Free 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.

CPO‐Fe3O4@mTiO2 nanocomposite with integrated magnetic separation and enzymatic and photocatalytic activities in efficient degradation of organic contaminants in wastewater

AbstractBACKGROUNDImmobilization of enzymes on a nanocarrier can improve their stability, but is often accompanied by a reduction of the initial catalytic activity. Meanwhile, inevitable mass loss during centrifugal separation inhibits reusability. It is still a challenge to design and prepare an immobilized enzyme with both improved catalytic activity and reusability simultaneously.RESULTSCore–shell magnetic microspheres (Fe3O4@mTiO2) with mesoporous structure were prepared and used to construct an enzymatic nanocomposite (CPO‐Fe3O4@mTiO2). This material can combine magnetic separability, photocatalytic property of TiO2 and enzymatic activity of chloroperoxidase (CPO) when applied in the degradation of aqueous organic contaminants diphenylamine and pesticide residue isoproturon. Moreover, CPO‐Fe3O4@mTiO2 showed good stability and reusability. It can keep 78.0% and 66.8% of its initial activity after incubation at 80 and 90 °C for 1 h, respectively, while free CPO only retained 18.3% and 6.9% of its original activity for the same conditions. After being used for eight cycles, CPO‐Fe3O4@mTiO2 can retain over 82.2% of activity. When applied in the degradation of isoproturon/diphenylamine in artificial wastewater, 60 μmol L−1 isoproturon can be completely degraded in 30 min; 82.2% of diphenylamine can be degraded at a concentration of 10 μmol L−1. Compared with the removal of aqueous contaminants using a porous adsorbent, CPO‐Fe3O4@mTiO2 can be regenerated ‘online’, obviating complicated procedures for regeneration.CONCLUSIONSThe results indicate that CPO‐Fe3O4@mTiO2 has potential for practical application in the treatment of wastewater. © 2021 Society of Chemical Industry

Amino modified magnetic halloysite nanotube supporting chloroperoxidase immobilization: enhanced stability, reusability, and efficient degradation of pesticide residue in wastewater.

Halloysite nanotube (HNT) is a natural bio-compatible and stable nanomaterial available in abundance at low-cost. In this work, HNT was modified by two strategies to make it suitable for supporting immobilization of chloroperoxidase (CPO). Firstly, Fe3O4 nanoparticles were deposited on HNT, so magnetic separation can be used instead of centrifugation. Then, the magnetic HNT was modified by 3-aminopropyltriethoxysilane (APTES), which can provide amine group on surface of HNT and meanwhile inhibit the agglomeration of magnetic HNT. Then, HNT-Fe3O4 -APTES was linked with branched polyethyleneimine (PEI) to provide more amino for binding with enzyme. The so-prepared CPO@HNT-Fe3O4-APTES-PEI showed enhanced enzyme loading, reusability, improved thermal stability and tolerance to organic solvents than free CPO. For example, after 10 repeated uses, CPO@HNT- Fe3O4-APTES-PEI can maintain 92.20% of its original activity compared with 65.12% of activity of CPO@HNT-APTES-PEI and 45.69% of activity of CPO@HNT. The kinetic parameters indicated the affinity and specificity of immobilized enzyme to substrate was increased. CPO@HNT-Fe3O4-APTES-PEI was very efficient when it was applied in the degradation of pesticides mesotrione in wastewater. The degradation efficiency can reach 90% within 20min at range of 5-40μmol·L-1. These results ensure the potential practical application of this bio-materials in wastewater treatment.

Construction of nano-composites by enzyme entrapped in mesoporous dendritic silica particles for efficient biocatalytic degradation of antibiotics in wastewater

Dendritic silica particles (DSP) with center-radial vertical pore were prepared for entrapping chloroperoxidase (CPO) to construct CPO@DSP. This enzymatic composite is very stable in the presence of heat and organic solvents, remaining 90.74% activity at 80 °C after 3 h while the free CPO can keep only 14.28% activity at the same condition. Moreover, it was found the pore size of DSP can influence the catalytic activity of the composite. The pore should keep the accessible internal areas for the substrates besides accommodating the enzyme itself. A novel amyloid-like protein nanofilm formed by phase transition lysozyme (PTL) was coated around the surface of CPO@DSP (expressed as PTL-CPO@DSP), and then the PTL was cross-linked with another layer of CPO through glutaraldehyde to construct CPO@PTL-CPO@DSP. In this way, the CPO was widely distributed both in mesoporous channel and on the surface of DSP simultaneously. Both the enzyme loading and the reuse of CPO@PTL-CPO@DSP was greatly improved compared with CPO@DSP. After 20 cycle of use, CPO@PTL-CPO@DSP can maintain over 80% of its original activity. CPO@PTL-CPO@DSP were very efficient when applied in the degradation of the antibiotic levofloxacin and rifaximin in wastewater. Over 88% of both antibiotics can be degraded within 30 min at concentration of 20 μg·mL−1 in wasterwater.

