Chloroperoxidase Research Articles

Visible-light-driven photobiocatalytic synthesis of chiral sulfoxides over immobilized chloroperoxidase on magnetic yolk–shell Fe3O4@Cu-H-TiO2 photocatalyst

Chloroperoxidase (CPO) is promising for the asymmetric synthesis of sulfoxides from sulfides, owing to its high enantioselectivity under mild reaction conditions. However, suffering from the stability and recyclability issue, the application of free CPO is heavily restricted in current stage. In this work, an integrated photobiocatalyst was designed and fabricated by immobilizing CPO on a magnetically recyclable photocatalyst, Fe3O4@Cu-H-TiO2, which can supply H2O2 for CPO in situ continuously and suppress the destruction of CPO caused by excess H2O2. The obtained CPO@Fe3O4@Cu-H-TiO2 can catalyze the synthesis of enantiopure (R)-sulfoxides from sulfides with high yield up to 88% and high ee value up to >99% over this TiO2-based photobiocatalyst. The enhanced performance of the integrated photobiocatalyst is attributed to the controllable supply of H2O2, the improved stability of immobilized CPO and the enhanced proximity effect between photo- and bio- catalytic modules.

In-Situ Generation of Hydrogen Polysulfides (H2Sn) with Thioglucose, Glucose Oxidase, and Chloroperoxidase.

Hydrogen polysulfides (H2Sn) have emerged as critical physiological mediators that are closely associated with hydrogen sulfide (H2S) signaling. H2Sn exhibit greater nucleophilicity than H2S while also having electrophilic characteristics, enabling unique activities such as protein S-persulfidation. Despite their physiological importance, mechanisms and reactivities of H2Sn remain inadequately explored due to their inherent instability in aqueous environments. Consequently, there is a need to develop biocompatible methods for controlled H2Sn generation to elucidate their behaviors in biological contexts. Herein, we present a dual enzyme system (containing glucose oxidase (GOx) and chloroperoxidase (CPO)) with thioglucose as the substrate to facilitate the controlled release of H2Sn. Fluorescence measurements with SSP4 and the trapping studies allowed us to confirm the production of H2Sn. Such a method may be useful in elucidating the reactivity of hydrogen polysulfides in biological systems as well as provide a potential delivery of H2Sn to target sites for biological applications.

Neutrophil-Mimicking Nanozyme with Cascade Catalytic Releasing Nitric Oxide and Signet Oxygen Property for Synergistic Bimodal Therapy of Methicillin-Resistant Staphylococcus Aureus Infections.

Recently, chloroperoxidase (CPO)-mediated enzyme dynamic therapy (EDT) by mimicking the antipathogen function of neutrophils via generating highly active signet oxygen (1O2) has attracted great interest in biomedical applications. However, the therapeutic efficiency of EDT is largely restricted by the low CPO delivery efficiency and insufficient hydrogen peroxide (H2O2) supply. In the present work, a neutrophil-mimicking nanozyme of MGBC with high CPO delivery efficiency, H2O2 self-supply, and enzyme-cascade catalytic properties is designed for high-efficient treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. In the infection microenvironment, MGBC can effectively catalyze glucose to self-supply substantial H2O2, which enables long-lasting 1O2 generation via the CPO-mediated catalytic reaction. At the meantime, MGBC can also catalyze H2O2 to sustainably release NO for gas therapy (GT), which synergistically strengthens the therapeutic effect of EDT. As a result, MGBC displayed effective MRSA-killing and MSRA biofilms-eradicating properties, and high efficiency in treating both MRSA infected full-thickness excision wounds and subcutaneous MRSA infection by exerting the synergistic bimodal EDT/GT therapeutic effects. In-depth mechanism study revealed that the synergistic EDT/GT antibacterial effects of MGBC can attenuate the drug resistance and toxicity of MRSA by significantly downregulating quorum sensing, multidrug efflux, virulence, and biofilm formation-related genes.

Controlled Delivery of H2O2: A Three-Enzyme Cascade Flow Reactor for Peroxidase-Catalyzed Reactions.

