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The electrochemical nitrite reduction reaction provides an alternative approach to offer sustainable ammonia source routes for repairing imbalances in the global nitrogen cycle. In this work, electrocatalysis is combined with enzymatic catalysis to provide an efficient and clean process for recoverable ammonia production. NO2− is reduced to NH3 by electroenzymatic cascade reduction reaction on a bioconjugate, in which 1‐butyl‐3‐methylimidazolium bromide (ILBMB) modified chloroperoxidase (CPO) is fixed on polyethyleneimine (PEI) modified multi‐walled carbon nanotubes (MWCNT) to from bioconjugate (CPO‐ILBMB/MWCNT‐PEI). 15N and 14N isotope labeling reveal that the NH3 species is derived from NO2− reduction. Density functional theory calculations identify that the FeII species in heme center of CPO serve as the key active site for NO2− reduction. The amino groups derived from MWCNT‐PEI not only serve as a bridge to covalently immobilize CPO but also enrich the NO2− ion at electrode/solution interface through electrostatic interactions. The low energy barrier of NO2− reduction and low adsorption free energy of the intermediate result in high Faradaic efficiency (96.4%), NH3 yield (112.7 mg h−1 mgCPO−1), and high selectivity in pH 5.0 solution. The highly efficient electroenzymatic reaction ensurespromising applications in the conversion of NO2− to NH3. [ABSTRACT FROM AUTHOR]

A novel nitrite electrochemical biosensor is constructed by glassy carbon electrodes (GC) coated by chloroperoxidase (CPO) -Au nanoparticles-MoS2 nanoflowers in the presence of ionic liquid, 1-ethyl-3-methylimidazolium bromide (EMB-IL). Au-MoS2 nanoflowers modified nanocomposites are synthesized using sodium carboxymethyl cellulose as reducing agent and stabilizer. The EMB-IL can effectively promote direct electron transfer between CPO and electrode surface without redox mediator. Herein, the surface concentration of CPO on the electrode surface is 1.36 × 10–10 mol cm−2, and the electron transfer coefficient and the rate constant of CPO at electrode surface are 0.87 and 2.03 s−1, respectively. The proposed nitrite electrochemical biosensor shows a wide linear range, low detection limit, and high sensitivity. The biosensor was applied to the determination of nitrite in pickled cabbage with a good accuracy and recovery. [ABSTRACT FROM AUTHOR]

A multitude of industrial processes are catalyzed by two or more enzymes working together in a cascade way. However, designing efficient enzymatic cascade reactions is still a challenge. In this work, a TiO2 thin film with mesoporous pores was prepared and used as carrier for co-immobilization of chloroperoxidase (CPO) and glucose peroxidase (GOx). By adjusting the dosage of hexadecyltrimethylammonium bromide (CTAB) and the ratio of the two enzymes, CPO and GOx were well distributed and positional orientated to their own appropriate pores to form an ordered "occupation" based on a "feet in right shoes" effect. Moreover, when the pore size was controlled around 12 nm, the enzymes aggregation was inhibited so as to avoid the decrease of activity of enzyme; The catalytic performance of TiO2–GOx and CPO composites was evaluated by the application of decolorization of Orange G dye in a cascaded manner. The oxidant H2O2 needed by CPO is generated in situ through glucose oxidation by GOx. Upon co-immobilization of CPO and GOx on the same carrier, a large increase in the initial catalytic efficiency was detected when compared to an equimolar mixture of the free enzymes, which was four times greater. Moreover, the affinity of the enzyme toward substrate binding was improved according to the kinetic assay. The thermal stability of TiO2–GOx and CPO composites were greatly improved than free enzymes. The TiO2–GOx and CPO composites can be easily separated from the reaction media which facilitate its recycle use. [ABSTRACT FROM AUTHOR]

