Appl Microbiol Biotechnol DOI 10.1007/s00253-014-5938-6
BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Biogenic magnetic nanoparticles from Burkholderia sp. YN01 exhibiting intrinsic peroxidase-like activity and their applications Yu Pan & Na Li & Jianshuai Mu & Runhong Zhou & Yan Xu & Daizong Cui & Yan Wang & Min Zhao
Received: 21 May 2014 / Revised: 2 July 2014 / Accepted: 5 July 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract A novel bacterial strain containing biogenic magnetic nanoparticles (BMNPs) was isolated from the sediments of Songhua River in Harbin, China, and was identified as Burkholderia sp. YN01. Extracted BMNPs from YN01 were characterized as pure face-centered cubic Fe3O4 with an average size of 80 nm through transmission electron microscope (TEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The hysteresis parameters of the BMNP samples such as Bc and Bcr and ratios Mrs/Ms were deduced as 35.6 mT, 43.2 mT, and 0.47, respectively, indicating that the BMNPs exhibit a ferromagnetic behavior. This is the first report concerning on biogenic Fe3O4 NPs produced in Burkholderia genus. Significantly, the BMNPs were proved to possess intrinsic peroxidase-like activity that could catalyze the o xidation of p eroxidase substrate 3,3′ ,5 ,5′ tetramethylbenzidine (TMB) in the presence of H2O 2. Kinetic analysis indicates that the catalytic behavior is in accord with typical Michaelis–Menten kinetics and follows ping-pong mechanism. The catalytic constants (Kcat) were 6.5×104 s−1 and 0.78×104 s−1 with H2O2 and TMB as substrate, respectively, which was higher than that of horseradish
Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-5938-6) contains supplementary material, which is available to authorized users. Y. Pan : N. Li : R. Zhou : Y. Xu : D. Cui : M. Zhao (*) College of Life Science, Northeast Forestry University, No. 26, Hexing Road, Harbin 150040, China e-mail: [email protected] J. Mu : Y. Wang Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, Harbin 150001, China Y. Pan School of Environment and Chemical Engineering, Heilongjiang University of Science and Technology, Harbin 150022, China
peroxidase (HRP). Electron spin resonance (ESR) spectroscopy experiments showed that the BMNPs could catalyze H2O2 to produce hydroxyl radicals. The origin of peroxidase-like activity is also associated with their ability to transfer electron between electrode and H2O2 according to an electrochemical study. As a novel peroxidase mimetic, the BMNPs were employed to offer a simple, sensitive, and selective colorimetric method for H2O2 and glucose determination, and the BMNPs could efficiently catalyze the degradation of phenol and Congo red dye. Keywords Biogenic magnetic nanoparticles . Burkholderia . Peroxidase mimetic . Glucose determination . Degradation
Introduction Nanomaterials have attracted considerable attention over the past few decades due to their unique size, shape, optical electric, chemical, and mechanical properties (Lee et al. 2006; Khalavka et al. 2009). Recently, many nanomaterials have been evaluated as oxidase, peroxidase, or catalase mimetics with low cost, increased stability, high tunability, sustainable electrocatalytic activity, and robustness to harsh environments (Wei and Wang 2013), which can be potentially used in bioassays and environmental chemistry (Gao et al. 2007; Dai et al. 2009; André et al. 2010; Mu et al. 2012; Chaudhari et al. 2012; Song et al. 2010; Shi et al. 2011a, b). Among known nanomaterials with enzyme activities, magnetic nanoparticles (MNPs) are of particular interest in magnetic resonance imaging (MRI) (Brahler et al. 2006), magnetic biosensor (Chemla et al. 2000), and wastewater treatment (Ambashta and Sillanpaa 2010), or as detection tools (Liu et al. 2012; Yua et al. 2009) because of their dual functionality as a peroxidase mimetic and a magnetic separation agent. And, ferruginous bodies containing magnetite within ferritin
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coating were also verified to exert peroxidase-like activity (Borelli et al. 2012). Various synthetic methods have been described for the synthesis of Fe3O4 NPs, such as the sol-gel method, the reverse micellar method, and the thermal decomposition of organometallic iron precursors using organic solvents at high temperatures. These synthetic approaches involve high temperatures and toxic reagents, which are both expensive and environmentally undesirable (Seabra et al. 2013). In recent years, green nanotechnology has attracted increasing attention because of the possibility to reduce or eliminate toxic substance; therefore, microbial synthesis of MNPs is considered as an eco-friendly process, since it occurs in water, at room temperature and pressure, and very close to neutral pH (Durán and Seabra 2012). Many studies revealed that biogenic MNPs (BMNPs) can be formed by numerous microorganisms, such as bacteria and fungi (Seabra et al. 2013; Mandal et al. 2006; Roh et al. 2001). Bacterial magnetosomes are natural inorganic ferromagnetic nanoparticles within the single-domain size range of 35~120 nm biomineralized in magnetotactic bacteria (Blakemore 1975; Bazylinski and Frankel 2004; Alphandéry et al. 2012). Besides magnetotactic bacteria, BMNPs were also found in non-magnetotactic bacteria (Vainshtein et al. 2002). This biogenic MNPs would be expected to show superior performances of low environmental cost, high chemical purity, fine and uniform particle size, good dispersity, and biocompatibility without surface modification (Knopp et al. 2009), which make them a promising magnetic nanomaterial in biomolecule immobilization, drug and gene targeting, and wastewater treatment (Matsunaga et al. 2007; Dutz et al. 2009). However, only very few bacteria strains that can produce MNPs are available in pure culture owing to their fastidious growth requirements and strong metabolic diversity (Flies et al. 2005). Many studies focus on biomineralization process in cells, magnetism of magnetosomes (Bazylinski and Frankel 2004; Calugay et al. 2004; Gorby et al. 1988), and their ability to eliminate intracellular reactive oxygen species (Guo et al. 2012); however, to date, the potential of BMNPs from bacteria cells as enzyme mimetics and their applications have not been explored. In this paper, the purification and characterization of biogenic Fe3O4 MNPs from a newly isolated bacteria strain (designated Burkholderia sp. YN01) were described. Significantly, these BMNPs were proved to possess peroxidase-like activity, and the catalytic mechanisms and origin of their intrinsic peroxidase-like activity were studied by kinetic and electrochemical analysis and electron spin resonance (ESR) technique. The BMNPs were also tested for their peroxidase-like activity in glucose determination and degradation of phenol and Congo red dye.