Peroxidases are promising catalysts for oxidation reactions, yet their practical utility has been hindered by the fact that they require hydrogen peroxide (H2O2), which at high concentrations can cause deactivation of enzymes. Practical processes involving the use of peroxidases require the frequent addition of low concentrations of H2O2. In situ generation of H2O2 can be achieved using oxidase-type enzymes. In this study, a three-enzyme cascade system comprised of a H2O2 generator (glucose oxidase (GOx)), H2O2-dependent enzymes (chloroperoxidase (CPO) or horseradish peroxidase (HRP)), and a H2O2 scavenger (catalase (CAT)) was deployed in a flow reactor. Immobilization of the enzymes on a graphite rod was achieved through electrochemically driven physical adsorption, followed by cross-linking with glutaraldehyde. Modeling studies indicated that the flow in the reactor was laminar (Reynolds number, R e < 2000) and was nearly fully developed at the midplane of the annular reactor. Immobilized CAT and GOx displayed good stability, retaining 79% and 84% of their initial activity, respectively, after three cycles of operation. Conversely, immobilized CPO exhibited a considerable reduction in activity after one use, retaining only 30% of its initial activity. The GOx-CAT-GRE system enabled controlled delivery of H2O2 in a more stable manner with a 4-fold enhancement in the oxidation of indole compared to the direct addition of H2O2. Using CPO in solution coupled with GOx-CAT-GRE yields of 90% for the oxidation of indole to 2-oxyindole and of 93% and 91% for the chlorination of thymol and carvacrol, respectively.

Unspecific Peroxygenase (UPO) can be Tuned for Oxygenation or Halogenation Activity by Controlling the Reaction pH.

Unspecific Peroxygenases (UPOs) are increasingly significant enzymes for selective oxygenations as they are stable, highly active and catalyze their reactions at the expense of only hydrogen peroxide as the oxidant. Their structural similarity to chloroperoxidase (CPO) means that UPOs can also catalyze halogenation reactions based upon the generation of hypohalous acids from halide and H2O2. Here we show that the halogenation and oxygenation modes of a UPO can be stimulated at different pH values. Using simple aromatic compounds such as thymol, we show that, at a pH of 3.0 and 6.0, either brominated or oxygenated products respectively are produced. Preparative 100 mg scale transformations of substrates were performed with 60-72 % isolated yields of brominated products obtained. A one-pot bromination-oxygenation cascade reaction on 4-ethylanisole, in which the pH was adjusted from 3.0 to 6.0 at the halfway stage, yielded sequentially brominated and oxygenated products 1-(3-bromo-4-methoxyphenyl)ethyl alcohol and 3-bromo-4-methoxy acetophenone with 82 % combined conversion. These results identify UPOs as an unusual example of a biocatalyst that is tunable for entirely different chemical reactions, dependent upon the reaction conditions.

Natural Disinfection-like Process Unveiled in Soil Microenvironments by Enzyme-Catalyzed Chlorination.

Substantial natural chlorination processes are a growing concern in diverse terrestrial ecosystems, occurring through abiotic redox reactions or biological enzymatic reactions. Among these, exoenzymatically mediated chlorination is suggested to be an important pathway for producing organochlorines and converting chloride ions (Cl-) to reactive chlorine species (RCS) in the presence of reactive oxygen species like hydrogen peroxide (H2O2). However, the role of natural enzymatic chlorination in antibacterial activity occurring in soil microenvironments remains unexplored. Here, we conceptualized that heme-containing chloroperoxidase (CPO)-catalyzed chlorination functions as a naturally occurring disinfection process in soils. Combining antimicrobial experiments and microfluidic chip-based fluorescence imaging, we showed that the enzymatic chlorination process exhibited significantly enhanced antibacterial activity against Escherichia coli and Bacillus subtilis compared to H2O2. This enhancement was primarily attributed to in situ-formed RCS. Based on semiquantitative imaging of RCS distribution using a fluorescence probe, the effective distance of this antibacterial effect was estimated to be approximately 2 mm. Ultrahigh-resolution mass spectrometry analysis showed over 97% similarity between chlorine-containing formulas from CPO-catalyzed chlorination and abiotic chlorination (by sodium hypochlorite) of model dissolved organic matter, indicating a natural source of disinfection byproduct analogues. Our findings unveil a novel natural disinfection process in soils mediated by indigenous enzymes, which effectively links chlorine-carbon interactions and reactive species dynamics.