Chloroperoxidase (CPO) was immobilized on graphene oxide (GO) nanosheets via site-specific interactions of concanavalin (ConA) with saccharide groups on the CPO surface to make GO-ConA-CPO nano-architectonics. Enzymatic oxidative decolorization of malachite green was used to investigate the catalytic performance of GO-ConA-CPO. The GO-ConA-CPO showed high activity (based on the decolorization efficiency), i.e., 93.68% in 15 min. Moreover, GO-ConA-CPO showed better thermostability and remained higher activity against pH change and high concentrations of oxidant HO compared with GO-CPO and the free enzyme. When incubated at 60 °C for 1.5 h, 63.02% of the activity of GO-ConA-CPO and 35.75% of GO-CPO remained compared with that at 25 °C, while free CPO remained only 8.46% in the same condition. The tolerance of GO-ConA-CPO to HO improved from 2.5 to 6 mmol L, and the suitable pH range enlarged from 2.5-3.0 to 2.0-4.5. After 8 cycles, the GO-ConA-CPO can keep 52% activity of that in the first run. The enzymatic kinetic constants indicated that introducing CPO into GO-ConA-CPO bioarchitecture improved the diffusion of reactants and products. [ABSTRACT FROM AUTHOR]

The operational stability of chloroperoxidase (CPO) was considerably enhanced by coupling with glucose oxidase (GOx) because H 2 O 2 could be generated in situ from glucose and oxygen. In this paper, a CPO–GOx nanoreactor was fabricated on the surface of Au@Fe 3 O 4 nanoparticles through layer-by-layer assembly using the specific avidin–biotin interaction. The X-ray diffraction data indicated the presence of both Fe and Au in the Au@Fe 3 O 4 carrier. The Au@Fe 3 O 4 displayed a uniform core/shell nanostructure, whereas the nanoparticles of the bienzymatic reactor were larger than the carrier. The catalytic activity of CPO was highly dependent on the structure of the enzymatic nanoreactor. The activity of Au@Fe 3 O 4 –GOx (inner)–CPO (outer) was 15.5% higher than that of Au@Fe 3 O 4 –CPO (inner)–GOx (outer). Moreover, Au@Fe 3 O 4 –GOx–CPO exhibited better thermostability. Au@Fe 3 O 4 –GOx–CPO retained 53.2% of its initial activity after incubation for 1.0 h at 60 °C and 30.4% of its initial activity after 18 h at 50 °C. Au@Fe 3 O 4 –GOx–CPO had good reusability. It retained more than 62.4% of its activity after the 12th cycle. This was attributed to the cage-like structure in Au@Fe 3 O 4 –GOx–CPO, which could effectively prevent the removal of the enzyme molecules from the carrier. [ABSTRACT FROM AUTHOR]

BACKGROUND Sulfamethoxazole is found in surface and ground waters because it is usually not metabolized and simply passes through the human body after ingestion. This drug is difficult to degrade by conventional treatments and may cause environmental concerns. RESULTS A rapid and efficient enzymatic degradation of sulfamethoxazole by a heme peroxidase, chloroperoxidase ( CPO), was investigated in this work. The degradation efficiency of sulfamethoxazole reached 98.64% within 20 min at room temperature, atmospheric pressure and environment-friendly reaction conditions with an enzyme concentration of approximately 10−9 mol L−1. High performance liquid chromatography-mass spectrometry analysis was employed to identify the intermediates and final products. Moreover, chemical oxygen demand ( COD) measurements showed that pre-treatment by CPO-H2O2 oxidative degradation followed by existing bioremediation technologies can effectively improve the efficiency and thoroughness of decontaminating this drug from wastewater. The degradation efficiency was enhanced from 76.5% to 84.47%, and the COD removal increased from 29.72% to 51.32%. The eco-toxicity test using a green algae, Chlorella pyrenoidos, showed that the degraded products had lower toxicity than the parent drug. CONCLUSION CPO catalyzed oxidative degradation is a promising alternative for treatment of wastewater containing sulfonamide drugs. This enzymatic method is also suitable for treatment of other sulfonamide antibiotics. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

