Pig Pepsin Research Articles

Heterogenous expression and enzymatic property analysis of microbial-derived pectinases in pig PK15 cells

As one of plant cell wall components, pectin is the main anti-nutritional factor in livestock and poultry feeds and has an adverse effect on utilization efficiency of feed energy and nitrogen. Pectinases, which are widely found in microorganisms such as bacteria, yeast and filamentous fungi in nature,can improve feed efficiency by relieving the anti-nutritional effect of pectin through promoting the hydrolysis reaction of feed pectin. To explore the feasibility of expressing microbial-derived pectinase genes in pig cells, we introduced microbial-derived pectinase genes pg5a, pgI, pga3A, and pgaA into porcine PK 15 cells by lipofection for heterogenous expression. Enzymatic activities of the pectinases encoded by these genes were analyzed using the 3,5 dinitrosalicylic acid (DNS) method. Results showed that all four pectinase genes were able to be transcribed into mRNAs in porcine PK 15 cells, but only pg5a and pgI were adapted to the porcine cell expression system. Among them, the maximum activity of pectinase PG5A was 0.95 U/mL, the optimum pH was pH 4.0, and the enzymatic activity was maintained above 46% in the range of pH 4.6 to 6.0. Pectinase PGI obtained the highest enzymatic activity at pH 5.0, which was 0.30 U/mL, and maintained more than 35% of the activity in the range of pH 4.0 to 6.0. The results of digestive protease tolerance test showed that PG5A and PGI were highly resistant to pepsin and trypsin. After treatment with 1 mg/mL pig pepsin for two hours, the residual enzymatic activities of PG5A and PGI were 76% and 71%, respectively. And after two hours treatment with 1 mg/mL of pig trypsin, the remaining enzymatic activities of PG5A and PGI were 44% and 93%, respectively. In summary, pectinase PG5A and PGI can be effectively expressed in pig cells, and have strong tolerance to pig intestinal pH environment and digestive proteases. Therefore, both pg5a and pgI can be used as candidate genes for production of transgenic pigs.

Purification and characterization of a novel aspartic protease from basidiomycetous yeast Cryptococcus sp. S-2

An aspartic protease (Cap1) was purified from basidiomycetous yeast Cryptococcus sp. S-2 (FERM ABP-10961) using HiTrap DEAE FF column and HiTrap Q HP column chromatography with azocasein as a substrate. Cap1 has a molecular mass of 34 kDa on SDS-PAGE. It was stable up to 50°C with maximum activity at 30°C. Maximum proteolytic activity was observed at pH 5.0. Cap1 was stable in the pH range 3.0-7.0. Its enzyme activity was strongly inhibited by pepstatin A, an inhibitor of aspartic proteases, indicating that Cap1 is an aspartic protease. Cap1 hydrolyzed protein substrates, including BSA, hemoglobin, α-casein, β-casein, and κ-casein. It showed activity on synthetic substrates, such as MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH₂ and MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH₂. Hydrolysis of the oxidized insulin B chain followed by amino acid sequencing analysis of the cleavage products revealed that 9 of its 30 peptide bonds were hydrolyzed by Cap1. This result was similar to that observed with pig pepsin A and human pepsin A. Cap1 also exhibited milk-clotting activity. We cloned the cDNA of CAP1 gene, which contained a 1254 bp open reading frame encoding a protein of 417 amino acid residues. Homology search in the NCBI database revealed that the amino acid sequence of Cap1 showed less than 39% identity to other known proteins. Therefore, we proposed that Cap1 is a novel aspartic protease.

α 67-106 of bovine hemoglobin: a new family of antimicrobial and angiotensin I-converting enzyme inhibitory peptides

Protein hydrolysates are of a significant interest, due to their potential application as a source of bioactive peptides in nutraceutical and pharmaceutical domains. The present study was focused on bovine hemoglobin hydrolysate obtained with pig pepsin in the presence of 30% ethanol. This hydrolysate was fractioned by reversed-phase high-performance liquid chromatography (RP-HPLC) into 12 major fractions (F1–F12). All fractions were analyzed by ESI/MS and ESI/MS/MS, in order to characterize and identify the peptides in these fractions. This hydrolysis permitted to generate a new serial of bioactive peptides with both antimicrobial and ACE inhibitory activities. Identified peptides were TKAVEHLDDLPGALSELSDLHAHKLRVDPVNFKLLSHSLL, LDDLPGALSELSDLHAHKLRVDPVNFKLLSHSL, KLLSHSL, and LLSHSL corresponding respectively to the 67-106, 73-105, 99-105, and 100-105 fragments of the α chain of bovine hemoglobin. They were the first found from bovine hemoglobin. These purified peptides have an antibacterial activity against four bacteria strains: Kocuria luteus A270, Listeria innocua, Escherichia coli, and Staphylococcus aureus with a MIC between 187.1 and 35.2 μM. On the other hand, these peptides displayed at the same time ACE inhibitory activity with an IC50 range from 42.55 to 1,095 μM.

