Chloroperoxidase (CPO) from Caldariomyces fumago could mediate a very efficient oxidative decolorization/degradation of aqueous azo dye. The decolorization efficiencies for Congo Red and Alizarin Yellow R reached 88.25% in 3 min and 92.38% in 5 min, respectively, under mild conditions at enzyme concentrations of 0.025 and 0.03 μmol L–1, respectively. Compared with the typical mechanism involved in oxidative decolorization of azo dyes catalyzed by other peroxidases, a different enzymatic catalytic cycle was found in this work. The oxidative decolorization was carried out in bulk solution, which would get rid of the restriction of substrate required by the channel access to the substrate pocket in the enzymatic active site. Nine degradation products of Congo Red were detected by HPLC–MS analysis, and two types of degradation pathways were proposed accordingly. The strong toleration of the typical salt species in industrial effluents by this system ensured that it has potential applications in treatment of industrial wastewater.

•Chloroperoxidase(CPO)-TiO2 composite combines enzyme and photo catalysis in one pot.A chloroperoxidase (CPO) -TiO2 thin film type enzyme reactor was constructed by single chloroperoxidase molecule immobilization on TiO2 mesoporous thin film. The CPO-TiO2 enzyme reactor showed an enzymatic-photocatalytic synergetic effect, 1 + 1 > 2, when applied in the decolorization of azo dye crocein orange G. Moreover, the enzyme reactor made it possible that the photocatalytic catalysis was carried out under natural sunlight instead of ultraviolet radiation. The synergetic effect was studied by PL emission spectroscopy with terephthalic acid used as a probe molecule to investigate the formation of ·OH radicals.The mesoporous TiO2, prepared by a sol-gel and dip-coating method, being spherical form without interconnectivity in a 3D pore systems of 8 nm ~ 15 nm pore size, matched the size of CPO molecules. Such an architecture was suitable for single CPO molecule immobilization, which enabled easy access of the substrates to enzyme active sites.The CPO-TiO2 enzyme reactor was very efficient. 98.61% decolorization efficiency of crocein orange G was achieved in 5 min. It can be reused for seven cycles with no loss in activity. Compared with free CPO, the stability of CPO-TiO2 enzyme reactor against thermal denaturation and deactivation by high concentration of oxidants or in a strong acid-base environment was enhanced.Download high-res image (205KB)Download full-size image

•RhCo/CB nanohybrids are synthesized by a co-precipitation/co-reduction method.•RhCo nanocrystals show the high alloying degree and good dispersion.•RhCo/CB nanohybrids exhibit a composition-dependent catalytic activity for the MOR.•Rh3Co1/CB nanohybrids reveal the higher MOR activity than Pt/C electrocatalyst.Although both Pd and Rh fall into the Pd-group Pt-group elements (PPGEs), the electrocatalytic property of Rh nanocrystals for the methanol oxidation reaction (MOR) in alkaline media is rarely explored till now. In this work, we develop a surfactant-free co-precipitation/co-reduction method to synthesize the carbon ball supported RhCo alloy nanocrystals (RhCo/CB) nanohybrids with different Rh/Co atomic ratios and investigate their electrocatalytic acitvity for the MOR in alkaline media. Cyclic voltammetry and chronoamperometry measurements show Rh3Co1/CB nanohybrids have exceptional electrocatalytic activity and long-term stability for the MOR in alkaline media, much higher than commercial Pd/C and Pt/C electrocatalysts, demonstrating that Rh-based alloy nanocrystals may be highly promising Pt-alternative electrocatalyst for the MOR in alkaline media.Download high-res image (686KB)Download full-size image

Norfloxacin is often found in wastewater treatment plants, groundwater, and even drinking water causing environmental concerns because of its potential undesirable effects on human health or aquatic ecosystems. However, conventional treatments cannot deal with norfloxacin efficiently. This work proposes an efficiently enzymatic degradation of norfloxacin by chloroperoxidase (CPO). 82.18% degradation efficiency of norfloxacin was achieved after 25 min reaction time at pH 5.0 with an enzyme concentration of 1.5 × 10−9 mol L−1. HPLC–MS was used to determine the intermediates or final products. The product analysis and determination of the chemical oxygen demand indicated if the enzymatic degradation by CPO was carried out before the usually existing bioremediation techniques (usually activated sludge) in sewage treatment plant, the effluent containing norfloxacin can be decontaminated more efficiently and thoroughly than that only by activated sludge treatment. The eco-toxicity tests using a green algae, Chlorella pyrenoidosa, indicated that the toxicity of degraded products of norfloxacin was lower than the parent norfloxacin molecule. CPO-catalyzed degradation of norfloxacin is a promising alternative for treating effluent containing norfloxacin.