Materials and methods Materials All chemicals used in this work were of analytical grade and used as received without further purification. Bacterial DNA kit and gel extraction kit were purchased from Omega Bio-Tek (Norcross, GA, USA). Taq DNA polymerase, primers, and pMD 18-T vector were obtained from TaKaRa (Dalian, China). 3,3′,5,5′-Tetramethylbenzidine (TMB), horseradish peroxidase (EC 1.11.1.17, 250–330 U/mg using pyrogallol, type VI-A), glucose oxidase (GOx, from Aspergillus niger, ≥100 U/mg), and DMPO (5,5-dimethyl-1-pyrroline N-oxide) were purchased from Sigma-Aldrich (St. Louis, USA). H2O2 and other regents were obtained from Boyue Biological Reagent Co. (Harbin, China). Glucose, fructose, lactose, and maltose were purchased from Beijing Chemical Reagent Company (Beijing, China). Isolation and identification of MNP-producing strain Samples of freshwater and sediment are collected from Songhua River, which is located in Harbin, China. Sediment and water in proportion of 1:3 (vol/vol) were stored in loosely capped bottles. Each liter of the sample was supplemented with 1.0 g sodium succinate, 0.1 g NaNO3, 0.2 g sodium acetate, 0.05 g sodium thioglycollate, 3.5 mL 0.01 M ferric citrate solution, 10 mL Wolfe’s vitamin solution, and 5.0 mL Wolfe’s mineral solution, and then was left undisturbed for 1 month at room temperature under dim light for enrichment culture. After enrichment, the bacteria culture was precipitated by centrifugation then resuspended in distilled water. Then, 0.5-mL cell suspension was placed on the sterile flat dish. Then, sterile semi-solid isolation medium (IM) supplemented with 0.74 g sodium succinate, 0.1 g NaNO3, 0.2 g sodium acetate, 0.05 g sodium thioglycollate, 5.0 mL 0.01 M ferric citrate solution, 10 mL Wolfe’s vitamin solution, 5.0 mL Wolfe’s mineral solution, and 1.2 % (g/vol) agar was poured to each plate, mixed equably, and cultivated for 7 days at 28 °C. The monocolonies isolated by picking up colonies from the agar plates were inoculated in IM, followed by stab-cultivating in semi-solid IM in tubes for 7 days at 28 °C. The process was repeated two times before monocolonies were considered as pure. The purified bacteria cells were investigated under transmission electron microscopy (TEM, FEI/Philips TCNAI G2) at an accelerating voltage of 80 kV and direct magnification of×30,000. 16S ribosomal DNA (16S rDNA) genes of the obtained bacteria were amplified between positions 27 and 1492 (Escherichia coli 16S rRNA gene sequence numbers), using primers 27 F (5′-AGAGTYTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTI‘GTI’ ACGACTT-3′) by polymerase chain reaction (PCR) carried out with the following cycle: an
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initial denaturing step at 94 °C for 5 min, followed by 25 cycles of 1 min at 94 °C, 45 s at 50 °C, and 1 min at 72 °C, and a final extension step of 10 min at 72 °C. Then, the PCR products were cloned using a commercially available pGEM-T vector cloning kit and subsequently sequenced by Shenggong Company in Shanghai. The obtained sequences of the 16S rDNA gene (about 1.5 kb) were analyzed using aligning tool of BLAST program (http://www.ncbi.nlm.nih. gov/BLAST/).
Preparation and characterization of BMNPs The purified bacterial cells were inoculated in a 250-mL bottle containing 200-mL growth medium. After 2-day incubation, the culture from medium was harvested as described above and lysed by ultrasonication for 15 min. The magnetic particles released from the bacterial cells were collected by permanent magnet. The resulting black solid products were washed with water three times then separated and collected by magnet for characterization. The composition and phase of BMNPs were identified by powder X-ray diffraction (XRD) on an D/max-rB X-ray diffractometer (Rigaku, Japan) using Cu Kα radiation (λ=1.5418 Å). The morphology and size of BMNPs were examined by transmission electron microscopy (TEM, FEI/Philips TCNAI G2) at an accelerating voltage of 80 kV and direct magnification of×80,000. X-ray photoelectron spectroscopy (XPS) analysis was conducted with an AXIS ULTRA PLD spectrometer (Kratos Co.) using Al as the exciting source at 1,486.4 eV. The polluted carbon was used in charged correction of binding energy of the BMNP sample at 284.6 eV. Room temperature magnetic experiments were performed on a vibrating sample magnetometer (Model 3900, Princeton Measurements Corporation, sensitivity is 5.0×10−10 Am2). The hysteresis loop was measured between +500 and −500 mT with an average time of 400 ms. Saturation magnetization (Ms) and saturation remanence (Mrs) were determined after correction for paramagnetic phases.
ESR spectroscopy measurements ESR technique was used to detect hydroxyl radicals (•OH) formed during the decomposition of H 2O2 induced by BMNPs. Because of its diamagnetic property, 5,5-dimethyl1-pyrroline N-oxide (DMPO) is capable of trapping these short-lived •OH and readily forming stable spin adducts DMPO/•OH. BMNP sample was mixed with DMPO in the standard buffer of pH 3.8, and the reaction was triggered by addition of H2O2, then the sample mixtures were transferred into a glass capillary and placed in the ESR cavity. The spectra subtraction between the sample mixtures with and without H2O2 solution was conducted to obtain ESR spectra signal of spin adducts DMPO/•OH. ESR measurements were carried out using Bruker EMX ESR spectrometer (Billerica, MA) at ambient temperature with 20-mW microwave power. Electrochemical analysis of BMNP-modified electrodes The BMNPs (30 mg) were dispersed into distilled water (10 mL) to obtain a suspension of 3 mg mL−1. The glassy carbon electrodes (GCEs, 3.0 mm in diameter) were polished with 0.3- and 0.05-mm alumina slurry, respectively, followed by thoroughly rinsing with water. The sample suspension (100 μL) was mixed with 3 % polytetrafluoroethylene (PTFE), then the colloidal solution (5 μL) was dropped on the pretreated GCE surface and allowed to dry under ambient conditions for 3 h to obtain BMNP-modified electrodes. Cyclic voltammetric and amperometric measurements were performed on CHI 660D (Chenhua, China). A three-electrode system was used, which comprise a platinum wire as auxiliary, a saturated calomel electrode as reference, and the BMNPmodified electrode as working electrodes. All experimental solutions were deoxygenated by bubbling highly pure nitrogen to maintain the nitrogen atmosphere during the measurements process. In amperometric experiments, the current–time data were recorded after a constant residual current and addition of H2O2 solution successively into the buffer solution, which was carried out by applying a potential of −0.6 V on a stirred cell at room temperature.
Kinetic analysis H2O2 and glucose detection Unless otherwise stated, steady state kinetic measurements were carried out in time-drive mode by monitoring the absorbance change at 652 nm on a Lambda 750 UV-Visible-nearinfrared (UV-Vis-NIR) spectrophotometer (PerkinElmer, USA). Experiments were carried out using 30 μg/mL BMNPs in 3-mL reaction buffer solution (200 mM Na2HPO4 ·12H2O and 100 mM citric acid, pH 3.8, 30 °C) in the presence of 0.5 mM TMB and 200 mM H2O2 as substrate. The Michaelis–Menten constant was calculated using Lineweaver–Burk plots of the double reciprocal of the Michaelis–Menten equation, 1/v=(Km /Vmax)·(1/[S])+1/Vmax.