The optically active C3 synthetic blocks are remarkably versatile intermediates for the synthesis of numerous pharmaceuticals and agrochemicals. This work provides a simple and efficient enzymatic synthetic route for the environment-friendly synthesis of C3 chiral building blocks. Chloroperoxidase (CPO)-catalyzed enantioselective halo-hydroxylation and epoxidation of chloropropene and allyl alcohol was employed to prepare C3 chiral building blocks in this work, including ( R)-2,3-dichloro-1-propanol (DCP*), ( R)-2,3-epoxy-1-propanol (GLD*), and ( R)-3-chloro-1-2-propanediol (CPD*). The ee values of the formed C3 chiral building blocks DCP*, CPD*, and glycidol were 98.1, 97.5, and 96.7%, respectively. Moreover, the use of small amount of imidazolium ionic liquid enhanced the yield efficiently due to the increase of solubility of hydrophobic organic substrates in aqueous reaction media, as well as the improvement of affinity and selectivity of CPO to substrate. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:724-729, 2015 [ABSTRACT FROM AUTHOR]

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 M 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. [ABSTRACT FROM AUTHOR]

Abstract: 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 150min. 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 K m while an increase of the second order rate constants k cat/K m. The strategy reported in this work is environment-friendly, straightforward and applicable to large scale preparation. [Copyright &y& Elsevier]

[Display omitted] The chiral epoxidation of styrene and its derivatives is an important transformation that has attracted considerable scientific interest in the chemical industry. Herein, we integrate enzymatic catalysis and electrocatalysis to propose a new route for the chiral epoxidation of styrene and its derivatives. Chloroperoxidase (CPO) functionalized with 1-ethyl-3-methylimidazolium bromide (IL EMB) was loaded onto cobalt nitrogen-doped carbon nanotubes (CoN@CNT) to form a biohybrid (CPO-IL EMB /CoN@CNT). H 2 O 2 species were generated in situ through a two-electron oxygen reduction reaction (2e–ORR) at CoN@CNT to initiate the following enzymatic epoxidation of styrene by CPO. CoN@CNT had high electroactivity for the ORR to produce H 2 O 2 at a more positive potential, prohibiting the conversion of FeIII to FeII in the heme of CPO to maintain enzymatic activity. Meanwhile, CoN@CNT could serve as an ideal carrier for the immobilization of CPO-IL EMB. Hence, the coimmobilization of CPO-IL EMB and CoN@CNT could facilitate the diffusion of intermediate H 2 O 2 , which achieved 17 times higher efficiency than the equivalent amounts of free CPO-IL EMB in bulk solution for styrene epoxidation. Notably, an enhancement (∼45%) of chiral selectivity for the epoxidation of styrene was achieved. [ABSTRACT FROM AUTHOR]

Molecular orientation of chloroperoxidase (CPO) on surface of Fe 3 O 4 magnetic nanoparticles (MNPs) was achieved through layer-by-layer (LBL) controlled assembly by site specific interactions of avidin and biotin. Enzymatic oxidative decolorization of soluble aniline blue was employed to evaluate the catalytic performance of immobilized CPO (I-CPO). The decolorization efficiency of dye by I-CPO was higher than 90% within 10 min. Moreover, I-CPO displayed improved thermostability and kept higher activity in wider pH range and in the presence of high concentration of oxidant (H 2 O 2 ) compared to that of free enzyme. When incubated at 70 °C for an hour, 42.35% of the activity of immobilized enzyme was reserved compared with that at 25 °C, whereas free CPO remained only 8.46%. The tolerance to H 2 O 2 was improved from 2 mmol L −1 to 4 mmol L −1 , and the suitable pH range become wider. The relative activities of I-CPO in the 20 times use maintained 62% of that in the first run. The enzymatic kinetic constants confirmed that the entrapping CPO into this well-defined architecture could reduce diffusional limitation of reactants and products. [ABSTRACT FROM AUTHOR]

