Preparation and catalytic properties of catalase-inorganic hybrid nanoflowers / 生物工程学报

Catalase is widely used in the food, medical, and textile industries. It possesses exceptional properties including high catalytic efficiency, high specificity, and environmental friendliness. Free catalase cannot be recycled and reused in industry, resulting in a costly industrial biotransformation process if catalase is used as a core ingredient. Developing a simple, mild, cost-effective, and environmentally friendly approach to immobilize catalase is anticipated to improve its utilization efficiency and enzymatic performance. In this study, the catalase KatA derived from Bacillus subtilis 168 was expressed in Escherichia coli. Following separation and purification, the purified enzyme was prepared as an immobilized enzyme in the form of enzyme-inorganic hybrid nanoflowers, and the enzymatic properties were investigated. The results indicated that the purified KatA was obtained through a three-step procedure that included ethanol precipitation, DEAE anion exchange chromatography, and hydrophobic chromatography. Then, by optimizing the process parameters, a novel KatA/Ca3(PO4)2 hybrid nanoflower was developed. The optimum reaction temperature of the free KatA was determined to be 35 ℃, the optimum reaction temperature of KatA/Ca3(PO4)2 hybrid nanoflowers was 30-35 ℃, and the optimum reaction pH of both was 11.0. The free KatA and KatA/Ca3(PO4)2 hybrid nanoflowers exhibited excellent stability at pH 4.0-11.0 and 25-50 ℃. The KatA/Ca3(PO4)2 hybrid nanoflowers demonstrated increased storage stability than that of the free KatA, maintaining 82% of the original enzymatic activity after 14 d of storage at 4 ℃, whereas the free KatA has only 50% of the original enzymatic activity. In addition, after 5 catalytic reactions, the nanoflower still maintained 55% of its initial enzymatic activity, indicating that it has good operational stability. The Km of the free KatA to the substrate hydrogen peroxide was (8.80±0.42) mmol/L, and the kcat/Km was (13 151.53± 299.19) L/(mmol·s). The Km of the KatA/Ca3(PO4)2 hybrid nanoflowers was (32.75±2.96) mmol/L, and the kcat/Km was (4 550.67±107.51) L/(mmol·s). Compared to the free KatA, the affinity of KatA/Ca3(PO4)2 hybrid nanoflowers to the substrate hydrogen peroxide was decreased, and the catalytic efficiency was also decreased. In summary, this study developed KatA/Ca3(PO4)2 hybrid nanoflowers using Ca2+ as a self-assembly inducer, which enhanced the enzymatic properties and will facilitate the environmentally friendly preparation and widespread application of immobilized catalase.

Immobilization of horseradish peroxidase on Fe3O4 magnetic nanoparticles

Background: Iron magnetic nanoparticles have attracted much attention. They have been used in enzyme immobilization because of their properties such as product is easily separated from the medium by magnetic separation. The present work was designed to immobilize horseradish peroxidase on Fe3O4 magnetic nanopraticles without modification. Results: In the present study, horseradish peroxidase (HRP) was immobilized on non-modified Fe3O4 magnetic nanoparticles. The immobilized HRP was characterized by FT-IR spectroscopy, scanning electron microscopy, and energy dispersive X-ray. In addition, it retained 55% of its initial activity after 10 reuses. The optimal pH shifted from 7.0 for soluble HRP to 7.5 for the immobilized HRP, and the optimal temperature shifted from 40°C to 50°C. The immobilized HRP is more thermostable than soluble HRP. Various substrates were oxidized by the immobilized HRP with higher efficiencies than by soluble HRP. Km values of the soluble and immobilized HRP were 31 and 45 mM for guaiacol and 5.0 and 7.0 mM for H2O2, respectively. The effect of metals on soluble and immobilized HRP was studied. Moreover, the immobilized HRP was more stable against high concentrations of urea, Triton X-100, and isopropanol. Conclusions: Physical immobilization of HRP on iron magnetic nanoparticles improved the stability toward the denaturation induced by pH, heat, metal ions, urea, detergent, and water-miscible organic solvent.

Kinetic constraints and features imposed by the immobilization of enzymes onto solid matrices: A key to advanced biotransformation.

The kinetics of immobilized enzymes can not be analyzed by means of the simple Michaelis-Menten concept, which generally fails to describe the immobilized state due to both its probable barriers, and because the active concentration of the enzyme approaches, or even exceeds this of its substrate(s). In such cases, the various experimental data are usually treated by complex rate equations comprising too many parameters acquiring different natures and meanings, depending on both the properties of the immobilization state and the experimental conditions; thus, more likely, only apparent values of the Michaelis-Menten kinetic parameters can be estimated experimentally. Likewise, immobilization is often a key method in optimizing the operational performance of enzymes, in both laboratory and industrial scale, and affects considerably the kinetics in non-aqueous and non-conventional media due to several issues as the structural changes of the enzyme molecule, the heterogeneity of the system, and the partial or total absence of water. In this work a theoretical approach is described on the formulation of simplified rate equations, reflecting also the actual mass balances of the reactants, in the case where esterification synthetic reactions are catalyzed by immobilized lipases, in either a non-aqueous organic solvent or in a non-solvent system.