A typical colorimetric analysis was carried out as follows: 60 μL of 25 mM TMB, 100 μL of 0.3 mg/mL BMNPs, and 200 μL of H2O2 with different concentrations were added into 2,640 μL of buffer (200 mM Na2HPO4 ·12H2O and 100 mM citric acid, pH 3.8). Then, the mixed reaction solution was determined using adsorption spectroscopy measurement in time-drive mode. Glucose detection was carried out in two stages: First, 20 μL of 1.0 mg/mL GOx and 20 μL of glucose in different concentrations were added into 100 μL of Na2HPO4 buffer
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solution (pH 7.0), and the glucose reaction solution was incubated at 37 °C for 30 min; second, 200 μL of 5 mM TMB, 200 μL of 0.3 mg/mL BMNPs, and 1,460 μL of buffer solution (200 mM Na2HPO4 ·12H2O and 100 mM citric acid, pH 3.8) were added into the above solution, and the mixed solution was incubated at room temperature for 30 min and determined using adsorption spectroscopy measurement. In control experiments, 5 mM maltose, 5 mM lactose, and 5 mM fructose were determined following the similar way.
Phenol and Congo red dye degradation Phenol degradation was carried out in the following stages. (1) The standard curve determination: 0, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50, 4.00, 4.50, and 5.00 mL of phenol solution of 1 g/L were added in a group of 50-mL colorimetric tubes, respectively, to obtain a series of diluent with 0~100 mg/L; 2 mL phenol dilution was shifted from each tube to 25-mL volumetric flask, followed by adding 2 mL NH4Cl buffer solution, 2 mL 2 % 4-aminoantipyrine reagent (4-AAP), and 1 mL 8 % potassium ferricyanide solution; the mixture was mixed thoroughly after shaking that was allowed to stand for 10 min, then the reaction solution absorbance was determined using adsorption spectroscopy measurement at wavelength of 510 nm with a 1-cm cuvette, setting the blank reagent solution as control. The standard curve was obtained according to the graph of concentration (mg/L) and the corresponding absorbance. (2) Phenol degradation by BMNPs: Experiments were carried out using 30 μg mL−1 BMNPs in 3-mL reaction buffer solution (200 mM Na2HPO4 ·12H2O and 100 mM citric acid, pH 3.8, 30 °C) in the presence of 100 mg/L phenol and 50 mM H2O2 as substrate. The measured samples were taken out from the reaction solution every 2 min and then added into the above phenol test system to obtain the absorbance at the wavelength of 510 nm. The phenol concentration after degradation by BMNPs was determined according to the standard curve. Congo red dye degradation measurement by BMNPs were carried out in time-drive mode by monitoring the absorbance change at 500 nm on a Lambda 750 UV-Vis-NIR spectrophotometer (PerkinElmer, USA). Experiments were carried out using 30 μg mL−1 BMNPs in 3-mL reaction buffer solution (200 mM Na2HPO4 ·12H2O and 100 mM citric acid, pH 3.8, 30 °C) in the presence of 100 mg/L Congo red dye and 50 mM H2O2 as substrate. Degradation rate was determined according to the change in absorbance. When the degradation experiments were finished, BMNPs were separated from the reaction system using the permanent magnet and then reused in the next degradation reaction. The process was repeated seven cycles to characterize the recycling property of the BMNPs.
Nucleotide sequence accession number The newly determined nucleotide sequence in this study is available from GenBank under the accession number KJ162573.
Collection and strain number The newly isolated bacteria strain in this study is available in China Center of Industries Culture Collection (CICC) that belongs to WDCM under the deposited number CICC 10828.
Results MNP-producing strain isolation and identification Transmission electron microscope (TEM) observation (Fig. 1) shows that the cells are short rod in morphology with a mean width of 0.5 μm and a mean length of 1.2 μm. They were uniflagellate bacteria with several flagella at one pole. And, one or two nanoparticles were identified in each cell. The cell yield of YN01 is about 1.14×1011 cells/L (growth medium). Chromosomal DNA was prepared from the bacterial culture, and 16S rDNA genes were amplified for identification as described previously (Schüler et al. 1999). According to a sequence homology analysis of Genbank by BLAST program, this novel MNP-producing bacteria strain was identified as Burkholderia sp., named as YN01.
Fig. 1 TEM image of the bacteria cells of Burkholderia sp. YN01
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Preparation and characterization of the BMNPs extracted from YN01 The magnetic nanoparticles in bacterial cells were separated and purified following procedures of ultrasonication, ultracentrifugation, and magnet adsorption. The BMNP productivity is about 1.89 mg/L (growth medium). The BMNPs were identified as pure face-centered cubic phase of magnetite (Fe3O4) (JCPDS 019-0629) by X-ray diffraction (XRD) shown in Fig. 2a. TEM image (Fig. 3) reveals that the size distribution of the crystals is uniform and the average size is about 80 nm. X-ray photoelectron spectroscopy (XPS) was employed to further explore the composition of the BMNPs purified from YN01. As shown in Fig. 4a, there are three major elements that are Fe 2p, O 1 s, and C 1 s in the surface of BMNPs according to the wide spectrum. The photoelectron peaks at 711.2 and 724.5 eV are the characteristic doublets of Fe 2p3/2 and Fe 2p1/2 according to the narrow spectrum of Fe
Fig. 3 TEM image of the BMNPs
2p (Fig. 4b). The charactersitic peaks at 532.2 and 284.6 eVof O 1 s and C 1 s are shown in Fig. 4c, d. The hysteresis loop of the BMNP samples is potbellied as shown in Fig. 5, and the values of hysteresis parameters such as Bc and Bcr and ratios Mrs/Ms were deduced as 35.6 mT, 43.2 mT, and 0.47, respectively. Peroxidase-like activity of the BMNPs As illustrated in Fig. 6, we found that BMNPs could catalyze the oxidation of peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2 to produce a blue color reaction with maximum absorbance of the reaction mixture at 652 nm (Josephy et al. 1982). In contrast, both the solution of TMB and H2O2 in the absence of BMNPs and the solution of TMB and BMNPs in the absence of H2O2 showed no oxidation reaction, showing that both the components are required for the oxidative reaction, as observed for horseradish peroxidase (HRP) (Josephy et al. 1982; Chattopadhyay and Mazumdar 2000). Additionally, the TMB oxidation rate catalyzed by BMNPs is dependent on their concentration; the reaction rate increased with increasing BMNPs concentration as shown in Fig. 7a. The composition and phase of BMNPs remained unchanged after the peroxidase reaction according to XRD patterns (Fig. 2b). The BMNPs were incubated in the reaction buffer for 10 min and then removed from the solution using a magnet to prepare a leaching solution. Online Resource 1 showed that the leaching solution had no activity. Effect of pH, temperature, and H2O2 concentration on BMNP activity
Fig. 2 XRD patterns of BMNPs before a and after b the catalytic reaction
The peroxidase-like activity of BMNPs was measured while varying the pH from 1 to 8, the temperature from 25 to 60 °C, and H2O2 concentration from 0.001 to 2 M. The maximum
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Fig. 4 XPS of the BMNPs. a XPS wide spectrum of BMNPs. b XPS narrow spectrum of Fe 2p. c XPS narrow spectrum of O 1 s. d XPS narrow spectrum of C 1 s
catalytic activity of BMNPs was obtained under the following optimal conditions: pH 3.8, 30 °C, 200 mM H2O2; the reaction time was set as 5 min. (Fig. 7b–d). Kinetic analysis of BMNPs activity The peroxidase-like catalytic activity of BMNPs was investigated using steady state kinetics, and the typical Michaelis– Menten curves are shown in Fig. 8a, b. The kinetic data were obtained by varying one substrate concentration while keeping the other substrate concentration constant. A series of initial reaction rates were calculated and applied to the double reciprocal of the Michaelis–Menten equation, 1/v=(Km / Vmax)·(1/[S])+1/Vmax, where v is the initial velocity, [S] is the concentration of the substrate, Km is the Michaelis– Menten constant, and Vmax is the maximal reaction velocity. The Km, Vmax, and Kcat values (Online Resource 6) were obtained using Lineweaver–Burk plots. To further investigate the mechanism of BMNP catalysis, their activity over a range of TMB and H2O2 concentrations was measured. The doublereciprocal plots of initial velocity against the concentration of one substrate were obtained over a range of concentrations of the other (Fig. 8c, d).