Biodegradable copper peroxide decorated hollow polydopamine nanocomposite as effective chloroperoxidase delivery carrier for enhancing enzyme dynamic therapy via self-activated cascade reactions

Recently, enzyme dynamic therapy (EDT) has gained enormous attraction in cancer therapy. However, the efficiency of EDT is still largely restricted by the inefficient delivery efficiency of chloroperoxidase (CPO) and the insufficient hydrogen peroxide (H2O2) content in the tumor microenvironment (TME). In the present work, a biodegradable cupper peroxide decorated hollow polydopamine nanocomposite (HPDCP) with surface functionalization of PEGlated tumor targeting peptide RGD (PEG-RGD) was designed for effective CPO delivery. The resultant nanosystem of HPDCP@CPO@PEG-RGD displayed high CPO delivery efficiency to breast tumors without comprising the enzyme activity. Moreover, the nanosystem can dissociate rapidly in acidic TME by supplying substantial H2O2 and simultaneously releasing Cu2+ and CPO, followed by initiating self-activated cascade reactions. At the one hand, the self-supplied H2O2 greatly enhanced the singlet oxygen (1O2) generation via the CPO-mediated enzyme dynamic reaction. At the other hand, substantial hydroxyl radical (•OH) was produced via the Cu2+-driven Fenton reaction for chemodynamic therapy (CDT). Therefore, the anticancer efficiency of EDT was significantly enhanced due to the synergistic EDT/CDT effect. Mechanism study revealed that 1O2/•OH generated by HPDCP@CPO@PEG-RGD synergistically inhibited breast cancer cells by firstly causing severe lipid peroxidation, followed by triggering mitochondria-mediated apoptosis and causing irreversible DNA damage by inhibiting the expression of DNA repair enzyme poly (ADP-ribose) polymerase 1 (PARP-1).

Electroenzymatic cascade reaction on a biohybrid boosts the chiral epoxidation reaction

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 (ILEMB) was loaded onto cobalt nitrogen-doped carbon nanotubes (CoN@CNT) to form a biohybrid (CPO-ILEMB/CoN@CNT). H2O2 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 H2O2 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-ILEMB. Hence, the coimmobilization of CPO-ILEMB and CoN@CNT could facilitate the diffusion of intermediate H2O2, which achieved 17 times higher efficiency than the equivalent amounts of free CPO-ILEMB in bulk solution for styrene epoxidation. Notably, an enhancement (∼45%) of chiral selectivity for the epoxidation of styrene was achieved.

Synergistic catalysis and detection of hydrogen peroxide based on a 3D-dimensional molybdenum disulfide interspersed carbon nanotubes nanonetwork immobilized chloroperoxidase biosensor

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 (H2O2). The molybdenum disulfide nanoflowers (MoS2) is self-assembled on carboxylated carbon nanotubes (CNT) to form a three-dimensional conductive network (3D-CNT@MoS2), which is modified with 1-ethyl-3-methylimidazolium bromide (ILEMB), and followed by anchoring chloroperoxidase (CPO) onto the nanocomposite (3D-CNT@MoS2/ILEMB) through covalent binding to form a bioconjugate (3D-CNT@MoS2/ILEMB/CPO). The ILEMB modified 3D-CNT@MoS2/ILEMB 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@MoS2/ILEMB/CPO bioconjugate modified electrode has a high catalytic efficiency for H2O2. Through electroenzymatic synergistic catalysis for H2O2 detection by 3D-CNT@MoS2/ILEMB/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@MoS2/ILEMB/CPO-GCE displayed exceptional stability, selectivity, and reproducibility. Furthermore, 3D-CNT@MoS2/ILEMB/CPO-GCE is suitable for sensing of H2O2 in human urine s with good recovery, suggesting its potential application for the detection of H2O2 in biomedical field.

