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

Abstract

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.

Keywords: electrostatic interactions; enzyme protonation; lipase hybrid nanoflowers.

Figure 1

Relative activity of hNF-lipases relative to the free enzyme, using pNFA as the substrate, at pH 7.4, for different buffer concentration (10–100 mM) (p < 0.01).

Figure 2

The activity of nanoflowers concerning the free enzyme (yellow), with pNFA as the substrate. Different concentrations of buffer concentration, 25 mM (pink), 50 mM (blue), and 100 mM (green), at different pHs (6–8) (p < 0.01).

Figure 3

Electrostatic charge at the BCL surface, calculated for pHs 5 to 9. Red–white–blue scale refers to minimum (−5 kT/e, red) and maximum (5 kT/e, blue) surface potential.

Figure 4

All molecular docking poses of BCL with Cu²⁺. Lid domain in green and catalytic triad amino acids (SER87, ASP264, and HIS286) in purple.

Figure 5

SEM images of hNF-lipases at different pHs (6–8) and at different buffer concentrations: (a–d) 25 mM, (e–h) 50 mM, and (i–l) 100 mM.

Figure 6

FTIR spectrum for hNF-lipase synthesized at pH 7.4 (100 mM) compared with free lipase and PBS-Cu²⁺.