Study of free radical formation by ESR ESR technique was employed to investigate the possible mechanism by determining free radical formation induced by BMNPs in the presence of H2O2. In the results of this study, the ESR spectra in the presence of BMNPs displayed a fourfold characteristic peak of the typical DMPO/•OH adduct with an intensity ratio of 1:2:2:1. There is no DMPO/•OH adduct signal intensity in the control experiment in the absence of BMNPs (Fig. 9).
Electrochemical analysis of the origin of BMNP activity The electrocatalytic behavior of BMNP-modified glassy carbon electrode (GCE) toward reduction of H2O2 was examined using cyclic voltammetry in standard conditions, demonstrating clearly that no obvious current was found in the absence of H2O2, but an obvious current was observed in the presence of H2O2 (Fig. 10a). The amperometric response of the BMNPmodified GCE to H2O2 is shown in Fig. 10b. The reduction current increased steeply to reach a steady state value upon addition of an aliquot of H2O2 to the buffer solution.
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Fig. 5 Room temperature hysteresis loop of the BMNPs
Comparison of stability of peroxidase activity of BMNPs and HRP To examine and compare the robustness of peroxidase activity of BMNPs and HRP, both BMNPs and HRP were exposed to
a range of values of pH and a range of temperatures for 2 h, and then their activities under standard conditions (pH 3.8 and 30 °C) were measured. The catalytic activity of HRP was largely inhibited after treatment at pH lower than 4 or temperature higher than 50 °C (Online Resource 2a, b). In contrast, the catalytic activity of BMNPs remained stable over a wide range of pH from 2 to 12 and temperature from 4 to 70 °C (Online Resource 2a, b). H2O2 and glucose detection
Fig. 6 Time-dependent absorbance changes at 652 nm of TMB in different reaction systems: TMB+H2O2 (a), TMB+BMNPs (b), and TMB+BMNPs+H2O2 (c). Reaction conditions were 0.5 mM TMB, 20 μg mL−1 BMNPs, 200 mM Na2HPO4 ·12H2O, and 100 mM citric acid buffer, pH 3.8 at 30 °C
The absorbance at 652 nm is proportional to H2O2 concentrations from 0.01 to 8 mM with a detection limit (DL) of 0.005 mM (Online Resource 3a). Because H2O2 is the main product of glucose oxidase (GOx)-catalyzed reaction, when the catalytic reaction by BMNPs is combined with the glucose catalytic reaction by GOx, the proposed colorimetric method could be used to determine glucose. The linear range for glucose is from 0.01 to 5 mM and the DL is 0.005 mM (Online Resource 3b), which can be potentially applied to detect glucose in diluted serum. The specificity to glucose of the proposed colorimetric method was investigated using fructose, lactose, and maltose. The results demonstrated that these glucose analogs were even at a concentration as high as 5 mM, and no detectable signals of the control samples was observed compared with that of glucose (Online Resource 4).
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Fig. 7 Dependency of peroxidase-like activity on BMNP concentration (a), pH (b), temperature (c), and H2O2 concentration (d). a The oxidation activity rate of BMNPs was determined in 200 mM Na2HPO4 ·12H2O and 100 mM citric acid buffer (pH 3.8) with a series of BMNP concentrations that are 0, 10, 15, 20, 25, 30 μg mL−1, respectively (from a to f);
TMB (0.5 mM ) and H2O2 (150 mM) were added to initiate the reaction. b–d Experiments were carried out using 20 μg mL−1 BMNPs in the reaction buffer above with 0.5 mM TMB as substrate. The H2O2 concentration was 150 mM at 30 °C unless otherwise stated. The maximum point in each curve was set as 100 %
Phenol and Congo red dye degradation
Discussion
As shown in Online Resource 5a, the BMNPs were able to efficiently degrade phenol at pH 3.8 and temperature 30 °C; about 68 % of phenol was degraded after 25 min by 100 mg/L BMNPs, and the phenol degradation rate increased with increasing time, and the time to achieve the same degradation rate was significantly reduced with the increasing of BMNP concentration. Online Resource 5b demonstrated that nearly 60 % degradation for Congo red dye by 200 mg/mL BMNPs was observed during 10 min, and the degradation rate was also BMNP concentration dependent. The effect of BMNP recycling times on degradation rate was demonstrated in Online Resource 5c; the degradation rate gradually decreased with increasing BMNP recycling time. When the BMNPs had been reused for seven times, the degradation rate of both phenol and Congo red decreased by about 15 % compared with that in the first time, which might be due to the loss of BMNPs in the recycling and washing processes.
