Chemically Bonded Pepsin via Its Inert Center to Diazo Functionalized Silica Gel through Multipoint Attachment Mode: A Way of Restoring Biocatalytic Sustainability over “Wider pH” Range

Proteolytic enzymes play a pivotal role in the industry. Still, because of denaturation, the extensive applicability at their level of best catalytic efficiency over a more comprehensive pH range, particularly in alkaline conditions over pH 8, has not been fully developed. On the other hand, enzyme immobilization following a suitable protocol is a long pending issue that determines the conformational stability, specificity, selectivity, enantioselectivity, and activity of the native enzymes at long-range pH. As a bridge between these two findings, in an attempt at a freezing temperature 273–278 K at an alkaline pH, the diazo-functionalized silica gel (SG) surface has been used to rapidly diazo couple pepsin through its inert center, the O-carbon of the phenolic −OH of surface-occupied Tyr residues in a multipoint mode: when all the various protein groups, viz., amino, thiol, phenol, imidazole, carboxy, etc., in the molecular sequence including those belonging to the active sites, remain intact, the inherent inbuilt interactions among themselves remain. Thereby, the macromolecule’s global conformation and helicity preserve the status quo. The dimension of the SG-enzyme conjugate confirms as {Si­(OSi)4 (H2O)1.03} n {−O–Si­(CH3)2–O–C6H4–NN+}4·{pepsin}·yH2O; where the values of n and y have been determined respectively as 347 and 188. The material performs the catalytic activity much better at 7–8.5 than at pH 2–3.5 and continues for up to six months without any appreciable change.