Purification and characterization of pepsins A1 and A2 from the Antarctic rock cod Trematomus bernacchii

The Antarctic notothenioid Trematomus bernacchii (rock cod) lives at a constant mean temperature of -1.9 degrees C. Gastric digestion under these conditions relies on the proteolytic activity of aspartic proteases such as pepsin. To understand the molecular mechanisms of Antarctic fish pepsins, T. bernacchii pepsins A1 and A2 were cloned, overexpressed in Escherichia coli, purified and characterized with a number of biochemical and biophysical methods. The properties of these two Antarctic isoenzymes were compared to those of porcine pepsin and found to be unique in a number of ways. Fish pepsins were found to be more temperature sensitive, generally less active at lower pH and more sensitive to inhibition by pepstatin than their mesophilic counterparts. The specificity of Antarctic fish pepsins was similar but not identical to that of pig pepsin, probably owing to changes in the sequence of fish enzymes near the active site. Gene duplication of Antarctic rock cod pepsins is the likely mechanism for adaptation to the harsh temperature environment in which these enzymes must function.

Secretion of total pepsin and pepsin 1 in healthy volunteers in response to pentagastrin and to insulin-induced hypoglycaemia

Objective. Human gastric juice contains a multiplicity of proteinases, and one enzyme (pepsin 1) is increased in patients with peptic ulcer disease. However, little is known about its secretion in health. The purpose of this study was to determine the pepsin 1 content in relation to the total pepsin of human gastric juice in healthy volunteers and the effect of different stimuli on secretion. Material and methods. Human gastric juice from healthy volunteers was collected at 10-min intervals over a period of 30 min basally and up to 60 min after continuous intravenous pentagastrin administration (n=13) or insulin-induced hypoglycaemia (n=11). Pepsin 1 was measured by semiquantitative agar gel electrophoresis and the total pepsin as the proteolytic activity calibrated against pig pepsin A. Results. Pepsin 1 was present in 23 out of 24 basal gastric juice samples at a mean concentration of approximately 12 µg/ml. During continuous intravenous pentagastrin to 11 subjects for 60 min, the mean concentration and secretion rates per minute rose to 24 µg/ml and 122 µg/min at 60 min; the proportion of pepsin 1 to total pepsin was 2.8% in basal secretions increasing to 7.8% in the 60 to 70-min samples. Following intravenous insulin administration to 11 subjects, the mean concentration and secretion rates of pepsin 1 rose to 33 µg/ml and 127 µg/min at 60 min: the proportion of pepsin 1 to total pepsin was 0.8% in basal secretions and 3.1% in the 50 to 60-min samples. Conclusions. Both pentagastrin and hypoglycaemia caused similar maximal secretion rates of pepsin 1, but hypoglycaemia caused greater secretion rates of total pepsin. The maximum concentrations and secretion rates of pepsin 1 occurred at different times from those of total pepsin and there is a threshold of total pepsin secretion below which pepsin 1 was not released.

Corrigendum

Alimentary Pharmacology & TherapeuticsVolume 24, Issue 10 p. 1506-1506 Free Access Corrigendum This article corrects the following: Review article: human pepsins – their multiplicity, function and role in reflux disease N. B. ROBERTS, Volume 24Issue s2Alimentary Pharmacology & Therapeutics pages: 2-9 First Published online: August 21, 2006 First published: 27 October 2006 https://doi.org/10.1111/j.1365-2036.2006.03178.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat In [1], the following error occurred on p. 7: Figure 2 Figure 2Open in figure viewerPowerPoint The distribution of different pepsins using high performance ion exchange chromatography (HPIEC) in healthy controls and patients with reflux oesophagitis. Gastric juice was collected basally over 30 min and post pentagastrin (6 μg/kg i.m.) over 60 min in controls (n = 11, age 23–31 median 24 years) and patients (grades 2/3) (n = 20, age 29–74 median 57 years). Pepsin was recorded as concentration in mg/mL using pig pepsin A as a calibrant.10,15 The chart shows the median and the bar the upper value. There was no difference in any of the pepsins as measured by HPIEC. The x axis is incorrectly labelled. The corrected Figure appears below. Reference 1 Roberts N. Review article: human pepsins – their multiplicity, function and role in reflux disease. Aliment Pharmacol Ther 2006; 24 (Suppl. 2): 2– 9. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Volume24, Issue10November 2006Pages 1506-1506 FiguresReferencesRelatedInformation