It is well known that magnetic nanoparticles (MNPs) are of particular interest because of their dual functionality as a peroxidase mimetic and a magnetic separation agent (Gao et al. 2007). Recently, biosynthesis of MNPs has become a useful technique in the place of traditional chemical procedures which are known to employ high temperatures and pressure, and hazardous organic solvents. In contrast, the development of eco-friendly and green synthesis approaches to synthesize MNPs using microorganisms has gained considerable attention although microbial synthesis still has some challenges to overcome and some issues to explore, such as the better control of nanoparticle sizes and shapes (Durán and Seabra 2012; Seabra et al. 2013). Some microorganisms are believed to produce ferromagnetic nanoparticles with singledomain and uniform particle size, for instance, magnetotactic bacteria (Bazylinski and Frankel 2004; Matsunaga et al. 2007; Dutz et al. 2009) and non-magnetotactic bacteria (Vainshtein
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Fig. 8 Steady state kinetic assay of BMNPs. a The concentration of TMB was 0.5 mM and the H2O2 concentration was varied. b The concentration of H2O2 was 150 mM and the TMB concentration was
varied. c, d Double-reciprocal plots of activity of BMNPs at a fixed concentration of one substrate versus varying concentration of the second substrate for H2O2 and TMB
et al. 2002). Compared with AMNPs, the biogenic MNPs would be expected to show superior performances (Knopp et al. 2009). Here, we have isolated a novel MNP-producing bacteria strain identified as Burkholderia sp. YN01; extracted MNPs from YN01 were characterized as face-centered cubic 80 nm Fe3O4 with ferromagnetic behavior. According to the XRD patterns of the BMNPs from YN01, the seven characteristic diffraction peaks in the range of 15
BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Biogenic magnetic nanoparticles from Burkholderia sp. YN01 exhibiting intrinsic peroxidase-like activity and their applications Yu Pan & Na Li & Jianshuai Mu & Runhong Zhou & Yan Xu & Daizong Cui & Yan Wang & Min Zhao
Received: 21 May 2014 / Revised: 2 July 2014 / Accepted: 5 July 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract A novel bacterial strain containing biogenic magnetic nanoparticles (BMNPs) was isolated from the sediments of Songhua River in Harbin, China, and was identified as Burkholderia sp. YN01. Extracted BMNPs from YN01 were characterized as pure face-centered cubic Fe3O4 with an average size of 80 nm through transmission electron microscope (TEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The hysteresis parameters of the BMNP samples such as Bc and Bcr and ratios Mrs/Ms were deduced as 35.6 mT, 43.2 mT, and 0.47, respectively, indicating that the BMNPs exhibit a ferromagnetic behavior. This is the first report concerning on biogenic Fe3O4 NPs produced in Burkholderia genus. Significantly, the BMNPs were proved to possess intrinsic peroxidase-like activity that could catalyze the o xidation of p eroxidase substrate 3,3′ ,5 ,5′ tetramethylbenzidine (TMB) in the presence of H2O 2. Kinetic analysis indicates that the catalytic behavior is in accord with typical Michaelis–Menten kinetics and follows ping-pong mechanism. The catalytic constants (Kcat) were 6.5×104 s−1 and 0.78×104 s−1 with H2O2 and TMB as substrate, respectively, which was higher than that of horseradish
Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-5938-6) contains supplementary material, which is available to authorized users. Y. Pan : N. Li : R. Zhou : Y. Xu : D. Cui : M. Zhao (*) College of Life Science, Northeast Forestry University, No. 26, Hexing Road, Harbin 150040, China e-mail: [email protected] J. Mu : Y. Wang Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, Harbin 150001, China Y. Pan School of Environment and Chemical Engineering, Heilongjiang University of Science and Technology, Harbin 150022, China
peroxidase (HRP). Electron spin resonance (ESR) spectroscopy experiments showed that the BMNPs could catalyze H2O2 to produce hydroxyl radicals. The origin of peroxidase-like activity is also associated with their ability to transfer electron between electrode and H2O2 according to an electrochemical study. As a novel peroxidase mimetic, the BMNPs were employed to offer a simple, sensitive, and selective colorimetric method for H2O2 and glucose determination, and the BMNPs could efficiently catalyze the degradation of phenol and Congo red dye. Keywords Biogenic magnetic nanoparticles . Burkholderia . Peroxidase mimetic . Glucose determination . Degradation
Introduction Nanomaterials have attracted considerable attention over the past few decades due to their unique size, shape, optical electric, chemical, and mechanical properties (Lee et al. 2006; Khalavka et al. 2009). Recently, many nanomaterials have been evaluated as oxidase, peroxidase, or catalase mimetics with low cost, increased stability, high tunability, sustainable electrocatalytic activity, and robustness to harsh environments (Wei and Wang 2013), which can be potentially used in bioassays and environmental chemistry (Gao et al. 2007; Dai et al. 2009; André et al. 2010; Mu et al. 2012; Chaudhari et al. 2012; Song et al. 2010; Shi et al. 2011a, b). Among known nanomaterials with enzyme activities, magnetic nanoparticles (MNPs) are of particular interest in magnetic resonance imaging (MRI) (Brahler et al. 2006), magnetic biosensor (Chemla et al. 2000), and wastewater treatment (Ambashta and Sillanpaa 2010), or as detection tools (Liu et al. 2012; Yua et al. 2009) because of their dual functionality as a peroxidase mimetic and a magnetic separation agent. And, ferruginous bodies containing magnetite within ferritin
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coating were also verified to exert peroxidase-like activity (Borelli et al. 2012). Various synthetic methods have been described for the synthesis of Fe3O4 NPs, such as the sol-gel method, the reverse micellar method, and the thermal decomposition of organometallic iron precursors using organic solvents at high temperatures. These synthetic approaches involve high temperatures and toxic reagents, which are both expensive and environmentally undesirable (Seabra et al. 2013). In recent years, green nanotechnology has attracted increasing attention because of the possibility to reduce or eliminate toxic substance; therefore, microbial synthesis of MNPs is considered as an eco-friendly process, since it occurs in water, at room temperature and pressure, and very close to neutral pH (Durán and Seabra 2012). Many studies revealed that biogenic MNPs (BMNPs) can be formed by numerous microorganisms, such as bacteria and fungi (Seabra et al. 2013; Mandal et al. 2006; Roh et al. 2001). Bacterial magnetosomes are natural inorganic ferromagnetic nanoparticles within the single-domain size range of 35~120 nm biomineralized in magnetotactic bacteria (Blakemore 1975; Bazylinski and Frankel 2004; Alphandéry et al. 2012). Besides magnetotactic bacteria, BMNPs were also found in non-magnetotactic bacteria (Vainshtein et al. 2002). This biogenic MNPs would be expected to show superior performances of low environmental cost, high chemical purity, fine and uniform particle size, good dispersity, and biocompatibility without surface modification (Knopp et al. 2009), which make them a promising magnetic nanomaterial in biomolecule immobilization, drug and gene targeting, and wastewater treatment (Matsunaga et al. 2007; Dutz et al. 2009). However, only very few bacteria strains that can produce MNPs are available in pure culture owing to their fastidious growth requirements and strong metabolic diversity (Flies et al. 2005). Many studies focus on biomineralization process in cells, magnetism of magnetosomes (Bazylinski and Frankel 2004; Calugay et al. 2004; Gorby et al. 1988), and their ability to eliminate intracellular reactive oxygen species (Guo et al. 2012); however, to date, the potential of BMNPs from bacteria cells as enzyme mimetics and their applications have not been explored. In this paper, the purification and characterization of biogenic Fe3O4 MNPs from a newly isolated bacteria strain (designated Burkholderia sp. YN01) were described. Significantly, these BMNPs were proved to possess peroxidase-like activity, and the catalytic mechanisms and origin of their intrinsic peroxidase-like activity were studied by kinetic and electrochemical analysis and electron spin resonance (ESR) technique. The BMNPs were also tested for their peroxidase-like activity in glucose determination and degradation of phenol and Congo red dye.
