When is pepsin most active

These interactions make pepsin active in an acidic environment with optimum pH between the values of 1. In our study, the optimum pH values for native pepsin were in the range of 2. The pepsin preparation immobilized on the silica gel support exhibited a pH optimum in the range of 2. Similarly, in the case of the cellulose-based supports, we observed that immobilized pepsin did not lose its activity in the pH range from 2.

At pH 6. Altum and Cetinus reported that pepsin immobilized on chitosan beads had no activity at pH 7. This is probably due to reduced conformational flexibility after immobilization.

Additionally, the interactions ionic, polar, or hydrogen bonding between the enzyme molecule and the carrier microspheres can change the ionisation of immobilized enzyme molecules Hu et al. During the immobilization process different groups of the carrier react with amine groups in pepsin, including the N-terminus, but these interactions do not disturb the proteolytic activity of pepsin.

Figure 3 Activity under various conditions of pH for native enzyme and pepsin immobilized on silica gel carriers and acrylic support carrier 2 Fig. The value of pH 3. The results obtained confirm that inorganic supports have greater dimensional stability than organic ones. The process of pepsin immobilization can increase the scope of its activity over a broader range of pH values in comparison to the native enzyme.

Furthermore, the immobilization process probably resulted in formation of two different enzyme forms with different optima for temperature and the value of pH Fig. This feature may facilitate using immobilized pepsin in the digestion of proteins soluble in slightly alkaline solutions.

The operational stability, which specifies the time after which half of the initial activity of enzyme is lost, is the basic technological parameter allowing evaluation of the desirability of applying the chosen immobilization method. This parameter is very important for preparative and industrial use of different form of enzymes.

In our study, the operational stability of the immobilized pepsin was tested in consecutive batch processes Fig. Figure 4 Operational stability for pepsin immobilized on cellulose-based carriers.

Figure 5 Operational stability for pepsin immobilized on silica-gel carriers and acrylic carriers carrier 2. Altun and Cetinus Altun, G. The ability of immobilized pepsin to maintain the enzymatic activity for several operating cycles gives it advantages over native, non-immobilized enzyme. Three different groups of carriers acrylic, cellulose-based, and silicagel supports were tested for immobilization of pepsin from hog stomach.

The results showed that only the cellulose-based carriers activated by glutaraldehyde or carbodiimide and silicagel activated by glutaraldehyde may be used to bind pepsin effectively. Very significant improvement in the stability of the immobilized pepsin over a broader pH range was observed, in comparison to the native enzyme.

Promising results for thermal and operational stability of the immobilized pepsin were also shown, compared to the free enzyme. The results obtained are promising due to the possibility of industrial application of immobilized pepsin. Abrir menu Brasil. Brazilian Journal of Chemical Engineering. Abrir menu. Abstract Pepsin was immobilized via covalent bonds on different carriers: a silica gel carrier, acrylic beads, and a cellulose-based carrier - Granocel.

Table 2 Efficiency and storage stability of pepsin immobilized on the tested carriers. Ahn, J. Altun, G. Anson, M. Bahar, T. Bryjak, J. Cooper, J. Dharmapuri, A. Dumitriu, S. Immobilization of pepsin on N -[4-carboxyphenylcarbamoylmethyl]-cellulose. Esawy, M. Gamze, D. Gray, S. Han, W. Hu, J. Kamatari, Y. Konkular, G. Kurimoto, E. Labus, K. Li, S. Li, B. Liese, A.

Line, W. Lowry, O. Mateo, C. Rea, D. Serys, A. Shukla, S. Stigter, E. Ticu, E. Zynek, K. Publication Dates Publication in this collection Apr-Jun This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Figures 5 Tables 2. Abbreviations: S BET - specific surface area,. The enzyme activity unit AU was defined as the change of absorbance of 0. Google Google Scholar. Acrylic carriers. Silica gels. SEL 10 5 Da. In contrast, pepsin and the cleavage at low pH seemed to be extremely efficient in the digestion of both linear and tightly folded structures Figure 1. Pretreatment of the substrates by lowering the pH, which would occur if the food passes through the stomach before entering the duodenum, did also not seems to induce a significant opening of the structure for more efficient digestion by the pancreatic serine proteases.