Mesoporoussilica material was prepared to immobilize chloroperoxidase(CPO) from Caldariomyces fumagointhis work. The mesoporous material (FDU-12-140) with 15–18nm pore entrances was found to be a very suitable support not onlybecause it allowed the entry of only a few CPO molecules (diameteraround 6.2 nm) so as to avoid the aggregation of enzyme moleculesthat may result in a decline in its apparent activity, but also becauseit provided enough flip space for the enzyme during its catalyticaction. Enzymatic oxidative decolorization of Crocein Orange G wasemployed to evaluate the catalytic performance of CPO. The decolorizationefficiency of dye by immobilized CPO reached 90% within 70 min. Moreover,it displayed improved thermostability and maintained higher activityin organic solvents compared to that of free enzyme. When incubatedat 80 °C for 1 h, 42% of the activity of immobilized enzyme wasreserved compared with that at 20 °C, whereas free CPO maintainedonly 8.5%. Even in the most hydrophilic N,N-dimethylformamide(log p= −1.0), the decolorizationefficiency remained30% by immobilized CPO, but no decolorization was observed by freeCPO at the same conditions. The relative activity of immobilized CPOin the 20th reuse maintained 61% of that in the first run in decolorizationof the dye. [ABSTRACT FROM AUTHOR]

Chloroperoxidase(CPO) from Caldariomyces fumagocouldmediate a very efficient oxidative decolorization/degradationof aqueous azo dye. The decolorization efficiencies for Congo Redand Alizarin Yellow R reached 88.25% in 3 min and 92.38% in 5 min,respectively, under mild conditions at enzyme concentrations of 0.025and 0.03 μmol L–1, respectively. Comparedwith the typical mechanism involved in oxidative decolorization ofazo dyes catalyzed by other peroxidases, a different enzymatic catalyticcycle was found in this work. The oxidative decolorization was carriedout in bulk solution, which would get rid of the restriction of substraterequired by the channel access to the substrate pocket in the enzymaticactive site. Nine degradation products of Congo Red were detectedby HPLC–MS analysis, and two types of degradation pathwayswere proposed accordingly. The strong toleration of the typical saltspecies in industrial effluents by this system ensured that it haspotential applications in treatment of industrial wastewater. [ABSTRACT FROM AUTHOR]

Abstract: The decolorization/degradation of aqueous azo dyes generated by textile photography industries are a main issue in wastewater treatment. Despite many significant efforts and numerous systems have been exploited, the problem is still unsolved due to intrinsically low activity or use of non-convenient chemical reagents. Biodegradation appears to be one of the most efficient ways, however, the initial step of biotransformation usually involves a reductive cleavage of the azo bond, which under anaerobic conditions leads to the formation of aromatic amines known to be carcinogenic. This study shows that applying an oxidative process of H2O2 catalyzed by chloroperoxidase (CPO) for decolorization/degradation of aqueous azo dyes (Orange G and Sunset Yellow) in buffer solution is a very efficient system. The effect of initial pH, dosage of enzyme, H2O2 concentration, reaction temperature and time, and suitable initial dye concentration on the decolorization efficiency were investigated in detail and optimized accordingly. At optimum reaction condition, the decolorization efficiency of Orange G reached 98.72% in 5min, and 77.25% in 10min for Sunset Yellow. The dosage of CPO was below ppm level. This enzymatic reaction is an ideal potential alternative for application on decolorization/degradation of aqueous azo dyes. Furthermore, the elucidation of degradation pathways is of special interest considering healthy and environmental priorities. In this work, LC–MS was used to determine the structures of intermediates arising from Orange G by CPO–H2O2 oxidation. Eight transformation products were identified and two types of cleavage were proposed for the degradation pathway. [Copyright &y& Elsevier]

Abstract: Asymmetric epoxidation of 3-chloropropene can be catalyzed by chloroperoxidase (CPO) from Caldariomyces fumago to prepare (R)-epichlorohydrin (ECH) in homogenous phosphate buffer/ionic liquid mixtures using t-butyl hydroperoxide (TBHP) as O2 donor. Reaction conditions were optimized by the investigation of the choice of oxidants, the presence of ionic liquids (ILs), pH effect and CPO consumption. The best ECH yield reached 88.8% within a duration of 60min with high enantiomeric excesses (e.e. 97.1%) at pH 5.5 and room temperature, using 1-ethyl-3-methylimidazolium [EMIM][Br] as co-solvent. The ILs with shorter carbon chain was more efficient on chiral ECH preparation. [Copyright &y& Elsevier]