Materials and methods Materials All chemicals used in this work were of analytical grade and used as received without further purification. Bacterial DNA kit and gel extraction kit were purchased from Omega Bio-Tek (Norcross, GA, USA). Taq DNA polymerase, primers, and pMD 18-T vector were obtained from TaKaRa (Dalian, China). 3,3′,5,5′-Tetramethylbenzidine (TMB), horseradish peroxidase (EC 1.11.1.17, 250–330 U/mg using pyrogallol, type VI-A), glucose oxidase (GOx, from Aspergillus niger, ≥100 U/mg), and DMPO (5,5-dimethyl-1-pyrroline N-oxide) were purchased from Sigma-Aldrich (St. Louis, USA). H2O2 and other regents were obtained from Boyue Biological Reagent Co. (Harbin, China). Glucose, fructose, lactose, and maltose were purchased from Beijing Chemical Reagent Company (Beijing, China). Isolation and identification of MNP-producing strain Samples of freshwater and sediment are collected from Songhua River, which is located in Harbin, China. Sediment and water in proportion of 1:3 (vol/vol) were stored in loosely capped bottles. Each liter of the sample was supplemented with 1.0 g sodium succinate, 0.1 g NaNO3, 0.2 g sodium acetate, 0.05 g sodium thioglycollate, 3.5 mL 0.01 M ferric citrate solution, 10 mL Wolfe’s vitamin solution, and 5.0 mL Wolfe’s mineral solution, and then was left undisturbed for 1 month at room temperature under dim light for enrichment culture. After enrichment, the bacteria culture was precipitated by centrifugation then resuspended in distilled water. Then, 0.5-mL cell suspension was placed on the sterile flat dish. Then, sterile semi-solid isolation medium (IM) supplemented with 0.74 g sodium succinate, 0.1 g NaNO3, 0.2 g sodium acetate, 0.05 g sodium thioglycollate, 5.0 mL 0.01 M ferric citrate solution, 10 mL Wolfe’s vitamin solution, 5.0 mL Wolfe’s mineral solution, and 1.2 % (g/vol) agar was poured to each plate, mixed equably, and cultivated for 7 days at 28 °C. The monocolonies isolated by picking up colonies from the agar plates were inoculated in IM, followed by stab-cultivating in semi-solid IM in tubes for 7 days at 28 °C. The process was repeated two times before monocolonies were considered as pure. The purified bacteria cells were investigated under transmission electron microscopy (TEM, FEI/Philips TCNAI G2) at an accelerating voltage of 80 kV and direct magnification of×30,000. 16S ribosomal DNA (16S rDNA) genes of the obtained bacteria were amplified between positions 27 and 1492 (Escherichia coli 16S rRNA gene sequence numbers), using primers 27 F (5′-AGAGTYTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTI‘GTI’ ACGACTT-3′) by polymerase chain reaction (PCR) carried out with the following cycle: an
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initial denaturing step at 94 °C for 5 min, followed by 25 cycles of 1 min at 94 °C, 45 s at 50 °C, and 1 min at 72 °C, and a final extension step of 10 min at 72 °C. Then, the PCR products were cloned using a commercially available pGEM-T vector cloning kit and subsequently sequenced by Shenggong Company in Shanghai. The obtained sequences of the 16S rDNA gene (about 1.5 kb) were analyzed using aligning tool of BLAST program (http://www.ncbi.nlm.nih. gov/BLAST/).
Preparation and characterization of BMNPs The purified bacterial cells were inoculated in a 250-mL bottle containing 200-mL growth medium. After 2-day incubation, the culture from medium was harvested as described above and lysed by ultrasonication for 15 min. The magnetic particles released from the bacterial cells were collected by permanent magnet. The resulting black solid products were washed with water three times then separated and collected by magnet for characterization. The composition and phase of BMNPs were identified by powder X-ray diffraction (XRD) on an D/max-rB X-ray diffractometer (Rigaku, Japan) using Cu Kα radiation (λ=1.5418 Å). The morphology and size of BMNPs were examined by transmission electron microscopy (TEM, FEI/Philips TCNAI G2) at an accelerating voltage of 80 kV and direct magnification of×80,000. X-ray photoelectron spectroscopy (XPS) analysis was conducted with an AXIS ULTRA PLD spectrometer (Kratos Co.) using Al as the exciting source at 1,486.4 eV. The polluted carbon was used in charged correction of binding energy of the BMNP sample at 284.6 eV. Room temperature magnetic experiments were performed on a vibrating sample magnetometer (Model 3900, Princeton Measurements Corporation, sensitivity is 5.0×10−10 Am2). The hysteresis loop was measured between +500 and −500 mT with an average time of 400 ms. Saturation magnetization (Ms) and saturation remanence (Mrs) were determined after correction for paramagnetic phases.
ESR spectroscopy measurements ESR technique was used to detect hydroxyl radicals (•OH) formed during the decomposition of H 2O2 induced by BMNPs. Because of its diamagnetic property, 5,5-dimethyl1-pyrroline N-oxide (DMPO) is capable of trapping these short-lived •OH and readily forming stable spin adducts DMPO/•OH. BMNP sample was mixed with DMPO in the standard buffer of pH 3.8, and the reaction was triggered by addition of H2O2, then the sample mixtures were transferred into a glass capillary and placed in the ESR cavity. The spectra subtraction between the sample mixtures with and without H2O2 solution was conducted to obtain ESR spectra signal of spin adducts DMPO/•OH. ESR measurements were carried out using Bruker EMX ESR spectrometer (Billerica, MA) at ambient temperature with 20-mW microwave power. Electrochemical analysis of BMNP-modified electrodes The BMNPs (30 mg) were dispersed into distilled water (10 mL) to obtain a suspension of 3 mg mL−1. The glassy carbon electrodes (GCEs, 3.0 mm in diameter) were polished with 0.3- and 0.05-mm alumina slurry, respectively, followed by thoroughly rinsing with water. The sample suspension (100 μL) was mixed with 3 % polytetrafluoroethylene (PTFE), then the colloidal solution (5 μL) was dropped on the pretreated GCE surface and allowed to dry under ambient conditions for 3 h to obtain BMNP-modified electrodes. Cyclic voltammetric and amperometric measurements were performed on CHI 660D (Chenhua, China). A three-electrode system was used, which comprise a platinum wire as auxiliary, a saturated calomel electrode as reference, and the BMNPmodified electrode as working electrodes. All experimental solutions were deoxygenated by bubbling highly pure nitrogen to maintain the nitrogen atmosphere during the measurements process. In amperometric experiments, the current–time data were recorded after a constant residual current and addition of H2O2 solution successively into the buffer solution, which was carried out by applying a potential of −0.6 V on a stirred cell at room temperature.
Kinetic analysis H2O2 and glucose detection Unless otherwise stated, steady state kinetic measurements were carried out in time-drive mode by monitoring the absorbance change at 652 nm on a Lambda 750 UV-Visible-nearinfrared (UV-Vis-NIR) spectrophotometer (PerkinElmer, USA). Experiments were carried out using 30 μg/mL BMNPs in 3-mL reaction buffer solution (200 mM Na2HPO4 ·12H2O and 100 mM citric acid, pH 3.8, 30 °C) in the presence of 0.5 mM TMB and 200 mM H2O2 as substrate. The Michaelis–Menten constant was calculated using Lineweaver–Burk plots of the double reciprocal of the Michaelis–Menten equation, 1/v=(Km /Vmax)·(1/[S])+1/Vmax.