In contrast, heat treatment was relatively efficient in making the substrates more accessible for these enzymes. Both of these findings stand to reason; food digestion at low pH, and by an enzyme that has its optimum at this low pH, is very common among multicellular organisms, indicating that it has been an evolutionary successful strategy for food uptake.

Heat denaturation of food has also been claimed to have been a major step in human development, to be able to better absorb the nutrients of protein-rich food and thereby a key factor in the development of a large and energy dependent brain Carmody et al.

What is then the role of the pancreatic serine proteases and the carboxy-peptidases if pepsin does the major work in protein digestion? Amino acid and peptide transporters of the small intestine have been found to only take up peptides of a size of four amino acids or smaller, and even the size of four amino acids is less efficiently absorbed compared to shorter peptides making the subsequent cleavage of small peptides generated by pepsin into very short peptides or single amino acids very important.

It is therefore possible that the major function of the pancreatic proteases is to complete the job performed by pepsin.

The need for multiple primary specificities and a combination of endo- and exopeptidases does seem logical. We would need an array of different primary specificities to cleave these small peptides where some may not contain a positively charged amino acid and thereby not cleaved by trypsin. Some would not contain an aromatic amino acid and thereby not cleaved by chymotrypsin and some would not contain an aliphatic amino acid and thereby not cleaved by the elastase.

However, by a combination of these three, and together with the exopeptidases, most peptides would be suitable targets for one or more of these enzymes. The final step is then carried out by membrane-bound brush border peptidases of the intestinal mucosa and cytoplasmic proteases of the epithelial cells. The brush border enzymes are a number of integral membrane proteins that convert the small peptides to single amino acids or very small peptides Goodman ; Hooton et al.

The free amino acids or the very short peptides are then imported into the epithelial cells by sodium-dependent amino acid transporters, one each for basic, acidic, and neutral amino acids Goodman ; Spanier and Rohm These transporters bind the amino acids only after also binding sodium. Following binding of both sodium and peptide, the transporter goes through a conformational change and pumps in both the sodium and the amino acid into the cytoplasm of the enterocyte.

Following uptake of the di- and tripeptides, these peptides are further digested by cytoplasmic proteases within the enterocyte. The free amino acids are then transported into the blood by another transporter that sits at the basolateral membrane of the enterocyte. This transporter does not need a sodium gradient. Only a very small number of the peptides enter the blood.

In conclusion, pepsin, which was very efficient in cleaving even tightly folded proteins, seems to very important for the initial step in the absorption of food proteins and to be an evolutionary old and successful strategy for food uptake Janiak The possibility to act at a low denaturing pH appeared to be important for efficient cleavage of the otherwise tightly folded proteins, which seemed to be difficult to access by the pancreatic enzymes.

This pattern is very similar to what we previously observed for the hematopoietic serine proteases, which were highly dependent on accessibility of the potential cleavage sites for efficient cleavage Fu et al. The pancreatic enzymes may therefore have a major function to perform the second step in the digestion of the food proteins by reducing the size of the peptides generated by pepsin.

Both hematopoietic and pancreatic endopeptidases thereby seem to show many similarities concerning the effect of folding on the efficiency of substrate cleavage. As a third and fourth step, the brush border enzymes and the cytoplasmic proteases of the enterocytes finish the sequence by cleaving the small peptides into single amino acids for final transport into the blood.

We could also show that some proteins are remarkable resistant to the cleavage by pepsin at pH 1. However, we could also see that after heat treatment ovalbumin was relatively efficiently cleaved by a combination of the pancreatic enzymes indicating that the combination of the different digestive enzymes, pepsin and the pancreatic enzymes, are of importance for the cleavage of the majority of proteins of the food.

It is also important to say that although pepsin is very efficient in cleaving tightly folded proteins and that an acidic environment and proteases that are able to act efficiently at this low pH persons with complete gastrectomy can live a relatively normal life indicating pepsin hydrolysis is not absolutely necessary for survival and that a combination of the pancreatic enzymes can be sufficient when acting together for our survival Goodman Chymotrypsin and trypsin were dissolved with PBS, while pepsin was dissolved with 0.