To degrade enzymatically bisphenol A (BPA) that causes serious environmental concerns and is difficult to be degraded by chemical or physical methods.BPA (150 mg l−1) was completely degraded by chloroperoxidase (CPO)/H2O2 within 7 min at room temperature, atmospheric pressure with the enzyme at 6 μg CPO ml−1. The degradation products were identified by HPLC–MS, which suggested involvement of multiple steps. Enzymatic treatment followed by existing bioremediation technologies (activated sludge) enhanced removal of COD from 9 to 54 %. Using an ecotoxicity evaluation with Chlorella pyrenoidosa, the degradation products had a lower toxicity than BPA.BPA can be degraded rapidly and efficiently under mild conditions with chloroperoxidase at 6 μg ml−1. The degradation products had a lower toxicity than BPA.

Mesoporous silica material was prepared to immobilize chloroperoxidase (CPO) from Caldariomyces fumago in this work. The mesoporous material (FDU-12-140) with 15–18 nm pore entrances was found to be a very suitable support not only because it allowed the entry of only a few CPO molecules (diameter around 6.2 nm) so as to avoid the aggregation of enzyme molecules that may result in a decline in its apparent activity, but also because it provided enough flip space for the enzyme during its catalytic action. Enzymatic oxidative decolorization of Crocein Orange G was employed to evaluate the catalytic performance of CPO. The decolorization efficiency of dye by immobilized CPO reached 90% within 70 min. Moreover, it displayed improved thermostability and maintained higher activity in organic solvents compared to that of free enzyme. When incubated at 80 °C for 1 h, 42% of the activity of immobilized enzyme was reserved compared with that at 20 °C, whereas free CPO maintained only 8.5%. Even in the most hydrophilic N,N-dimethylformamide (log p = −1.0), the decolorization efficiency remained 30% by immobilized CPO, but no decolorization was observed by free CPO at the same conditions. The relative activity of immobilized CPO in the 20th reuse maintained 61% of that in the first run in decolorization of the dye.

The effect of M2+ (Zn2+, Cu2+, Cd2+, Mn2+, Pb2+) and M3+ (Cr3+, La3+, Fe3+, Ce3+, Y3+, Al3+) metal ions on the activity and thermal stability of chloroperoxidase (CPO) was investigated in this work. It was found that the lower concentration of metal ions was favorable to CPO activity whereas the higher concentration reversed the results. CPO activity could be increased to 116.4–127.1 % in the presence of a trace amount of these M2+/M3+ metal ions at a concentration range of 0–25 μmol L−1 after 2 h of incubation at 25 °C. The activating effect of M3+ is better than that of M2+, and Cr3+ was mostly efficient. The thermal stability of the enzyme was also improved significantly. Only 30.3 % of CPO activity was retained at 50 °C whereas 82.6 % of CPO activity was maintained in the presence of Cr3+ after 2 h of incubation at the same temperature. The activation of CPO by metal ions at their low concentration was studied through intrinsic fluorescence, circular dichroism (CD), and UV–Vis spectra assay. A favorable environment around the active site was achieved in the presence of metal ions. Intrinsic fluorescence and CD spectra indicated that the α-helix structure of CPO was strengthened in metal ion-contained media. More exposure of the heme ring was achieved for easy access of the substrate, which was suggested by UV–Vis spectrum analysis. This strategy for enhancing CPO activity is very simple and useful. It will be favorable to the practical application of this enzyme.