A typical colorimetric analysis was carried out as follows: 60 μL of 25 mM TMB, 100 μL of 0.3 mg/mL BMNPs, and 200 μL of H2O2 with different concentrations were added into 2,640 μL of buffer (200 mM Na2HPO4 ·12H2O and 100 mM citric acid, pH 3.8). Then, the mixed reaction solution was determined using adsorption spectroscopy measurement in time-drive mode. Glucose detection was carried out in two stages: First, 20 μL of 1.0 mg/mL GOx and 20 μL of glucose in different concentrations were added into 100 μL of Na2HPO4 buffer
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solution (pH 7.0), and the glucose reaction solution was incubated at 37 °C for 30 min; second, 200 μL of 5 mM TMB, 200 μL of 0.3 mg/mL BMNPs, and 1,460 μL of buffer solution (200 mM Na2HPO4 ·12H2O and 100 mM citric acid, pH 3.8) were added into the above solution, and the mixed solution was incubated at room temperature for 30 min and determined using adsorption spectroscopy measurement. In control experiments, 5 mM maltose, 5 mM lactose, and 5 mM fructose were determined following the similar way.
Phenol and Congo red dye degradation Phenol degradation was carried out in the following stages. (1) The standard curve determination: 0, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50, 4.00, 4.50, and 5.00 mL of phenol solution of 1 g/L were added in a group of 50-mL colorimetric tubes, respectively, to obtain a series of diluent with 0~100 mg/L; 2 mL phenol dilution was shifted from each tube to 25-mL volumetric flask, followed by adding 2 mL NH4Cl buffer solution, 2 mL 2 % 4-aminoantipyrine reagent (4-AAP), and 1 mL 8 % potassium ferricyanide solution; the mixture was mixed thoroughly after shaking that was allowed to stand for 10 min, then the reaction solution absorbance was determined using adsorption spectroscopy measurement at wavelength of 510 nm with a 1-cm cuvette, setting the blank reagent solution as control. The standard curve was obtained according to the graph of concentration (mg/L) and the corresponding absorbance. (2) Phenol degradation by BMNPs: Experiments were carried out using 30 μg mL−1 BMNPs in 3-mL reaction buffer solution (200 mM Na2HPO4 ·12H2O and 100 mM citric acid, pH 3.8, 30 °C) in the presence of 100 mg/L phenol and 50 mM H2O2 as substrate. The measured samples were taken out from the reaction solution every 2 min and then added into the above phenol test system to obtain the absorbance at the wavelength of 510 nm. The phenol concentration after degradation by BMNPs was determined according to the standard curve. Congo red dye degradation measurement by BMNPs were carried out in time-drive mode by monitoring the absorbance change at 500 nm on a Lambda 750 UV-Vis-NIR spectrophotometer (PerkinElmer, USA). Experiments were carried out using 30 μg mL−1 BMNPs in 3-mL reaction buffer solution (200 mM Na2HPO4 ·12H2O and 100 mM citric acid, pH 3.8, 30 °C) in the presence of 100 mg/L Congo red dye and 50 mM H2O2 as substrate. Degradation rate was determined according to the change in absorbance. When the degradation experiments were finished, BMNPs were separated from the reaction system using the permanent magnet and then reused in the next degradation reaction. The process was repeated seven cycles to characterize the recycling property of the BMNPs.
Nucleotide sequence accession number The newly determined nucleotide sequence in this study is available from GenBank under the accession number KJ162573.
Collection and strain number The newly isolated bacteria strain in this study is available in China Center of Industries Culture Collection (CICC) that belongs to WDCM under the deposited number CICC 10828.
Results MNP-producing strain isolation and identification Transmission electron microscope (TEM) observation (Fig. 1) shows that the cells are short rod in morphology with a mean width of 0.5 μm and a mean length of 1.2 μm. They were uniflagellate bacteria with several flagella at one pole. And, one or two nanoparticles were identified in each cell. The cell yield of YN01 is about 1.14×1011 cells/L (growth medium). Chromosomal DNA was prepared from the bacterial culture, and 16S rDNA genes were amplified for identification as described previously (Schüler et al. 1999). According to a sequence homology analysis of Genbank by BLAST program, this novel MNP-producing bacteria strain was identified as Burkholderia sp., named as YN01.
Fig. 1 TEM image of the bacteria cells of Burkholderia sp. YN01
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Preparation and characterization of the BMNPs extracted from YN01 The magnetic nanoparticles in bacterial cells were separated and purified following procedures of ultrasonication, ultracentrifugation, and magnet adsorption. The BMNP productivity is about 1.89 mg/L (growth medium). The BMNPs were identified as pure face-centered cubic phase of magnetite (Fe3O4) (JCPDS 019-0629) by X-ray diffraction (XRD) shown in Fig. 2a. TEM image (Fig. 3) reveals that the size distribution of the crystals is uniform and the average size is about 80 nm. X-ray photoelectron spectroscopy (XPS) was employed to further explore the composition of the BMNPs purified from YN01. As shown in Fig. 4a, there are three major elements that are Fe 2p, O 1 s, and C 1 s in the surface of BMNPs according to the wide spectrum. The photoelectron peaks at 711.2 and 724.5 eV are the characteristic doublets of Fe 2p3/2 and Fe 2p1/2 according to the narrow spectrum of Fe
Fig. 3 TEM image of the BMNPs
2p (Fig. 4b). The charactersitic peaks at 532.2 and 284.6 eVof O 1 s and C 1 s are shown in Fig. 4c, d. The hysteresis loop of the BMNP samples is potbellied as shown in Fig. 5, and the values of hysteresis parameters such as Bc and Bcr and ratios Mrs/Ms were deduced as 35.6 mT, 43.2 mT, and 0.47, respectively. Peroxidase-like activity of the BMNPs As illustrated in Fig. 6, we found that BMNPs could catalyze the oxidation of peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2 to produce a blue color reaction with maximum absorbance of the reaction mixture at 652 nm (Josephy et al. 1982). In contrast, both the solution of TMB and H2O2 in the absence of BMNPs and the solution of TMB and BMNPs in the absence of H2O2 showed no oxidation reaction, showing that both the components are required for the oxidative reaction, as observed for horseradish peroxidase (HRP) (Josephy et al. 1982; Chattopadhyay and Mazumdar 2000). Additionally, the TMB oxidation rate catalyzed by BMNPs is dependent on their concentration; the reaction rate increased with increasing BMNPs concentration as shown in Fig. 7a. The composition and phase of BMNPs remained unchanged after the peroxidase reaction according to XRD patterns (Fig. 2b). The BMNPs were incubated in the reaction buffer for 10 min and then removed from the solution using a magnet to prepare a leaching solution. Online Resource 1 showed that the leaching solution had no activity. Effect of pH, temperature, and H2O2 concentration on BMNP activity
Fig. 2 XRD patterns of BMNPs before a and after b the catalytic reaction
The peroxidase-like activity of BMNPs was measured while varying the pH from 1 to 8, the temperature from 25 to 60 °C, and H2O2 concentration from 0.001 to 2 M. The maximum
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Fig. 4 XPS of the BMNPs. a XPS wide spectrum of BMNPs. b XPS narrow spectrum of Fe 2p. c XPS narrow spectrum of O 1 s. d XPS narrow spectrum of C 1 s
catalytic activity of BMNPs was obtained under the following optimal conditions: pH 3.8, 30 °C, 200 mM H2O2; the reaction time was set as 5 min. (Fig. 7b–d). Kinetic analysis of BMNPs activity The peroxidase-like catalytic activity of BMNPs was investigated using steady state kinetics, and the typical Michaelis– Menten curves are shown in Fig. 8a, b. The kinetic data were obtained by varying one substrate concentration while keeping the other substrate concentration constant. A series of initial reaction rates were calculated and applied to the double reciprocal of the Michaelis–Menten equation, 1/v=(Km / Vmax)·(1/[S])+1/Vmax, where v is the initial velocity, [S] is the concentration of the substrate, Km is the Michaelis– Menten constant, and Vmax is the maximal reaction velocity. The Km, Vmax, and Kcat values (Online Resource 6) were obtained using Lineweaver–Burk plots. To further investigate the mechanism of BMNP catalysis, their activity over a range of TMB and H2O2 concentrations was measured. The doublereciprocal plots of initial velocity against the concentration of one substrate were obtained over a range of concentrations of the other (Fig. 8c, d).
