Laccase-loaded magnetic dialdehyde inulin nanoparticles as an efficient heterogeneous natural polymer-based biocatalyst for removal and detoxification of ofloxacin.

An efficient heterogeneous natural polymer-based biocatalyst was fabricated through the immobilization of laccase onto dialdehyde inulin (DAI)-coated silica-caped magnetic nanoparticles (laccase@DAI@SiO2@Fe3O4⋅MNPs). The carrier was developed using SiO2@Fe3O4⋅MNPs and functionalized with DAI. The construction of immobilized laccase was confirmed by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. Immobilization yield and efficiency were calculated as 61.0 ± 0.3% and 93.0 ± 0.6%, respectively. The immobilized laccase maintained 50% and 85% of its relative activity after 25 repeated cycles and 20 days of storage at 4 °C, respectively. The prepared biocatalyst effectively eliminated ofloxacin, a fluoroquinolone-type antibiotic, with a 63% removal capacity. Besides, antimicrobial activity study on some soil microorganisms involved in the biodegradation of xenobiotics revealed that the laccase-treated ofloxacin resulted in less toxic metabolites. The obtained data indicated that the fabricated biocatalyst is promising for the removal of ofloxacin or other analogs of fluoroquinolones in the environment.

Hierarchically-structured laccase@Ni3(PO4)2 hybrid nanoflowers for antibiotic degradation: Application in real wastewater effluent and toxicity evaluation.

Laccase@Ni3(PO4)2 hybrid nanoflowers (HNFs) were prepared by the anisotropic growth of biomineralized nickel phosphate. The immobilization yield was 77.5 ± 3.6 %, and the immobilized enzyme retained 50 % of its initial activity after 18 reusability cycles. The immobilized and free enzymes lost 80 % of their activity after 18 and 6 h incubation in municipal wastewater effluent (MWWE), respectively. The increase in α-helix content (8 %) following immobilization led to a more rigid enzyme structure, potentially contributing to its improved stability. The removal of ciprofloxacin from MWWE by laccase@Ni3(PO4)2·HNFs/p-coumaric acid oxidation system was optimized using a Box-Behnken design. Under the optimized conditions [initial laccase activity (0.05 U mL-1), the concentration of p-coumaric acid (2.9 mM), and treatment time (4.9 h)], the biocatalyst removed 90 % of ciprofloxacin (10 mg L-1) from MWWE. The toxicity of ciprofloxacin against some G+ and G- bacteria was reduced by 35-70 %, depending on their strain. The EC50 of ciprofloxacin for the alga Raphidocelis subcapitata reduced from 3.08 to 1.07 mg L-1 (p-value <0.05) after the bioremoval. Also, the acute and chronic toxicity of identified biodegradation products was lower than ciprofloxacin at three trophic levels, as predicted by ECOSAR software.

Organic-inorganic hybrid nanoflowers: The known, the unknown, and the future.

Organic-inorganic hybrid nanoflowers (HNFs) are hierarchical flower-shaped microstructures that are assembled by nanoscale petal-like nanosheets composed of both organic and inorganic constituents. Generally, inorganic parts of HNFs are transition metal phosphates and organic components are mostly enzymes and proteins; however, non-protein molecules could be also used as organic phase in some types of newly described HNFs. Recent findings indicate that they are constructed through the coordination between organic and inorganic components. HNFs are mainly used for efficient biocatalysis and highly sensitive biosensing, while they have also some other noteworthy applications such as antimicrobial agents, antigen careers, and delivery platforms for anticancer drugs. It is believed that the high surface-to-volume ratio of HNFs could tackle mass transfer limitations leading to enhance the activity of their organic constituents. The environment-friendly route of synthesis and stabilization of biomolecules upon storage are the advantages of enzyme-based HNFs. In the present review, the focuses are on designs, preparations, formation mechanisms, and remarkable applications of the conventional forms and also magnetic, multi-component, and enzyme-free HNFs. Considering the fact that HNFs are in the early stages of development, the unknown aspects and future directions of research in this field are also discussed.

Instantaneous synthesis and full characterization of organic-inorganic laccase-cobalt phosphate hybrid nanoflowers.