In this work, it was found that some monosaccharides normally used to stabilize enzymes at high temperatures, however, actually caused a deactivation of chloroperoxidase (CPO). The red native CPO was converted to a stable pale species that lost enzymatic activity. This deactivation was irreversible and was sensitive to temperature. It was different from the general peroxide-mediated deactivation of CPO.Data from measurement of chlorination activity as well as UV–vis, fluorescent, and CD spectral analysis indicated that monosaccharide-induced deactivation can be attributed to precipitation of protein in the presence of monosaccharide and the interaction of the aldehyde group of sugar with amino groups, especially the terminal amino group, on proteins to form Schiff bases which then rearrange to the stable amino ketone.It is further noted that the deactivation efficiency depends on the stereostructure of monosaccharides. d-Glucose was the most efficient inactivating agents due to its aptitude for both of the interactions mentioned in the above paragraphs. The deactivation was specific to aldose. No deprivation of the heme iron was involved in this deactivation.Graphical abstractHighlights► Monosaccharides could cause a suicide inactivation of chloroperoxidase (CPO). ► The sixth axial ligand position of heme iron was occupied by monosaccharides. ► Absorption of Soret band decreased due to occurrence of precipitation of protein. ► Fluorescent and CD spectral analysis indicated a minor uncoiling of α-helix occurred. ► The stereostructure of monosaccharide is related with this enzymatic inactivation.

The catalytic performance of chloroperoxidase (CPO) in peroxidation of 2, 2′-azinobis-(-3 ethylbenzothiazoline-6-sulfononic acid) diammonium salt (ABTS) and oxidation of indole in a reverse micelle composed of surfactant-water-isooctane-pentanol was investigated and optimized in this work. Some positive results were obtained as follows: the peroxidation activity of CPO was enhanced 248% and 263%, while oxidation activity was enhanced 215% and 222% in cetyltrimethylammonium bromide (CTABr) reverse micelle medium and dodecyltrimethylammonium bromide (DTABr) medium, respectively. Thermostability was also greatly improved in reverse micelle: at 40°C, CPO essentially lost all its activity after 5 h incubation, while 58–76% catalytic activity was retained for both reactions in the two reverse micelle media. At 50°C, about 44–75% catalytic activity remained for both reactions in reverse micelle after 2 h compared with no observed activity in pure buffer under the same conditions. The enhancement of CPO activity was dependent mainly on the surfactant concentration and structure, organic solvent ratio (Vpentanol/Visooctane), and water content in the reverse micelle. The obtained kinetic parameters showed that the catalytic turnover frequency (kcat) was increased in reverse micelle. Moreover, the lower Km and higher kcat/Km demonstrated that both the affinity and specificity of CPO to substrates were improved in reverse micelle media. Fluorescence, circular dichroism (CD) and UV–vis spectra assays indicated that a catalytically favorable conformation of enzyme was achieved in reverse micelle, including the strengthening of the protein α-helix structure, and greater exposure of the heme prosthetic group for easy access of the substrate in bulk solution. These results are promising in view of the industrial applications of this versatile biological catalyst.

The Gibbs energy interaction parameters of RbCl with some monosaccharides (d-glucose, d-galactose, d-xylose, and d-arabinose) in water, gES, were obtained from electromotive force (emf) measurements of the electrochemical cell without liquid junction and containing two ion-selective electrodes (ISE): K-ISE∣RbCl(mE)∣ISE-Cl and K-ISE∣RbCl(mE),saccharide (mS)∣ISE-Cl, at 298.15 K. The enthalpy interaction parameters of RbCl with these monosaccharides in water, hES, are determined according to the McMillan–Mayer theory from the measurements of the enthalpies of mixing of aqueous RbCl solutions with aqueous monosaccharide solutions, as well as the enthalpies of dilution of RbCl and monosaccharide solutions in pure water at 298.15 K by a calorimetric method. Furthermore, the entropy interaction parameters, sES, can be evaluated through gES and hES. The results suggest that the electrostatic interactions of these monosaccharides with RbCl in water are predominant compared with structural interactions, and these parameters are controlled primarily by the stereochemical structure of the monosaccharides in water.

Electromotive force (emf) of the chemical cell without liquid-junction K-ISE | RbCl/CsCl ( mE) | ISE-Cl and K-ISE | RbCl/CsCl (mE), amide (mN) | ISE-Cl, have been measured at 298.15 K, where mE = (0.005 to 0.5) mol kg− 1 and mN= (0.05 to 3.0) mol kg− 1. The activity coefficients of RbCl/CsCl in amide (acetamide, propanamide, and n-butanamide) + water mixture can be obtained from these electromotive force data and in the mean time the Gibbs free energy interaction parameters of RbCl/CsCl + amide pair in water, gEN, as well as the salt constant, kS, can be evaluated. The results show that both gEN > 0, kS > 0 at 298.15 K, and all the activity coefficients of electrolyte in amide + water mixture increase with increasing the mN, but it is a little complicated for the dependence of activity coefficients on mE. These thermodynamic parameters were discussed in terms of a model of the structural interaction and electrostatic interaction and the dependence of them on the number of carbon atoms in amide as well as the radius of metal ions were interpreted by the group additivity principle.