Study of free radical formation by ESR ESR technique was employed to investigate the possible mechanism by determining free radical formation induced by BMNPs in the presence of H2O2. In the results of this study, the ESR spectra in the presence of BMNPs displayed a fourfold characteristic peak of the typical DMPO/•OH adduct with an intensity ratio of 1:2:2:1. There is no DMPO/•OH adduct signal intensity in the control experiment in the absence of BMNPs (Fig. 9).
Electrochemical analysis of the origin of BMNP activity The electrocatalytic behavior of BMNP-modified glassy carbon electrode (GCE) toward reduction of H2O2 was examined using cyclic voltammetry in standard conditions, demonstrating clearly that no obvious current was found in the absence of H2O2, but an obvious current was observed in the presence of H2O2 (Fig. 10a). The amperometric response of the BMNPmodified GCE to H2O2 is shown in Fig. 10b. The reduction current increased steeply to reach a steady state value upon addition of an aliquot of H2O2 to the buffer solution.
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Fig. 5 Room temperature hysteresis loop of the BMNPs
Comparison of stability of peroxidase activity of BMNPs and HRP To examine and compare the robustness of peroxidase activity of BMNPs and HRP, both BMNPs and HRP were exposed to
a range of values of pH and a range of temperatures for 2 h, and then their activities under standard conditions (pH 3.8 and 30 °C) were measured. The catalytic activity of HRP was largely inhibited after treatment at pH lower than 4 or temperature higher than 50 °C (Online Resource 2a, b). In contrast, the catalytic activity of BMNPs remained stable over a wide range of pH from 2 to 12 and temperature from 4 to 70 °C (Online Resource 2a, b). H2O2 and glucose detection
Fig. 6 Time-dependent absorbance changes at 652 nm of TMB in different reaction systems: TMB+H2O2 (a), TMB+BMNPs (b), and TMB+BMNPs+H2O2 (c). Reaction conditions were 0.5 mM TMB, 20 μg mL−1 BMNPs, 200 mM Na2HPO4 ·12H2O, and 100 mM citric acid buffer, pH 3.8 at 30 °C
The absorbance at 652 nm is proportional to H2O2 concentrations from 0.01 to 8 mM with a detection limit (DL) of 0.005 mM (Online Resource 3a). Because H2O2 is the main product of glucose oxidase (GOx)-catalyzed reaction, when the catalytic reaction by BMNPs is combined with the glucose catalytic reaction by GOx, the proposed colorimetric method could be used to determine glucose. The linear range for glucose is from 0.01 to 5 mM and the DL is 0.005 mM (Online Resource 3b), which can be potentially applied to detect glucose in diluted serum. The specificity to glucose of the proposed colorimetric method was investigated using fructose, lactose, and maltose. The results demonstrated that these glucose analogs were even at a concentration as high as 5 mM, and no detectable signals of the control samples was observed compared with that of glucose (Online Resource 4).
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Fig. 7 Dependency of peroxidase-like activity on BMNP concentration (a), pH (b), temperature (c), and H2O2 concentration (d). a The oxidation activity rate of BMNPs was determined in 200 mM Na2HPO4 ·12H2O and 100 mM citric acid buffer (pH 3.8) with a series of BMNP concentrations that are 0, 10, 15, 20, 25, 30 μg mL−1, respectively (from a to f);
TMB (0.5 mM ) and H2O2 (150 mM) were added to initiate the reaction. b–d Experiments were carried out using 20 μg mL−1 BMNPs in the reaction buffer above with 0.5 mM TMB as substrate. The H2O2 concentration was 150 mM at 30 °C unless otherwise stated. The maximum point in each curve was set as 100 %
Phenol and Congo red dye degradation
Discussion
As shown in Online Resource 5a, the BMNPs were able to efficiently degrade phenol at pH 3.8 and temperature 30 °C; about 68 % of phenol was degraded after 25 min by 100 mg/L BMNPs, and the phenol degradation rate increased with increasing time, and the time to achieve the same degradation rate was significantly reduced with the increasing of BMNP concentration. Online Resource 5b demonstrated that nearly 60 % degradation for Congo red dye by 200 mg/mL BMNPs was observed during 10 min, and the degradation rate was also BMNP concentration dependent. The effect of BMNP recycling times on degradation rate was demonstrated in Online Resource 5c; the degradation rate gradually decreased with increasing BMNP recycling time. When the BMNPs had been reused for seven times, the degradation rate of both phenol and Congo red decreased by about 15 % compared with that in the first time, which might be due to the loss of BMNPs in the recycling and washing processes.
It is well known that magnetic nanoparticles (MNPs) are of particular interest because of their dual functionality as a peroxidase mimetic and a magnetic separation agent (Gao et al. 2007). Recently, biosynthesis of MNPs has become a useful technique in the place of traditional chemical procedures which are known to employ high temperatures and pressure, and hazardous organic solvents. In contrast, the development of eco-friendly and green synthesis approaches to synthesize MNPs using microorganisms has gained considerable attention although microbial synthesis still has some challenges to overcome and some issues to explore, such as the better control of nanoparticle sizes and shapes (Durán and Seabra 2012; Seabra et al. 2013). Some microorganisms are believed to produce ferromagnetic nanoparticles with singledomain and uniform particle size, for instance, magnetotactic bacteria (Bazylinski and Frankel 2004; Matsunaga et al. 2007; Dutz et al. 2009) and non-magnetotactic bacteria (Vainshtein
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Fig. 8 Steady state kinetic assay of BMNPs. a The concentration of TMB was 0.5 mM and the H2O2 concentration was varied. b The concentration of H2O2 was 150 mM and the TMB concentration was
varied. c, d Double-reciprocal plots of activity of BMNPs at a fixed concentration of one substrate versus varying concentration of the second substrate for H2O2 and TMB
et al. 2002). Compared with AMNPs, the biogenic MNPs would be expected to show superior performances (Knopp et al. 2009). Here, we have isolated a novel MNP-producing bacteria strain identified as Burkholderia sp. YN01; extracted MNPs from YN01 were characterized as face-centered cubic 80 nm Fe3O4 with ferromagnetic behavior. According to the XRD patterns of the BMNPs from YN01, the seven characteristic diffraction peaks in the range of 15
