor was hand shaken and allowed to react for 30 s prior to the termination of the reaction by the addition of 2 mL methanol. The mixture of reaction solution and methanol was then sampled for HPLC analysis. Three replicate reactors were prepared and tested for each reaction condition along with a blank reactor that was prepared at otherwise the same condition except for having enzyme absent.
Following the measurement of the substrate concentration in both the blank (S0) and reaction tubes (St), the initial reaction rate (v0) was calculated using the formulation v0= (S0 - St)/ Δt. The reaction time Δt was 30s for all experiments, which was determined in preliminary tests that enable to capture the pseudo-first-order rate behavior of the enzymatic reactions and to ensure reproducible handling of the reactors. The initial rate data were then fitted using non-linear regression to Michaelis-Menten equation v0 = vmax× S0 /(S0+KM) to obtain the maximum rate of enzymatic reaction vmax and the substrate’s Michaelis constant KM. We further assume the 30-seconds reaction time is sufficiently short such that the active enzyme concentration remain the same as the initial enzyme concentration [E0], and thus calculated the initial reaction rate constant according to kCAT = vmax /[E0] (Mao et al., 2009; Colosi et al., 2006).
SPE procedures
The product solution was extracted using C18 solid phase extraction (SPE) (500 mg, J. T. Baker Chemical Co., Phillipsburg, NJ). Prior to extraction, the cartridge was conditioned with 7mL methanol. The solution was passed through the column at a flow rate of approximately 1.5mLmin-1 followed by a rinse of 10mL DI water. The column was then blown to dryness for 10 min, after which the column was eluted with 2mL of methanol. Triplicate experiments of spiked sample were conducted to study the NP recovery efficiency of SPE. Recovery efficiency of SPE for NP is 97.2(± 2)%.
3D-fluorescence analysis
Experiments were performed to study the possible transformation of NOM in LiP/HRP-mediated NP oxidation systems. The reaction were conducted at room temperature in glass test tubes containing 2 mL buffer comprised of 0.15 U mL-1 enzyme, 5mg C L-1 NOM, 10 μM NP and 50 μMH2O2. The 3D-fluorescence spectra was recorded at reaction times of 0 and 90 min with a F-7000 Fluorescence Spectrophotometer (Hitachi High-Tech Corporation). The excitation wavelength ranged from 200 to 450 nm and the bandpass was 5 nm. The range of emission wavelengthwas from 250 to 600 nm. Controls with peroxidase or H2O2 absent were also prepared.
Sec. III. The data of total organic carbon (TOC) in the LiP/HRP-mediated NP reaction system
Figure SM-2. The change of TOC data in the LiP and HRP-mediated NP reaction systems. Experimental condition: [Enzyme]=10 nM, [NP]=10 µM.
Sec. IV. 3D-fluorescence spectra of NP reaction solutions with NOM presence
3D-fluorescence spectra of NP reaction solutions with NOM present are displayed in Figure S3. As shown in the FigureS3a and b, region A and B is respectively the fluorescence spectra of NP and NOM. It is the evident in Figure S3c that the fluorescence spectra of NP was dispeared and that of NOM has changed after the peroxidase-mediated reaction. As such, we proposed that NOM was likely to be the active substrates of LiP and HRP.
Figure SM-3. 3D-fluorescence spectra of NP reaction solutions having various components: H2O2 + NP + NOM (a), peroxidase + NP + NOM (b), and H2O2 + NP + NOM + peroxidase (c). All samples were incubated for 90 min priorto fluorescenceanalysis. The compositions of the samplesincluded the following: [NP] = 10 µM, [LiP/HRP] = 0.15 UmL-1, [H2O2]= 50μM, [NOM] = 5 mgL-1 as TOC.
S1