Trypsin-Based Chemoenzymatic Assay for Detection of Pollutants and Safety Assessment of Food Additives

Abstract

Chemoenzymatic assay systems are widely used to detect toxicants in various samples, including food and environment specimens. These methods are based on the ability of various types of toxicant to specifically inhibit/activate the functions of individual enzymes or enzyme systems. The present study examines the possibility of using the proteolytic enzyme trypsin as a specific marker to detect protease inhibitors in different samples. The study shows that trypsin activity is not affected by various heavy metals, pesticides, or quinones at levels considerably greater than their maximum allowable concentrations (MACs) in water bodies. At the same time, the IC50 value for the food preservative potassium sorbate (E202) is 15 mg/L, which is substantially lower than its acceptable daily intake (ADI). The quenching of trypsin fluorescence in the presence of potassium sorbate suggests that inhibition could occur due to the binding of the preservative to the enzyme in the region adjacent to the active center. The trypsin was immobilized in starch gel to ensure its stability in the enzyme inhibition based assay. Single-use reagents were prepared as dry starch disks that could be stored over long periods. Their sensitivity to copper (II) chloride, potassium sorbate, and chromium (III) chloride was similar to the sensitivity of the free trypsin.

Alternative Enzyme Inhibition Assay for Safety Evaluation of Food Preservatives

“…The IC 50 values for sorbic acid and potassium sorbate were calculated to be 1.6 and 12 mg/L, respectively [ 33 ]. A similar result was reported in [ 34 ], where the authors suggested that inhibition of the trypsin activity by potassium sorbate could occur due to the binding of the preservative to the enzyme in the region adjacent to the active center.…”

Enzymes Immobilized into Starch- and Gelatin-Based Hydrogels: Properties and Application in Inhibition Assay

“…The final step may include pipetting the mixture onto the support and drying (Figure 2). This is the way to produce single-use reagents containing the preset amount of enzyme (Figure 3) [57][58][59][60]. In this form, they can be used in cuvette-based instruments (spectrophotometer, luminometer).…”

“…However, the advantage of this method is that it saves on expensive enzyme preparations. The above-mentioned method of immobilizing enzymes by entrapping them in starch and gelatin hydrogels without any previous modifications was successfully used to produce stable preparations of butyrylcholinesterase (BChE), trypsin (Try), firefly luciferase (FLuc), and a bioluminescent bi-enzyme system of luminous bacteria: NAD(P)H:FMNoxidoreductase and luciferase (Red + Luc) [58][59][60].…”

“…The activity of the free Red + Luc enzymes in solution, however, dropped to zero in three days, even when it was stored at 8 • C [71]. Trypsin immobilized in starch gel stored at 8 • C for 4 months retained 80% of its activity [60]. The most impressive results were obtained for BChE immobilized (alone or together with the thiol group indicator-5,5 -dithiobis(2-nitrobenzoic acid)) in both starch gel and gelatin gel.…”

“…The sensitivity of trypsin immobilized in starch gel was assessed in experiments with pollutants and food additives [60]. The sensitivity of trypsin immobilized in starch gel to CuCl 2 , potassium sorbate, and CrCl 3 was comparable to the sensitivity of the free trypsin.…”