Pepsin immobilized

On-line hydrolysis of proteins catalyzed by trypsin or pepsin immobilized on monolithic silica beds was described by Kato et al. [86,195], whereas pepsin was encapsnlated into the silica-gel matrix (75 pm capillary column), without loss in enzymatic activity [195]. 

The work of Ferrier et al. (192) and Cheryan et al. (193) have definitely established the feasibility of using immobilized proteases, particularly immobilized pepsin, for the continuous clotting of milk. The major problem is the decay in enzymic activity apparently resulting from the coating of the immobilized pepsin with milk proteins creating a steric barrier. However, recently Lee (194) prepared a pepsin immobilized on protein-coated glass beads with a usable lifetime of about 200 hr defined as the time for the clotting time of the milk to decay from 1 min to 10 min. 

Ahn, J., Jung, M.C., Wyndham, K., et al. (2012) Pepsin immobilized on high-strength hybrid particles for continuous flow online digestion at 10,000 psi. Analytical Chemistry, 84 (16), 7256-7262. 

A protocol to improve pepsin immobilization was proposed by Busby et al. [17], Rather than directly using pepsin, they immobilized pepsinogen to POROS AL-20 support under optimal coupling conditions of pH 6.7 where pepsinogen is stable (in contrast with pepsin). Subsequently, they converted the coupled pepsinogen into active pepsin. Protein assays demonstrated that more enzyme was bound to the POROS AL-20 resin coupled with pepsinogen at pH 6.7 than pepsin at pH 5. 

Covalent linking of enzyme on fused silica capillaries was also demonstrated by Stigter [119]. Pepsin was covalently immobilized on dextran-modified capillaries as illustrated in Scheme 10.12. The applicability of pepsin-immobilized microreactor was tested with a number of proteins varying in molecular weight, isoelectric point, and sample composition. This open tubular microreactor demonstrated a flow-dependent digestion as represented by native hemoglobin. Complete digestion of... 

Kurimoto, E. Harada, T. Akiyama, A. Sakai, T., Kato, K. (2001) In vitro refolding of porcine pepsin immobilized on agarose beads. /. Biochem. (Tokyo).130, pp. 295-297. 

Equilibrate by washing 0.25 ml of immobilized pepsin (Thermo Fisher) with 4 X 1ml of 20 mM sodium acetate, pH 4.5 (digestion buffer). Finally, suspend the gel in 1ml of digestion buffer. 

Figure 1. Schematic representation of the relationships between proposed catalytic and inhibitory mechanisms. A. Postulated general acid-general base catalyzed mechanism for substrate hydrolysis by an aspartyl protease. The water molecule indicated is extensively hydrogen bonded to both aspartic acid residues plus other sites in the active site (see Reference 16 for details). Hydrogen bonds to water are omitted here. B. Kinetic events associated with the inhibition of pepsin by pepstatin. The pro-S hydroxyl group of statine displaces the enzyme immobilized water molecule shown in Figure lA. Variable aspartyl sequence numbers refer to penicillopepsin (pepsin, Rhizopus pepsin), respectively.

Mixed protein/polysaccharide micro-beads have also been found to be promising delivery vehicles for immobilized bifidobacteria (Guerin et al, 2003). Such micro-beads were made by a transacylation reaction involving the formation of amide bonds between protein and alginate (Levy and Edwards-Levy, 1996). This produces a membrane on the bead surface, protecting the immobilized bifidobacteria against both the very acidic conditions (pH 1-2) and the pepsin activity in the stomach. 

I Yab )2 (110 kDa) using immobilized Ficin at ImM cysteine or immobilized pepsin... 

Conclusion This preliminary work reveals whey proteins as a convenient material for immobilizing recombinant yeasts. Gel beads were resistant to acidification until pH 2 and pepsin attack, suggesting that they should cross the gastric barrier in humans. Moreover, the presence of the protein matrix seemed to create microconditions that favor the heterologous activity of entrapped yeasts. 

Fig. 4.7.4. (A) Capacity of immobilized inhibitor sorbent (e-aminocaproyl-L-Phe-D-Phe-OMe-Separon) in mg of pepsin per g of dry sorbent, and (B) portion of immobilized inhibitor molecules involved in specific complex formation (in %) with respect to immobilized inhibitor concentration (in pmol of inhibitor per g of dry sorbent). -0-, porcine pepsin chicken pepsin, and human pepsin.

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