Charge controlled immobilization of chloroperoxidase on both inner/outer wall of NHT: Improved stability and catalytic performance in the degradation of pesticide

Abstract 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 (Km, and kcat/Km) 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.

Improved Biodegradation of Synthetic Azo Dye by Anionic Cross-Linking of Chloroperoxidase on ZnO/SiO2 Nanocomposite Support.

A novel ZnO nanowire/macroporous SiO2 composite was used as a support to immobilize chloroperoxidase (CPO) by in situ cross-linking method. An anionic bi-epoxy compound was synthesized and used as a long-chained anionic cross-linker, and it was adsorbed on the surface of ZnO nanowires through static interaction before reaction with CPO, creating a new approach to change the structure, property, and catalytic performance of the produced cross-linking enzyme aggregates (CLEAs) of CPO. The immobilized CPO showed high activity in the decolorization of three azo dyes. The effect of various conditions such as the loading amount of CPO, solution pH, temperature, and dye concentration was optimized on the decolorization. Under optimized conditions, the decolorization percentage of Acid Blue 113, Direct Black 38, and Acid Black 10 BX reached as high as 95.4, 92.3, and 89.1%, respectively. The immobilized CPO exhibited much better thermostability and resistance to pH inactivation than free CPO. The storage stability and reusability were greatly improved through the immobilization. It was found from the decolorization of Acid Blue 113 that 83.6% of initial activity retained after incubation at 4°C for 60days and that 80.9% of decolorization efficiency retained after 12cycles of reuses.

Well-oriented bioarchitecture for immobilization of chloroperoxidase on graphene oxide nanosheets by site-specific interactions and its catalytic performance

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 H2O2 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 H2O2 improved from 2.5 to 6 mmol L−1, 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.

Enzymatic-photocatalytic synergetic effect on the decolorization of dyes by single chloroperoxidase molecule immobilization on TiO2 mesoporous thin film

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 8nm ~15nm 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 5min. 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.

Oxidative transformation of dibenzothiophene by chloroperoxidase enzyme immobilized on (1D)-γ-Al2O3 nanorods

(1D)-γ-Al2O3 nanorods (Al-nR) were synthesized, thermally treated and characterized by a variety of techniques. This material was used for chloroperoxidase (CPO) enzyme immobilization by physical adsorption and then assayed for the biocatalytic oxidation of dibenzothiophene (DBT). The textural properties of Al-nR nanostructured materials were successfully tuned to perform the CPO immobilization. The nanostructures exhibited a good stability throughout the thermal treatments since its crystallinity remained unchanged and the nanorods arrays were detected in all materials by XRD and HRTEM. A great affinity between the CPO and the nanorods was observed, due to favorable electrostatic interactions during the immobilization process. A remarkable increase on the dibenzothiophene oxidative biotransformation activity of immobilized CPO was obtained when compared with the free CPO. The morphological and textural properties of Al-nR-T material enhanced the enzymatic transformation of dibenzothiophene to form the sulfoxide derivative, especially when the Al-nR-700°C material was used. The CPO+Al-nR-700°C preparation exhibited almost twice the activity of the free CPO enzyme. The main product under our experimental conditions was DBT-sulfoxide. The potential application of biocatalytic nanorods for a fuel desulfurization process is discussed.