Abstract: Inactivation of chloroperoxidase (CPO) from Caldariomyces fumago by arginine was investigated. It was found that the red native CPO solution was turned into a stable green species with a concomitant shift of the Soret band from 398 to 425nm in the presence of arginine. The green CPO lost almost all of its catalytic activity, and this inactivation was irreversible. Differential UV–vis spectrophotometry was used to examine the binding properties of arginine to CPO. The formation of CPO-arginine (1:1) complex was highly pH-dependent. Fluorescence investigation revealed the exposure degree of prosthetic group increased. Kinetic analysis indicated that CPO has both a high affinity and specificity to arginine. This inactivation may be caused mainly by the binding of guanidinium group in arginine to the acid–base catalytic group Glu183 in CPO. The change of surrounding environment around heme induced by the interaction of heme propionates with arginine and the occupying of the sixth axial ligand position of heme iron by hydroxyl are also reasons bringing on this inactivation. [Copyright &y& Elsevier]

[Display omitted] • An efficient biosensor 3D-CNT@MoS 2 /IL EMB /CPO for H 2 O 2 detection. • 3D-CNT@MoS 2 increases the fixation site of the CPO while prevents it leaking. • CPO achieves direct electron transfer in the absence of a redox medium. • The biosensor is applied to the detection of H 2 O 2 in human urine. Enzyme-based electrochemical biosensors are promising for a wide range of applications due to their unique specificity and high sensitivity. In this work, we present a novel enzyme bioelectrode for the sensing of hydrogen peroxide (H 2 O 2). The molybdenum disulfide nanoflowers (MoS 2) is self-assembled on carboxylated carbon nanotubes (CNT) to form a three-dimensional conductive network (3D-CNT@MoS 2), which is modified with 1-ethyl-3-methylimidazolium bromide (IL EMB), and followed by anchoring chloroperoxidase (CPO) onto the nanocomposite (3D-CNT@MoS 2 /IL EMB) through covalent binding to form a bioconjugate (3D-CNT@MoS 2 /IL EMB /CPO). The IL EMB modified 3D-CNT@MoS 2 /IL EMB has good hydrophilicity and conductivity, which not only provides a suitable microenvironment for the immobilization of CPO but also facilitates the direct electron transfer (DET) of CPO at the electrode. The 3D-CNT@MoS 2 /IL EMB /CPO bioconjugate modified electrode has a high catalytic efficiency for H 2 O 2. Through electroenzymatic synergistic catalysis for H 2 O 2 detection by 3D-CNT@MoS 2 /IL EMB /CPO-GCE, a wide detection range of 0.2 μmol·L−1 to 997 μmol·L−1 and a low detection limit of 0.097 μmol･L−1 with high sensitivity of 1050 µA·mmol·L−1·cm−2 were achieved. Additionally, the 3D-CNT@MoS 2 /IL EMB /CPO-GCE displayed exceptional stability, selectivity, and reproducibility. Furthermore, 3D-CNT@MoS 2 /IL EMB /CPO-GCE is suitable for sensing of H 2 O 2 in human urine s with good recovery, suggesting its potential application for the detection of H 2 O 2 in biomedical field. [ABSTRACT FROM AUTHOR]

