Multipoint Immobilization at the Inert Center of Urease on Homofunctional Diazo-Activated Silica Gel: A Way of Restoring Room-Temperature Catalytic Sustainability for Perennial Utilization.

At present, enzyme immobilization is a big issue. It improves enzyme stability, activity, specificity, or selectivity, particularly the enantioselectivity compared to the native enzymes, and by solving the separation problem, it helps in recovering the catalyst with good reusability as desired in vitro. Motivated by these facts, in this work, Jack bean urease (JBU) is immobilized on three-dimensional (3D)-network silica gel (SG) via multipoint covalent bonding employing dimethyldichlorosilane (DMDCS) and -nitrophenol, respectively, as the second-generation silane-coupling reagent and spacer. The homofunctional diazo group appearing at the functionalized SG unit cell makes a diazo linkage at the inert center, the ortho position of the phenolic-OH of the tyrosine moiety, where all of the amino, thiol, phenol, imidazole, carboxy, etc., groups of the enzyme residues, including those that belong to the active site, remain intact. The coupling process, analyzed using field emission scanning electron microscopy (FESEM), energy-dispersive X-ray analysis (EDX), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR) spectroscopy, ultraviolet-visible spectroscopy (UV-vis), and fluorescence spectroscopy, occurs without molecular aggregation in borate buffer at pH 8.8 ± 0.4, which is much higher than the iso-electric point (pH 5.1) of the macromolecule where it becomes soluble. Eventually, the immobilization is maximize and also the native-enzyme activities are restored remarkably. The immobilized catalyst converts urea (0.0625-0.15 mmol L) to ammonia appreciably (94.50 ± 1.5%) at 27 °C, and the efficiency is well comparable to that of the native enzyme (93.0 ± 0.4%). The efficiency gradually diminishes, coming down to 50% at the 40th cycle, and the enzyme returns to its native conformation within 72 h in tris-EDTA borate buffer at 27 °C for the next 40 cycles of reuse and so on. The efficiency becomes hindered by 8-10% in every 5th subsequent reuse to reach 50% on the 30th reuse, resulting in room-temperature catalytic sustainability of 90 days. The catalytic performances are well restored in rice extract and coconut water.