Density measurements have been carried out at T=298.15 K for the CsCl–monosaccharide (d-galactose, d-xylose and d-arabinose)–water systems. The apparent molar volume of saccharides Vφ,S in the ternary solutions, the corresponding infinite dilution apparent molar volume Vφ,S∘, and the standard transfer volume ΔtVφ,S∘ of saccharides from water to aqueous CsCl solutions have been determined. The McMillan–Mayer theory was employed to relate the excess thermodynamic functions to a series of interaction parameters to obtain the volumetric interaction parameters of CsCl with monosaccharide in water. These parameters are interpreted by the group additivity principle and the stereochemistry of these monosaccharide molecules.

Biotransformation of cyclohexene catalyzed by chloroperoxidase (CPO) from Caldariomyces fumago was employed to prepare value-added oxygenated derivative: trans-1,2-cyclohexanediol (CHD) using H2O2 as oxidants. The conversion of substrate was enhanced to 89.2% in the presence of small quantities of quaternary ammonium salts (QAS) compared to that of only 48.1% in aqueous phosphate buffer after 150 min. The enhancement was dependent on the concentration and alkyl group length of QAS. QAS were found playing multiple functions in reaction media: phase transfer catalysis and tuning the composition of product. UV–vis, fluorescence and circular dichroism (CD) spectral assay were employed to investigate the effect of QAS on micro-environment around active center and structure of protein. The strengthening of α-helix structure of CPO and more exposure heme for easily access of substrate was found responsible for the improvement of catalytic performance of CPO. Moreover, the determined kinetic parameters indicated the affinity and selectivity of CPO to substrate was improved according to the observation of a decrease of Michaelis constant Km while an increase of the second order rate constants kcat/Km.The strategy reported in this work is environment-friendly, straightforward and applicable to large scale preparation.Graphical abstractDownload high-res image (192KB)Download full-size imageHighlights► Cyclohexene was turned to value-added derivative by CPO-catalyzed biotransformation. ► Small quantity of introduced quaternary ammonium salts played multiple functions. ► The strategy is environment-friendly, straightforward and applicable to large scale.

•Chloroperoxidase efficiently converts diclofenac and naproxen under mild conditions.•Complete conversion is reached in 9 min and 7 min respectively with 10−9 mol L−1 enzyme.•The enzymatic conversion pathways of the drugs was proposed according to MS and NMR assay.•Converted products are significantly less toxic than the parent drugs.•CPO-H2O2 conversion followed by activated sludge treatment greatly improved the drugs removal.Non-steroidal anti-inflammatory drugs diclofenac and naproxen are widely used for the treatment of arthritis, ankylosing spondylitis, and acute muscle pain. However, most of them are usually not metabolized and simply pass through human body. These drugs are difficult to be decomposed by general waste treatment strategies and have caused serious environmental concerns. We report a rapid and efficient conversion of diclofenac and naproxen by chloroperoxidase-catalyzed H2O2-oxidation, a heme protein isolated from Caldarimyces fumago. Complete conversion of diclofenac and naproxen was achieved in only 9 and 7 min respectively with 0.1 mmol L−1 H2O2 and nanomolar enzyme concentration at pH 3.0. The converted products were identified by HPLC–MS and NMR, suggesting involvement of multiple steps in CPO catalyzed conversion. Our work demonstrated that CPO treatment (with COD removal of 4.9%, 9.1% for diclofenac and naproxen, respectively) followed by existing bioremediation technologies (activated sludge) greatly improved the decontaminating these two drugs from waste water (COD removal was enhanced to 85% and 86%, respectively). The eco-toxicity evaluation according to the 72-h EC50 value using the green algae Chlorella Pyrenoidos as ecological indicators showed that the converted products of diclofenac and naproxen had lower toxicity than the original drugs.Chloroperoxidase efficiently degrades diclofenac and naproxen under mild conditions. Complete degradation is in 9 min and 7 min respectively using 10−9 mol L−1 enzyme.Download high-res image (100KB)Download full-size image