Trichloroacetic acid (TCA), as a by-product of chlorination disinfection, is a highly carcinogenic chemical. Due to the widespread use of chlorination disinfection, it is critical to detect TCA in drinking water to decrease the incidence of disease. In this work, we developed an efficient TCA biosensor via electroenzymatic synergistic catalysis. The porous carbon nanobowls (PCNB) are prepared and wrapped by an amyloid like proteins formed by phase-transitioned lysozyme (PTL-PCNB), then, chloroperoxidase (CPO) is abounding to PTL-PCNB owing to its strong adhesion. The ionic liquid of 1-ethyl-3-methylimidazolium bromide (IL EMB) is co-immobilized on PTL-PCNB to from CPO-IL EMB @PTL-PCNB nanocomposite to assist the direct electron transfer (DET) of CPO. The PCNB plays two roles here. In addition, to increasing the conductivity, it serves as an ideal support for holding CPO; The CPO-IL EMB @PTL-PCNB nanocomposite modified electrode presents high efficiency for sensing TCA. Through electroenzymatic synergistic catalysis, a wide detection range of 33 μmol L−1 to 98 mmol L−1 can be achieved with a low detection limit of 5.9 μmol L−1, and high stability, selectivity as well as reproducibility, which ensures its potential practical applicability. This work provides a new platform for the electro-enzyme synergistic catalysis in one pot. [Display omitted] • Porous carbon nanobowls for sensor of trichloroacetic acid by electroenzymatic catalysis. • Phase-transitioned lysozyme modified on carbon bowls for holding chloroperoxidase. • Ionic liquid assists direct electron transfer of chloroperoxidase without a redox mediator. • The nanocomposites modified electrode presents excellent performance for the TCA detection. [ABSTRACT FROM AUTHOR]

Graphical abstract Preparation and performance of CPO-GOx co-immobilized on MGO. Highlights • chloroperoxidase and glucose oxidase were co-immobilized onto the surface of MGO. • The catalytic efficiency of immobilized CPO was much superior to that of free CPO. • Immobilized CPO can be effectively and conveniently recovered and is ready to reuse. • Enzyme cascade helps to harness the power of enzymes requiring auxiliary proteins. Abstract Although chloroperoxidase (CPO) has long been recognized as a powerful and versatile catalyst, its applications have been thwarted by the difficulty in regenerating the active enzyme and substrate (peroxide) induced protein inactivation. In this paper, we describe the fabrication and characterization of chloroperoxidase (CPO) and glucose oxidase (GOx) on the surface of MGO. The performance of the immobilized CPO was considerably enhanced by coupling with GOx that provided the required H 2 O 2 in situ through glucose oxidation. The activity of MGO-GOx-CPO (96.6%) towards the decolorization of orange G was much superior to that of MGO-GOx+MGO-CPO (86.2%), probably as a result of reduced mass transfer resistance between CPO and H 2 O 2 generated from GOx molecules. Interestingly, MGO-GOx-CPO reached its peak activity (95.2%) when temperature was maintained between 42 °C and 50 °C compared to the optimal temperature of 35 °C for the free enzyme, possibly due to dispersion of the otherwise stacked MGO below 42 °C. Finally, MGO-GOx-CPO can be conveniently reused for its ease of recovery in the presence of an external magnetic field, with ∼38.5% activity remained after 6 cycles of applications. This work demonstrates the feasibility of co-immobilizing CPO and GOx onto MGO and the potential of MGO-GOx-CPO in environmental applications. [ABSTRACT FROM AUTHOR]

Halloysite nanotubes (HNT) is a kind of kaolin clay consisting of one alumina octahedron sheet and one silica tetrahedron sheet formed by rolling flat sheets into a hollow tubular structure. It is an ideal carrier for immobilization of enzyme due to its large surface area, biocompatibility, and chemical and mechanical stability besides abundance and low-cost. In order to make the most of this carrier for enzyme immobilization, in this work, two strategies were proposed for entrapping and embedding chloroperoxidase (CPO) on both inner/outer wall of HNT rather than in the hollow space alone by pH modulated electrostatic adsorption. Besides the enhanced loading amount, the thermal stability and tolerance to organic solvents of immobilized CPO (I-CPO) was greatly improved compared to the free enzyme. The free CPO can retain only 11.66% activity after 1 h incubation at 80 °C, while the I-CPO remained 87.63% activity at the same condition. Even after 1.5 h incubation at 90 °C, when the free CPO lost all its activity, the I-CPO can still remain 40.3% activity; Moreover, in the presence of organic solvent (ethyl acetate, acetonitrile, methanol, and DMF) with volume fraction of 10%, almost no loss of activity of I-CPO was observed, but free CPO can only remain 41.6%, 38.2%, and 23.5% of its initial activity in ethyl acetate, acetonitrile, methanol-water mixed system respectively, and even inactivated completely in DMF in the same condition. Furthermore, the enzymatic kinetic parameters ( K m , and k cat / K m ) suggested the affinity and specificity of I-CPO to the substrate was improved. I-CPO was very efficient when applied in the degradation of isoproturon in wastewater. The isoproturon with initial concentration of 26.7 μmol·L −1 can be completely degraded only in 10 min, indicating a potential practical application of I-CPO in treatment of wastewater containing pesticide. [ABSTRACT FROM AUTHOR]