The chloroperoxidase immobilized on porous carbon nanobowls for the detection of trichloroacetic acid by electroenzymatic synergistic catalysis

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 (ILEMB) is co-immobilized on PTL-PCNB to from CPO-ILEMB@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-ILEMB@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.

Spatial distribution and proximity effect-dependent multi-enzyme cascades catalysis operating in dendritic mesoporous silica nanoparticles

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 H2O2 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.

Highly Efficient Electroenzymatic Cascade Reduction Reaction For The Conversion of Nitrite to Ammonia

AbstractThe 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.

Rational design of Multi-model ROS regulation Nano-platform for enhanced Mild-temperature photothermal therapy

The tumor thermotolerance owing to the overexpression of heat shock proteins (HSPs) is the critical challenge in practical application of mild-temperature photothermal therapy (MPTT). Reactive oxygen species (ROS) can not only serve as a powerful weapon to kill cancer cells, but also a regulator to inhibit HSPs, thus improving the thermal sensitivity of tumors. Herein, a multi-model ROS regulation nano-platform based on TCLP nanocomposites consisting of polyacrylic acid encapsulated, chloroperoxidase (CPO) and β-Lapachone (β-Lap) loaded black mesoporous titanium (H-TiO2) has been constructed for the ROS-enhanced MPTT. In this nano-platform, the H-TiO2 acts as the NIR-Ⅱ light triggered photothermal agent and photosensitizer to contribute to deep tissue MPTT and PDT, the β-Lap acts as a H2O2 amplifier to improve the 1O2 production of CPO. The abundant ROS from TCLP NPs can limit the activity as well as the expression of HSPs, ultimately improving the antitumor efficacy of MPTT. Finally, excellent synergistic tumor inhibition based on TCLP NPs has been reached both in vitro and in vivo. This multi- model ROS enhanced MPTT nano-platform with low toxic side effects offers a promising strategy and enlightenments the clinical application of MPTT.

Genes related to osmoregulation and antioxidation play important roles in the response of Trollius chinensis seedlings to saline-alkali stress.

Saline-alkali stress is one of the main abiotic stress factors affecting plant growth and development. Trollius chinensis is a perennial herbal medicinal plant with high values for garden application. However, its response and tolerance to saline-alkali stress is unclear. In this study, we mixed four salts (NaCl: Na2SO4: NaHCO3: Na2CO3) with a concentration ratio of 1:9:9:1, and applied low (40 and 80 mM) and high (120 and 160 mM) saline-alkali stress to analyze osmotic regulation substances, antioxidant systems and the gene expression of T. chinensis. Along with higher saline-alkali stress, the leaf relative water content (RWC) started to decrease only from high stress, while the malondialdehyde (MDA) content in leaves decreased continuously, and the contents of proline (Pro), soluble sugar (SS) and soluble protein (SP) increased compared with control. The activities of antioxidant enzymes and the contents of non-enzymatic antioxidants were increased positively with the accumulation of superoxide anion (O2 •-) and hydrogen peroxide (H2O2). For instance, the ascorbic acid-glutathione (AsA-GSH) cycle was enhanced in T. chinensis seedling leaves subject to saline-alkali stress. Principal Component Analysis (PCA) indicates that MDA, Pro, SS, SP, H2O2, O2 •-, and GSH are important indexes to evaluate the response and tolerance of T. chinensis to saline-alkali stress. Through RNA-Seq, a total of 474 differentially expressed genes (DEGs) were found in plant under low saline-alkaline stress (40 mM, MSA1) vs. control. Among them, 364 genes were up-regulated and 110 genes were down-regulated. DEGs were extensively enriched in carbohydrate transport, transferase activity, zeatin biosynthesis, ABC transporters, and spliceosome. The transcription factor family MYB, BZIP, WRKY, and NAC were related to its saline-alkali tolerance. In addition, some DEGs encode key enzymes in the processes of osmoregulation and antioxidation, including betaine aldehyde dehydrogenase (BADH), inositol monophosphatase (IMP), chloroperoxidase (CPO), thioredoxin (Trx), and germin-like protein (GLPs) were found. Overall, these findings provide new insights into the physiological changes and molecular mechanism of T. chinensis to saline-alkali stress and lay a foundation for application of T. chinensis in saline-alkali environment.