A new type of recombinant substrate was used to study the importance of folding on the cleavage of dietary proteins. Two copies of the E. Between the two Trx molecules, a nine amino acid region was inserted with a sequence susceptible for cleavage by trypsin, chymotrypsin, or elastase respectively. For purification a His 6 -tag was also inserted in the C-terminal.

The sequences of the individual clones were verified after cloning by sequencing of both DNA strands. The plasmids were then transformed into the E. IPTG was then added to a final concentration of 1 mM. After incubation, the bacteria were pelleted by centrifugation at rpm for 12 min. The lysate was centrifuged at 13, rpm for 3 min and the supernatant was transferred to a new tube. Based on gel intensity the ratio between pepsin and target molecule seems to be close to 1—10 which is a slightly higher value compared to the value of the enzyme concentration given by the distributing company.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Conflict of interest statement: The authors declare that they have no conflict of interest regarding the contents of this article.

Carmody, R. Energetic consequences of thermal and nonthermal food processing. Search in Google Scholar. Chauncey, H. Comparative enzyme activity of saliva from the sheep, hog, dog, rabbit, rat, and human. Oral Biol. Fu, Z. Highly selective cleavage of cytokines and chemokines by the human mast cell chymase and neutrophil cathepsin G. Gallwitz, M. The extended substrate recognition profile of the dog mast cell chymase reveals similarities and differences to the human chymase. The extended cleavage specificity of human thrombin.

Goettig, P. Surface loops of trypsin-like serine proteases as determinants of function. Goodman, B. Insights into digestion and absorption of major nutrients in humans. Guyonnet, V. Purification and partial characterization of the pancreatic proteolytic enzymes trypsin, chymotrypsin, and elastase from the chicken. Hooton, D. The secretion and action of brush border enzymes in the mammalian small intestine.

Ishiguro, H. Physiology and pathophysiology of bicarbonate secretion by pancreatic duct epithelium. Nagoya J. Janiak, M. Digestive enzymes of human and nonhuman primates. Its primary structure is composed of 44 amino acids. Compared with pepsin, pepsinogen is stable in neutral and weak alkaline environment, but when exposed to hydrochloric acid in gastric juice, these 44 amino acids are removed in an autocatalytic manner and activated into pepsin.

Parietal cells of the gastric wall release hydrochloric acid HCl , pepsinogen can be activated by hydrochloric acid. Gastrin and vagus nerves trigger the release of pepsinogen and hydrochloric acid from the gastric wall when eating. Hydrochloric acid produces acidic environment, which makes pepsinogen unfold and cleave in an autocatalytic manner, thus producing pepsin. Pepsin cuts 44 amino acids in pepsinogen into more pepsin. Pepsin is a chain protein monomer composed of two similar folding domains separated by a deep cleft.

The catalytic site of pepsin is formed at the junction of the domain, each domain contains two aspartic acid residues, Asp32 and Asp Under the catalysis of pepsin, the water molecule helps the active carboxyl group to bear positive and negative charges with aspartic acid and aspartic acid 32, respectively, which breaks the peptide bond in the protein.

The activity of pepsin was the highest in pH2. Therefore, in the solution below pH8. The stability of pepsin at high pH value is of great significance to the diseases caused by pharynx and larynx reflux. Pepsin is one of the main causes of mucosal injury in pharynx and larynx reflux. Pepsin still stays in the pharynx and larynx after pharyngeal reflux. Although the enzyme is in a neutral environment, it can be reactivated in the subsequent acid reflux event.

After pepsin is activated, laryngeal mucosa is exposed to active pepsin, resulting in a decrease in the expression of protective proteins, thus increasing the susceptibility to laryngeal injury. In addition, pepsin may also cause mucosal damage in weak acid or non-acid reflux events.

Pepsin can be internalized in the upper airway by receptor-mediated endocytosis. When cells ingest pepsin, pepsin is stored in intracellular vesicles with a low pH value, at which the enzyme activity of pepsin recovers. The exposure of pepsin to neutral pH and the internalization of pepsin lead to changes in the expression of genes related to inflammation, which are the basis of signs and symptoms of reflux and tumor progression. Pepsin in airway specimens is considered to be a sensitive and specific marker for pharyngeal reflux.