A novel approach termed the "concentrated method" was developed for the instant fabrication of laccase@Co3(PO4)2•hybrid nanoflowers (HNFs). The constructed HNFs were obtained by optimizing the concentration of cobalt chloride and phosphate buffer to reach the highest activity recovery. The incorporation of 30 mM CoCl2 and 160 mM phosphate buffer (pH 7.4) resulted in a fast anisotropic growth of the nanomaterials. The purposed method did not involve harsh conditions and prolonged incubation of precursors, as the most reported approaches for the synthesis of HNFs. The catalytic efficiency of the immobilized and free laccase was 460 and 400 M-1S-1, respectively. Also, the enzymatic activity of the prepared biocatalyst was 113% of the free enzyme (0.5 U mL-1). The stability of the synthesized HNFs was enhanced by 400% at pH 6.5-9.5 and the elevated temperatures. The activity of laccase@Co3(PO4)2•HNFs declined to 50% of the initial value after 10 reusability cycles, indicating successful immobilization of the enzyme. Structural studies revealed a 32% increase in the α-helix content after hybridization with cobalt phosphate, which improved the activity and stability of the immobilized laccase. Furthermore, the fabricated HNFs exhibited a considerable ability to remove moxifloxacin as an emerging pollutant. The antibiotic (10 mg L-1) was removed by 24% and 75% after 24 h through adsorption and biodegradation, respectively. This study introduces a new method for synthesizing HNFs, which could be used for the fabrication of efficient biocatalysts, biosensors, and adsorbents for industrial, biomedical, and environmental applications.

Hybridization of laccase with dendrimer-grafted silica-coated hercynite-copper phosphate magnetic hybrid nanoflowers and its application in bioremoval of gemifloxacin.

Laccase was successfully hybridized with polyamidoamine (PAMAM) dendrimer-grafted silica-coated hercynite-copper phosphate magnetic hybrid nanoflowers (MHNFs) to increase the catalytic performance of the enzyme and apply in an effective bioremoval of gemifloxacin. For this purpose, the magnetic nanoparticles (MNPs) of hercynite were covered with a silica layer, and the core-shell SiO2@hercynite was then modified with PAMAM dendrimer to increase the surface area of the carrier for the enzyme attachment. Subsequently, the whole complex was hybridized with laccase and copper phosphate to attain a large surface area (104.3 m2 g-1). The fabricated MHNFs acquired the entrapment yield and efficiency of 90 ± 3% and 66 ± 5%, respectively. The catalytic activity of the fabricated biocatalyst was remained up to 50% after 13 reusability cycles. Approximately 90% of gemifloxacin was removed by the constructed MHNFs after 3 h incubation by adsorption and degradation mechanisms. The biotransformation products were then identified, and degradation pathways were proposed as defluorination, decarboxylation, elimination of a cyclopropyl group, and cleavage of the pyrrolidine moiety. Furthermore, the toxicity of gemifloxacin was effectively diminished against some bacterial strains.

Magnetic casein aggregates as an innovative support platform for laccase immobilization and bioremoval of crystal violet.

In this study, casein@CoFe2O4 was fabricated through a green synthesis methodology and applied to immobilize laccase. The constructed casein@CoFe2O4 exhibited porous structures with distinct cavities and suitable magnetic properties. The abundance of aromatic functional groups on the surface renneted casein and possible π-type interaction between laccase and para-κ-casein resulted in a successful immobilization. The biocatalyst retained 50% of its initial activity after 24 reusability cycles, indicating stable immobilization of laccase onto the casein microstructures. The stability of laccase after immobilization was improved by 300% in comparison with the free enzyme, especially in basic pH values. The constructed laccase@casein@CoFe2O4 was then incorporated to remove crystal violet (CV) as an environmentally harmful synthetic tri-phenylmethane dye. The prepared heterogeneous biocatalyst effectively diminished the antimicrobial activity of CV up to 81.3% in 40 min against some bacterial strains, resulting from the formation of more minor toxic metabolites identified by liquid chromatography coupled with mass spectroscopy after degradation procedure. The proposed green and feasible method for the preparation of magnetic casein aggregates has not been previously reported. The incorporation of casein, which acted as a molecular chaperon, resulted in a significant improvement in the enzymatic stability and exhibited appropriate reusability for the constructed biocatalytic system.

Fast anisotropic growth of the biomineralized zinc phosphate nanocrystals for a facile and instant construction of laccase@Zn3(PO4)2 hybrid nanoflowers.