Detection of ampicillin based on the fluorescence of a biocatalytic oxidation product

Antibiotics used in humans and farmed animals are an essential source of water and soil contamination. Ampicillin is a micropollutant commonly found in water, sludge, food, flora, and fauna. However, the methods used for its detection in environmental samples are often complicated and expensive. Therefore, developing more straightforward strategies to detect well-known target antibiotics is necessary. In this context, enzyme-based detection methods have been demonstrated to be selective, sensitive, rapid, and relatively simple. In this study, a fluorescent byproduct from the ampicillin oxidation using Chloroperoxidase (CPO) enzyme was used as a pointer compound to determine ampicillin concentration in environmental water samples. We oxidized 80% ampicillin for 1h, producing a fluorescent compound with m/z 274.2517. A response surface methodology (RSM) based on a central composite design (CCD) was used to evaluate and optimize the effects of hydrogen peroxide, enzyme concentration, and time as independent variables on the maximum fluorescence signal as the response function. The methodology proposes to build a calibration curve that relates the initial concentration of ampicillin with fluorescence intensity after the reaction with CPO, which helps detect ampicillin in the concentration range from 0.035 to 40 μM, with a limit of detection of 0.026 μM. The application of the method to fortified environmental water samples allowed percentages of recovery from 86 to 140%. The formation of the fluorescent compound was not affected by the presence of salts commonly found in wastewater; however, it was affected by other antibiotics. The proposed methodology was tested in the context of water from water bodies, urban, and WWTP effluents.

L-buthionine sulfoximine encapsulated hollow calcium peroxide as a chloroperoxidase nanocarrier for enhanced enzyme dynamic therapy

The appropriate design of multifunctional nanocarriers for chloroperoxidase (CPO) delivery and the simultaneous improvement of the efficiency of enzyme dynamic therapy (EDT) remain significant challenges. Herein, we report a facile one-step route to obtain a multifunctional nanocarrier for the formation of sodium hyaluronate-modified hollow calcium peroxide spheres with encapsulated L-buthionine sulfoximine (BSO), followed by delivery of CPO for enhanced EDT. After effective accumulation at the tumor sites, the nanocomposite rapidly decomposes and releases Ca2+, BSO molecules, CPO, and concurrently generates a large volume of hydrogen peroxide (H2O2) in the endogenous tumor microenvironment (TME). BSO molecules inhibit the biosynthesis of glutathione (GSH) by inactivating γ-glutamyl cysteine synthetase. Due to BSO-induced GSH depletion and self-supply of H2O2, the EDT efficiency of CPO was significantly enhanced to achieve high tumor therapy efficiency. Additionally, overloaded Ca2+ caused mitochondrial damage and amplified the oxidative stress. Moreover, calcification resulted from the unbalanced calcium transport channel caused by enhanced oxidative stress, accelerating tumor apoptosis and improving the efficacy of computed tomography (CT) imaging visual tumor therapy. This simple and efficient design for multifunctional nanocomposites will likely take an important place in the field of combined tumor therapeutics.

Ionic liquid modified chloroperoxidase is immobilized on gold nanoparticles-reduced graphene oxide nanocomposites for efficient sensoring of nitrite by electroenzymatic catalysis

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, ILBMB) modified chloroperoxidase (CPO) immobilized on gold nanoparticles (Au NPs) modified reduced graphene oxide (rGO). The ILBMB can facilitate the direct electron transfer (DET) of CPO on GCE without a redox mediator. The molecular docking indicates ILBMB 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 ILBMB. 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-ILBMB/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.

