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

Abstract

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 longrange pH. As a bridge between these two findings, in an attempt at a freezing temperature 273−278 K at an alkaline pH, the diazofunctionalized 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) 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.

Controlled Primary Amine-Enriched SG-Bonded Papain Surface: Synthesis, Characterization, and Extraction of Protonated Dichromate

“…The well-distinguished N 1s peak of lower binding energy (400.38 eV) confirms the primary amine (−NH 2 of lysine, arginine, asparagine, glutamine, etc.) and the secondary amine (NH of the tryptophan, proline residues). , On the other hand, the N 1s peak at higher binding energy (403.63 eV) identifies the imidazole nitrogen of the histidine residue and the presence of nitrogen in the diazo group. ,, The elemental populations of the constituent atoms (%) are shown in the inset in Figure a. Thus, the XPS studies confirm the protein residues within the synthesized compound.…”

“…The two well-defined peaks at 103.98 and 104.05 eV centered at 103.95 eV in high-resolution XPS (Figure b) characterized the Si 2p and appeared in two different chemical environments, {Si–O–Si} n in the SG core and the –Si(CH 3 ) 2 – fragment appeared in between the {SG} n -core and the m -nitroaniline. ,, The C 1s peak at 285.21 eV is deconvoluted into two components and centered at 284.29 eV (Figure c); the high intense XPS peak at 284.8 eV briefs the aliphatic and the acyclic carbon, while the low intense peak at 286.08 eV appeared for the aromatic and heterocyclic rings and the carbonyl carbon . From the O 1s peak 533.02 eV, the two deconvoluted peaks of 532.07 and 533.27 eV centered at 533.19 eV appeared (Figure d); the 532.07 eV XPS peak represents the {Si–O–Si} n in the SG core and {SG-core}–O–Si(CH 3 ) 2 –, while the high=-energy XPS peak at 533.27 eV briefs the carbonyl fragments. − Two distinct, well-separated, and deconvoluted XPS peaks (Figure e), at 400.38 and 406.12 eV, appear from the 400.14 eV N 1s XPS peak. The well-distinguished N 1s peak of lower binding energy (400.38 eV) confirms the primary amine (−NH 2 of lysine, arginine, asparagine, glutamine, etc.)…”