Organic-inorganic hybrid nanoflowers (HNFs) of laccase@Zn3(PO4)2 were fabricated through a facile, simple, and rapid one-step strategy. In this process, laccase was involved in nucleation and fast anisotropic growth reactions with Zn (II) and phosphate ions. The average pore size of the prepared HNFs was 54.5 nm, and its BET-specific surface area was 59.5 m2 g-1. In comparison with the free laccase, the entrapped enzyme activity in the constructed HNFs was 86.4%. In addition, the hybrid biocatalyst displayed a maximum rate of reaction (Vmax) of 1640.2 ± 3.6 µmol min-1 with respect to the native enzyme. The constructed HNFs maintained 45.1% and 60% of the original laccase activity after 12 successive reusability cycles and 30 days of storage at 4 °C, respectively. The as-obtained HNFs demonstrated a high bioremoval percentage of Direct blue-71 (94.1%) within a 10-h-treatment at 40 °C and 15 mg l-1 of the dye concentration. The pseudo-first order and second order were the best-fitted kinetic models for the dye removal using Zn3(PO4)2 nanoflakes and the fabricated HNFs, respectively. Besides, liquid chromatography-mass spectrometry (LC-MS) revealed biotransformation of the dye into less toxic metabolites as verified by testing on some bacterial strains.

A survey on the stabilizing effect of osmolytes on the ultrasound-irradiated lipase for efficient enzymatic hydrolysis of coconut oil.

The stabilizing effect of some osmolytes including betaine, mannitol, proline, sorbitol, and trehalose (each 0.5 M) was investigated on the ultrasound-irradiated (60 kHz and 138 W, for 240 min) lipase by determination of the enzyme half-life time, evaluation of the enzymatic reaction velocity (Vmax), and hydrolysis of coconut oil for production of lauric acid (the main saturated fatty acid of the oil). The enzyme conformational stability was also assessed by circular dichroism (CD) and fluorescence spectroscopy. The average half-life time of mannitol- and sorbitol-treated lipase under the ultrasound irradiation was 511 ± 3 min and 531 ± 2 min, respectively; 3-fold higher than the unirradiated enzyme. The Vmax value of the ultrasound-treated lipase increased from 100 ± 3 nmol min-1 in the absence of osmolyte to 500 ± 7 nmol min-1 and 500 ± 9 nmol min-1 in the presence of mannitol and sorbitol, respectively. CD and fluorescence spectra indicated that mannitol and sorbitol enhanced the rigidity of the lipase molecular conformational structure, increasing the enzyme stability against the ultrasonic field. The ultrasound-irradiated lipase was then used to hydrolyze coconut oil in the absence or presence of the selected osmolytes, which led to liberate 310 ± 6 mg g-1, 413 ± 7 mg g-1, and 420 ± 4 mg g-1 of lauric acid in the absence or presence of sorbitol and mannitol, respectively. In the absence of an ultrasonic field, the non-osmotically-treated lipase was able to liberate only 211 ± 5 mg g-1 of lauric acid. These promising results indicate that sorbitol and mannitol stabilize the structural conformation of lipase under an ultrasonic field which in turn could improve the enzymatic hydrolysis of coconut oil.

High efficiency of osmotically stable laccase for biotransformation and micro-detoxification of levofloxacin in the urea-containing solution: Catalytic performance and mechanism.

Laccase-catalyzed oxidation was applied in the biotransformation of levofloxacin (a potentially environmental antibiotic contamination); however, the enzyme may denature in urea-containing wastewater and lead to the formation of an inactive form followed by decreasing the yield of the bio-removal. In this study, the osmolytes-stabilized laccase was used to eliminate levofloxacin in the urea-containing solution. Sorbitol and proline 100 mM appeared to be the two most efficient laccase protectants against the urea-induced denaturation. In a 1-M urea solution, the maximum velocity (Vmax) of laccase was estimated to be 39.1 µmol min-1 mg-1. This value was improved to 101.7 and 51.8 µmol min-1 mg-1 in the presence of sorbitol and proline, respectively. In optimal conditions for the elimination of levofloxacin, sorbitol- and proline-treated laccase led to 82.9 % and 76.2 % bio-removal of the applied fluoroquinolone in 1 M urea solution, respectively. Biotransformation products of the parent antibiotic were spectroscopically analyzed that assigned to different reaction pathways including demethylation, defluorination, decarboxylation, deamination, and hydroxylation. A micro-toxicity study concerning the growth of some Gram+ and Gram- bacteria exhibited decreasing in inhibition of laccase-treated levofloxacin after a 10-h incubation at 37 °C.