Novel Application of Porous and Cellular Materials for Covalent Immobilization of Pepsin

Abstract

-Pepsin was immobilized via covalent bonds on different carriers: a silica gel carrier, acrylic beads, and a cellulose-based carrier -Granocel. All carriers were functionalized through the presence of -OH, -COOH, -NH 2 , or glycidyl groups on their surfaces. Three different cross-linkers were used for activation thereof. The results showed that Granocel activated by glutaraldehyde or carbodiimide and silica gel activated by glutaraldehyde were suitable carriers for the expression of enzyme activity. The optimum pH range for the native enzyme was 2.5-3.5 and this range was extended to the value 6.5 in the case of enzyme immobilized on the silica gel carrier and on Granocel. The optimum temperature values for the native and immobilized enzyme were in the range 37-40 °C and 40-50 °C, respectively. The activity of the immobilized pepsin at different values of pH and temperature was higher in comparison with the activity of the free enzyme.

Pepsin activity as a function of pH and digestion time on caseins and egg white proteins under static in vitro conditions

“…a bell-shaped curve with a rise around pH 3.5 and maximum around pH 2 (Figure 4). [13][14][15]30,45 The contrasted behaviours of substrate here reported might be of particular interest for the research community working in the field of dietary protein digestion, notably because the structure of the protein substrate and their extent of denaturation have been suggested to be the rate-determining factor for peptic digestion in weakly acidic conditions 16,17 . One should, indeed, not forget that the gastric pH after a meal decreases very slowly from about 6 to 2 before it remains about stable.…”

“…Lines are guides for the eyes and activity values were set arbitrary at 100% at pH = 2.0. Experimental data arise from the present study (means of OPA and pH-STAT data at 3 min) and the literature[13][14][15]30,45…”

Preparation of immobilized pepsin for extraction of collagen from bovine hide

“…The immobilization process can prevent molecular interaction and self-digestion, 20–22 as well as improving enzyme stability, activity, and selectivity. 23 Several methods have been used for enzyme immobilization, such as adsorption, trapping and covalent binding.…”

“…Immobilized enzymes can solve the problems of the high costs associated with using free enzymes, the timeconsuming separation of enzymolysis products from substrates, restriction of recovery and sample contamination, and are easy to use, separate and reuse. 11 The immobilization process can prevent molecular interaction and self-digestion, [20][21][22] as well as improving enzyme stability, activity, and selectivity. 23 Several methods have been used for enzyme immobilization, such as adsorption, trapping and covalent binding.…”

Pepsin immobilization on biochar by adsorption and covalent binding, and its application for hydrolysis of bovine casein

“…Since the second half of the 20 th Century, efforts have been made to develop substrates for enzyme immobilization, enabling their use in continuous processes. The immobilization of pepsin has been performed using different methods and insoluble polymeric supports, including silica gel, agarose bead, chitosan bead and porous and cellular materials. The use of immobilized enzymes allows reduction in operational costs, once it is possible to recover the enzyme from the reaction medium more efficiently by filtration, thus enabling the reuse of the enzyme and a more efficient control of large‐scale processes …”