Immobilization of a Bienzymatic System via Crosslinking to a Metal-Organic Framework

A leading biotechnological advancement in the field of biocatalysis is the immobilization of enzymes on solid supports to create more stable and recyclable systems. Metal-organic frameworks (MOFs) are porous materials that have been explored as solid supports for enzyme immobilization. Composed of organic linkers and inorganic nodes, MOFs feature empty void space with large surface areas and have the ability to be modified post-synthesis. Our target enzyme system for immobilization is glucose oxidase (GOx) and chloroperoxidase (CPO). Glucose oxidase catalyzes the oxidation of glucose and is used for many applications in biosensing, biofuel cells, and food production. Chloroperoxidase is a fungal heme enzyme that catalyzes peroxide-dependent halogenation, oxidation, and hydroxylation. These two enzymes work sequentially in this enzyme system by GOx producing peroxide, which activates CPO that reacts with a suitable substrate. This study focuses on using a zirconium-based MOF, UiO-66-NH2, to immobilize the enzyme system via crosslinking with the MOF’s amine group on the surface of the MOF. This study investigates two different crosslinkers: disuccinimidyl glutarate (DSG) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinidimide (NHS), providing stable crosslinking of the MOF to the enzymes. The two crosslinkers are used to covalently bond CPO and GOx onto UiO-66-NH2, and a comparison of the recyclability and enzymatic activity of the single immobilization of CPO and the doubly immobilized CPO and GOx is discussed through assays and characterization analyses. The DSG-crosslinked composites displayed enhanced activity relative to the free enzyme, and all crosslinked enzyme/MOF composites demonstrated recyclability, with at least 30% of the activity being retained after four catalytic cycles. The results of this report will aid researchers in utilizing CPO as a biocatalyst that is more active and has greater recyclability.

Glucose Induces Heme Leakage and Suppresses H2O2 Uptake of Chloroperoxidase in the Asymmetric Hydroxylation of Ethylbenzene

AbstractAs a basic feedstock for both natural life and chemical production, glucose is converted into various chemicals upon the help of peroxidase cascades composed of glucose oxidase (GOx) and peroxidases. However, little is known about the glucose effect on the structure and activity of peroxidases in a non‐natural environment. We report herein that glucose deactivates chloroperoxidase (CPO) used in the asymmetric hydroxylation of ethylbenzene into (R)‐1‐phenylethanol. Through both experiment and molecular simulation, we discover that glucose deactivates CPO through hydrophobic interaction with heme and hydrogen bonding with Glu183. These interactions lead to heme leakage and the reduction of H2O2 uptake thereby undermining the CPO activity. The mechanism appears generically applicable to other hemeproteins such as horseradish peroxidase, hemoglobin, and myoglobin. These findings may open new avenues for the design of peroxidase cascades for applications ranging from better diagnosis of diabetes to green synthesis of chemicals by direct functionalization of C−H bonds.

Toward Environmentally Benign Electrophilic Chlorinations: From Chloroperoxidase to Bioinspired Isoporphyrins.

Recent desires to develop environmentally benign procedures for electrophilic chlorinations have encouraged researchers to take inspiration from nature. In particular, the enzyme chloroperoxidase (CPO), which is capable of electrophilic chlorinations through the umpolung of chloride by oxidation with hydrogen peroxide (H2O2), has received lots of attention. CPO itself is unsuitable for industrial use because of its tendency to decompose in the presence of excess H2O2. Biomimetic complexes (CPO active-site mimics) were then developed and have been shown to successfully catalyze electrophilic chlorinations but are too synthetically demanding to be economically viable. Reported efforts at generating the putative active chlorinating agent of CPO (an iron hypochlorite species) via the umpolung of chloride and using simple meso-substituted iron porphyrins were unsuccessful. Instead, a meso-chloroisoporphyrin intermediate was formed, which was shown to be equally capable of performing electrophilic chlorinations. The current developments toward a potential method involving this novel intermediate for environmentally benign electrophilic chlorinations are discussed. Although this novel pathway no longer follows the mechanism of CPO, it was developed from efforts to replicate its function, showing the power that drawing inspiration from nature can have.