Silica Sol-Gel Entrapment of the Enzyme Chloroperoxidase

The enzyme chloroperoxidase (CPO) was immobilized in silica sol-gel beads prepared from tetramethoxysilane. The average pore diameter of the silica host structure (~3 nm) was smaller than the globular CPO diameter (~6 nm) and the enzyme remained entrapped after sol-gel maturation. The catalytic performance of the entrapped enzyme was assessed via the pyrogallol peroxidation reaction. Sol-gel beads loaded with 4 μg CPO per mL sol solution reached 9–12% relative activity compared to free CPO in solution. Enzyme kinetic analysis revealed a decrease inkcatbut no changes inKMorKI. Product release or enzyme damage might thus limit catalytic performance. Yet circular dichroism and visible absorption spectra of transparent CPO sol-gel sheets did not indicate enzyme damage. Activity decline due to methanol exposure was shown to be reversible in solution. To improve catalytic performance the sol-gel protocol was modified. The incorporation of 5, 20, or 40% methyltrimethoxysilane resulted in more brittle sol-gel beads but the catalytic performance increased to 14% relative to free CPO in solution. The use of more acidic casting buffers (pH 4.5 or 5.5 instead of 6.5) resulted in a more porous silica host reaching up to 18% relative activity.

Well-defined bioarchitecture for immobilization of chloroperoxidase on magnetic nanoparticles and its application in dye decolorization

Abstract 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.

Ordered Mesoporous Silica Matrix for Immobilization of Chloroperoxidase with Enhanced Biocatalytic Performance for Oxidative Decolorization of Azo Dye

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 hydroph...

Enhancing oxidation activity and stability of iso-1-cytochrome c and chloroperoxidase by immobilization in nanostructured supports

The immobilization of enzymes in inorganic materials has been widely used because it can produce an enhancement of the catalytic stability and enzymatic activity. In this article, the effect of the immobilization of iso-1-cytochrome c (CYC-Sc) from Saccharomyces cerevisiae and chloroperoxidase (CPO) from Caldariomyces fumago on the enzyme stability and catalytic oxidation of styrene was studied. The immobilization was carried out in three silica nanostructured supports with different pore size MCM-41 (3.3 nm), SBA-15 (6.4 nm) and MCF (12.1 nm). The adsorption parameters and leaching degree of immobilized enzymes were determined. Catalytic parameters of immobilized and free enzymes were determined at different temperatures (20–60 °C) and in different acetonitrile/water mixtures (15–85% of acetonitrile). The results show that there is low leaching of the enzymes in the three supports assayed and the adsorption capacity (qmax) was higher as the pore size of the support increased. The pore size also produces the enhancement of peroxidase activities on the styrene oxidation. Thus, CPO adsorption into SBA-15 and MCF showed remarkable thermal and solvent stabilities at 40 °C showing a total turnover numbers of 48,000 and 54,000 times higher than free CPO, respectively. The enhancement of activity and stability doubtless is interesting for the potential industrial use of peroxidases.

Recyclable Nanobiocatalyst for Enantioselective Sulfoxidation: Facile Fabrication and High Performance of Chloroperoxidase-Coated Magnetic Nanoparticles with Iron Oxide Core and Polymer Shell

Magnetic nanoparticles (MNPs) with a core diameter of 30 nm comprising several iron oxide crystals, a poly(glycidyl methacrylate) (PGMA) shell with a thickness of 30 nm, and a surface coated with chloroperoxidase (CPO) were facilely fabricated as a nanobiocatalyst for asymmetric sulfoxidation. The covalently bound CPO did not change the original conformation of the active site and showed the same catalytic activity and enantioselectivity as free CPO for the sulfoxidation of thioanisole to produce (R)-methyl phenyl sulfoxide in >99% ee. The thick PGMA shell significantly increased the stability of the nanobiocatalyst: no loss of the sulfoxidation activity was observed after 11 times of recycling and reuse of the catalyst. Thus, the nanobiocatalyst fabricated here showed the best performance among nanosized biocatalyst particles regarding both the retaining of free enzyme activity and the recycling of catalyst. This is also the first example of a nanobiocatalyst for asymmetric oxidation, and the concept could be generally applicable for fabricating active and recyclable nanobiocatalysts.

Improvement of chloroperoxidase stability by covalent immobilization on chitosan membranes

Chloroperoxidase (CPO) from Caldariomyces fumago was optimally covalently immobilized on chitosan membranes pretreated with 0.8 M glutaraldehyde at pH 3.5 to give 3.18 mg CPO g(-1) support. Using monochlorodimedone (MCD) as assay substrate, the immobilized-CPO retained 40% activity at 50 degrees C after 40 min whereas free CPO retained only 0.02%. The residual activity for immobilized-CPO was 99 and 58% compared with 68 and 43% for free CPO in the presence of 1.5 M urea and 300 microM H(2)O(2), respectively, after 20 h.