Urease has been covalently immobilized on a 3-D networking silica gel (SG) using dimethyldichlorosilane (DMDCS) as second generation silane coupling reagent and m-nitroaniline as linker component in a robust methodology and subsequently characterized as [{Si­(OSi)4(H2O)0.05}205.2] n=4{OSi­(CH3)2–NH–C6H4–NN-urease}·282.5H2O (molecular mass 263 445 g or 263.4 kDa). Selective coupling of tyrosine residue with an identifiable m-nitroaniline modified SG unit prevents enzyme–enzyme cross-linking leading to enhancement of enzymatic activity. The material worked at room temperature and its activity (luminescent and ammonia releasing efficiency) was enhanced by 3-fold (for both synthetic and real sample) compared to native enzyme values at neutral pH. Up to 30 days and 30 cycles, this 3-fold activity remains as such but reduces gradually to native enzyme level after 60 days and 60 cycles of reuse.

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 p-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 −1 ) 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.

No abstract

Although enzymes play a significant role in industrial applications, their potential usage at high-level efficiency, particularly above room temperature, has not yet been fully harnessed. It brings above room-temperature catalytic sustainability of an immobilized (imm.) bio-catalyst as a long pending issue to improve enzyme stability, activity, specificity, or selectivity, particularly the enantio-selectivity over the native-enzymes. At this juncture, in a robust methodology, a heterogeneous solid phase bio-catalyst, {Si­(OSi)4(H2O)1.03} n=328{OSi­(CH3)2–NH–C6H4–NN}4{papain}­(H2O)251, has efficiently been prepared by immobilizing papain on homo-functionalized SG (silica-gel) via multipoint covalent attachment. The bio-catalyst is easy to be recovered and reused multiple times. The homo-functional −NN+, which appears on the SG-surface, makes the multipoint diazo-links with the inert center of the tyrosine-moiety to couple the enzyme where all the amino, thiol, phenol, and so forth, groups of the protein, including those that belong to the active-site, remain intact. The immobilized enzyme (13.9 μmol g–1) swims in pore-water within the pore-channel, remains stable up to 70 ± 5 °C, and exhibits wider temperature adaptability in performing its hydrolyzing activities. The relative activity, 78 ± 2% at 27 °C, remains quantitative for 60 days and can be reused for 60 cycles with 53% activity at room-temperature. The thermal (relative activity: 87%; incubated at 70 ± 5 °C for 24 h) and mechanical (relative activity: 92%; incubated at 2500 rpm for 2 h at 27 °C) stability was outstanding.

No abstract