• A very efficient GOx-CPO cascade catalysis for HClO generation. • The spatial distribution and proximity effect account for the high cascade efficiency. • PTL coating plays mutil-functions including concentrating intermediate and improving dispersibility. • The prepared biocomposite PTL@GOx&CPO@DMSP is very stable at harsh conditions. • Efficient anti-tumor effect and effectively in application of fruit preservation. Enzymatic cascades catalysis engenders complex and selective intracellular transformations due to the organization of the enzymes in confined cellular environments. In this work, a biomimetic strategy is employed to develop a synthetic cell analogue for operating multi-enzymatic cascade catalysis in vitro. The glucose peroxidase (GOx) and chloroperoxidase (CPO) are co-encapsulated in the channel of dendritic mesoporous silica nanoparticles (DMSP) modified by phase-transitioned lysozyme (PTL@GOx&CPO@DMSP). The cascade efficiency is substantially enhanced compared with the diffusion mixtures of the free enzymes in bulk solution due to some nanoscale construction, such as confinement effect, proximity effect and special distribution mode of the enzymes. The high specific surface area and high concentration of mesoporous cages in DMSP is beneficial for encapsulation of enzyme. The cross-linked cavities ensure directional transfer of the intermediate from GOx to CPO, known as "substrate channel". PTL plays multiple functions including inhibiting leaking of enzyme from DMSP, concentrating intermediate H 2 O 2 at DMSP domains and keeping this biocomposite stable in aqueous environment by improving its dispersibility. The prepared biocomposite PTL@GOx & CPO@DMSP is very stable at harsh conditions. It can retain 42% of original catalytic activity at 90°C with incubation for 3 h while the retained activity of free enzymes is only 5% under the same conditions. Meanwhile, it can keep 92.4% of its original catalytic activity after 10 reuses. These characteristics ensure practical application of this biocatalytic material. The active HClO generated by GOx-CPO cascade has an efficient anti-tumor effect and is effectively applied to preserve fruit. [Display omitted] [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Enzymatic-inorganic hybrid microsphere was prepared in aqueous solution. • The formation of multilayer structures are studied by various methods. • The hybrid materials shows excellent bio-catalytic activity, stability and reusability. Abstract CPO, a versatile enzyme, has limitations in industrial applications due to its poor stability and the difficulty of reuse. The preparation of stable immobilized enzymes with high catalytic activity is therefore desirable, but remains a challenge. A facile preparation of a series of enzyme-inorganic hybrid micro-spheres [chloroperoxidase (CPO)-(Cu/Co/Cd) 3 (PO 4 ) 2 ] and its application in the decolorization of crystal violet is reported in this work. All the hybrid micro-spheres show a multilayer petal-like structure. The formation of hybrid micro-spheres were proposed via four stages: crystallization, in-situ coordination, self-assembly and size growth. The process was entropy-driven, and there was a competition between precipitation of phosphate and coordination of M2+ with amide groups of CPO. The introduction of excess of chloride ions retarded the phosphate from precipitating by forming [MCl 4 ]2− complexes, while simultaneously promoting the coordination of M2+ with amide groups. These hybrid materials showed high bio-catalytic activity in the decolorization of crystal violet. The decolorization efficiency reached 99.66%, 98.44% and 98.05% just within 3 min using CPO-Cu 3 (PO 4 ) 2 , CPO-Cd 3 (PO 4 ) 2 and CPO-Co 3 (PO 4 ) 2 respectively. They also have good thermal stability, and can keep 52.89% catalytic activity after 8 cycles of use. CPO-inorganic hybrid micro-spheres [chloroperoxidase (CPO)-(Cu/Co/Cd) 3 (PO 4 ) 2 ] is promising in the treatment of waste water containing crystal violet. It also has potential application for treatment of other soluble organic dyes in industrial wastewater. [ABSTRACT FROM AUTHOR]

