Experimental and Computational Analysis of Synthesis Conditions of Hybrid Nanoflowers for Lipase Immobilization.

This work presents a framework for evaluating hybrid nanoflowers using Burkholderia cepacia lipase. It was expanded on previous findings by testing lipase hybrid nanoflowers (hNF-lipase) formation over a wide range of pH values (5-9) and buffer concentrations (10-100 mM). The free enzyme activity was compared with that of hNF-lipase. The analysis, performed by molecular docking, described the effect of lipase interaction with copper ions. The morphological characterization of hNF-lipase was performed using scanning electron microscopy. Fourier Transform Infrared Spectroscopy performed the physical-chemical characterization. The results show that all hNF-lipase activity presented values higher than that of the free enzyme. Activity is higher at pH 7.4 and has the highest buffer concentration of 100 mM. Molecular docking analysis has been used to understand the effect of enzyme protonation on hNF-lipase formation and identify the main the main binding sites of the enzyme with copper ions. The hNF-lipase nanostructures show the shape of flowers in their micrographs from pH 6 to 8. The spectra of the nanoflowers present peaks typical of the amide regions I and II, current in lipase, and areas with P-O vibrations, confirming the presence of the phosphate group. Therefore, hNF-lipase is an efficient biocatalyst with increased catalytic activity, good nanostructure formation, and improved stability.

Practical and Rapid Membrane-Based Biosensor for Phenol Using Copper/Calcium-Enzyme Hybrid Nanoflowers.

Phenol, a pollutant frequently found in chemical industries effluents, is highly toxic even in low concentrations. This study reports a green, simple, and rapid method for qualitative phenol biosensing using horseradish peroxidase (HRP) hybrid nanoflowers made with copper (Cu2+-hNF) or calcium (Ca2+-hNF) ions. The enzyme was immobilized through protein-inorganic self-assembly into hybrid structures and subsequently supported onto a polyvinylidene fluoride (PVDF) membrane. SEM, EDS, FTIR, and XRD techniques sustained the effective enzyme encapsulation into hybrid structures. The protein concentration in the structures was 0.25 mg.mL-1 for both ions. The best temperature and pH were 60 °C and 7.4, respectively, for both hybrids and the free enzyme, suggesting that the immobilization did not affect the optimal conditions of the free HRP. Thermal stability from 25 to 70 °C and pH stability from 4.0 to 9.0 of the hybrids were also determined. Finally, using copper and calcium hybrids, both biosensors produced onto a PVDF membrane could detect phenol in concentrations ranging from 0.72 to 24.00 µmol.mL-1 in 1 min. In contrast, control biosensors produced with free enzyme have not presented a visible color change in the same conditions. The findings suggest a promising application of the developed biosensors in functional phenol detection.