STABILITY OF PEPSIN (EC 3.4.23.1) DURING IN VITRO PROTEIN DIGESTIBILITY ASSAY²

Abstract

To maximize the efficiency of utilization of pepsin and estimate the contamination of pepsin for in vitro protein digestibility assays, the specific activity decay and peptide bond hydrolysis of pepsin incubated at different pH and concentration were studied with the bovine hemoglobin method and the o‐phthaldialdehyde method, respectively. It was found that increase of pH and concentration of pepsin increased pepsin's half‐life for both specific activity decay and peptide bond hydrolysis. The half‐life for specific activity decay was not extended by the presence of a substrate protein. The results indicated the time needed to maximize pepsin utilization depended on pH and the concentration of pepsin. At the time when all specific activity of pepsin was lost, the average size of pepsin autolysates was between 6.9 and 12.1 amino acid residues, suggesting most peptic protein would be fractionated as digestible protein.

Stability of a Pancreatic Enzyme Cocktail During in Vitro Protein Digestibility Assays

“…In the absence of substrate, the pancreatic proteases such as trypsin and chymotrypsin lost activity more slowly at higher concentrations than at lower ones (Table 4). This trend was also observed in previous studies with pepsin (Qiao et al. 2002).…”

“…In addition, the half‐life of trypsin at the concentration of pancreatic enzyme cocktail of 0.05 mg/mL, being 1.6 h in the absence of substrate (Table 4) and 88.4 h in the presence of meat and bone (Table 5), seemed to suggest there was substrate protection for trypsin. As substrate protection was not observed for pepsin (Qiao et al. 2002), and in some cases, the enzyme may even be more stable without substrate than with substrate as observed with catalase (Haldane 1965), substrate protection for enzymes appears to be enzyme‐specific.…”

“…From this model, a nonlinear expression for enzymatic protein digestion in terms of degree of hydrolysis ( DH , %), is derived (Qiao et al. 2002):…”

“…The behavior of pepsin during in vitro digestion was reported previously (Qiao et al 2002). Although pancreatin is often used during in vitro protein digestibility assays, little is known about the kinetics of the loss of its enzymatic activities and the extent of the digestion of the pancreatic proteins.…”

Enzymatically Active Ultrathin Pepsin Membranes

“…[1] Thehigh enzyme specificity and activity are attractive for the isolation of peptides and in food upgrading processes.H owever, enzyme re-usage is complicated by self-cleavage-induced deactivation and difficulties associated with the recovery of dilute enzyme solutions used in relevant processes. [2] Immobilized enzymes can be used instead, potentially reducing deactivation and increasing conversion specificity and activity. [3] Enzyme mobility can be restricted by covalent coupling to asubstrate or by self-cross-linking of the enzyme.Enzyme immobilization allows for simultaneous enzymatic conversion and removal of the converted products while maintaining the enzyme activity of the immobilized proteins.…”