A chloroperoxidase (CPO) -TiO 2 thin film type enzyme reactor was constructed by single chloroperoxidase molecule immobilization on TiO 2 mesoporous thin film. The CPO-TiO 2 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 TiO 2 , 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-TiO 2 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-TiO 2 enzyme reactor against thermal denaturation and deactivation by high concentration of oxidants or in a strong acid-base environment was enhanced. [ABSTRACT FROM AUTHOR]

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 H 2 O 2 -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 H 2 O 2 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 EC 50 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. [ABSTRACT FROM AUTHOR]

Electroenzymatic biosensors have wide applications due to its fastness and sensitivity. Here we propose a bio-nanocomposite to be dropped on glassy carbon electrode (GCE) for nitrite sensoring by electroenzymatic catalysis. The composite is constructed by ionic liquids (1-butyl-3-methylimidazolium bromide, IL BMB) modified chloroperoxidase (CPO) immobilized on gold nanoparticles (Au NPs) modified reduced graphene oxide (rGO). The IL BMB can facilitate the direct electron transfer (DET) of CPO on GCE without a redox mediator. The molecular docking indicates IL BMB is located at the opening of substrate channel rather than occupy the active site of CPO, which is beneficial to its DET. Circular dichroic spectrum (CD) verifies the α-helix content of CPO is increased, and ultraviolet-visible spectrum (UV–vis) demonstrates the heme prosthetic group become more exposed by IL BMB. These changes result in the enhancement of CPO activity. The rGO-Au NPs plays two roles enhancing the detection signal and improving the loading of CPO on GCE. The sensor (CPO-IL BMB /rGO-Au NPs/GCE) provides two models for nitrite detection as cyclic voltammetry and amperometry. The detection limit is 0.25 μmol·L−1 and 0.049 μmol·L−1, respectively with corresponding detection range of 2.0−2500 μmol·L−1 and 0.5–300 μmol·L−1. The high sensitivity, stability, and selectivity of this electroenzymatic biosensor ensure its potential practical application. [Display omitted] • CPO-IL BMB /rGO-Au NPs/GCE for efficient sensoring of nitrite by electroenzymatic catalysis. • IL BMB can effectively assist the direct electron transfer of CPO without a redox mediator. • The rGO-Au NPs composite plays two roles as enhancing detection signal and improving CPO loading. • The enzymatic sensor shows a low detection limit in a wide range with high stability and selectivity. [ABSTRACT FROM AUTHOR]

A one-step asymmetric bio-synthesis of ( R )-enantiomer of the racemic drug modafinil was achieved by chloroperoxidase (CPO)-catalyzed enantioselective sulfoxidation of 2-(diphenylmethylthio) acetamide. Ionic liquids, quaternary ammonium salts or polyhydroxy compounds were introduced into the reaction media to improve productivity. The ( R )-modafinil yield of 40.8% and high enantiomeric excesses of 97.3% was obtained at pH 5.5 and room temperature in the presence of [EMIM][Br] ( V ILs / V buffer = 10%), and a low enzymatic concentration (0.013 mmol L −1 ) was required. UV–vis and circular dichroism spectral indicated that the α -helix of CPO was strengthened and the heme became more exposed for easier access of substrate to the active site in CPO in the presence of the above additives, and moreover, enzymatic kinetic data showed that the affinity and the specificity of CPO to substrate was improved. [ABSTRACT FROM AUTHOR]

Highlights: [•] Very efficient enzymatic decolorization for anthraquinone and triphenylmethane dye. [•] Products analysis indicated it is a good alternative for biodegradation of these compounds. [•] No restriction for dye species due to the active oxidants generated online in catalytic cycle. [•] Strong toleration to high saline wastewater was observed ensuring it potential application. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]