Bacterial killing in gastric juice – effect of pH and pepsin on Escherichia coli and Helicobacter pylori

The susceptibility of Escherichia coli and Helicobacter pylori to pH and the effect of pepsin-mediated proteolysis were investigated. This was to establish the relative importance of their bacterial killing properties in gastric juice. Solutions in the pH range 1.5-7.4 with or without pig pepsin A were used, together with seven gastric juice samples obtained from patients undergoing routine gastric collection. Escherichia coli C690 (a capsulate strain), E. coli K-12 (a rough mutant) and Helicobacter pylori E5 were selected as the test organisms. Suspensions of bacteria (1x10(6) E. coli ml-1 and 1x10(8) H. pylori ml-1) were pre-incubated with test solutions at 37 degrees C for up to 2 h, and then cultured to establish the effect on subsequent growth. Survival of bacteria was diminished at pHs of less than 3.5, whereas killing required a pH of less than 2.5. Pre-incubation with pig pepsin at 0.5, 1.0 and 2.0 mg ml-1 at pH 3.5 reduced viable counts by 100% for E. coli 690 and E. coli K-12 after 100 min incubation. With H. pylori, the viable counts decreased to 50% of the control after 20 min incubation in 1 mg pepsin ml-1 at pH 2.5, 3.0 and 3.5. The gastric juices showed bactericidal activity at pH 3.5, and the rate of killing was juice dependent, with complete death of E. coli 690 occurring between 5 and 40 min post-incubation. Thus, killing of E. coli and H. pylori occurs optimally at pHs of less than 2.5. At pH 3.5, little effect is observed, whereas addition of pepsin alone or in gastric juice causes a marked increase in bacterial susceptibility, suggesting an important role for proteolysis in the killing of bacteria.

A study of aspartyl proteases using intramolecularly quenched fluorogenic peptide substrates

A series of fluorogenic tetra-, penta-, and hexapeptide substrates of the general structure Abz-X-Phe-Phe-Y-Ded (or -pNa in place of -Ded), where X = Ala, Ala-Ala, or Val-Ala and Y = -, Ala, or Ala-Ala, were proposed. Kinetic parameters of hydrolysis of these substrates by pepsin, cathepsin D, human gastricsin, pig pepsin, calf chymosin, and aspergillopepsin A were determined. The compounds synthesized proved to be effective substrates for aspartyl proteases of diverse origins.

Penicillopepsin-JT2, a recombinant enzyme from Penicillium janthinellum and the contribution of a hydrogen bond in subsite S3 to k(cat).

The nucleotide sequence of the gene (pepA) of a zymogen of an aspartic proteinase from Penicillium janthinellum with a 71% identity in the deduced amino acid sequence to penicillopepsin (which we propose to call penicillopepsin-JT1) has been determined. The gene consists of 60 codons for a putative leader sequence of 20 amino acid residues, a sequence of about 150 nucleotides that probably codes for an activation peptide and a sequence with two introns that codes for the active aspartic proteinase. This gene, inserted into the expression vector pGPT-pyrG1, was expressed in an aspartic proteinase-free strain of Aspergillus niger var. awamori in high yield as a glycosylated form of the active enzyme that we call penicillopepsin-JT2. After removal of the carbohydrate component with endoglycosidase H, its relative molecular mass is between 33,700 and 34,000. Its kinetic properties, especially the rate-enhancing effects of the presence of alanine residues in positions P3 and P2' of substrates, are similar to those of penicillopepsin-JT1, endothiapepsin, rhizopuspepsin, and pig pepsin. Earlier findings suggested that this rate-enhancing effect was due to a hydrogen bond between the -NH- of P3 and the hydrogen bond accepting oxygen of the side chain of the fourth amino acid residue C-terminal to Asp215. Thr219 of penicillopepsin-JT2 was mutated to Ser, Val, Gly, and Ala. Thr219Ser showed an increase in k(cat) when a P3 residue was present in the substrate, which was similar to that of the wild-type, whereas the mutants Thr219Val, Thr219Gly, and Thr219Ala showed no significant increase when a P3 residue was added. The results show that the putative hydrogen bond alone is responsible for the increase. We propose that by locking the -NH- of P3 to the enzyme, the scissile peptide bond between P1 and P1' becomes distorted toward a tetrahedral conformation and becomes more susceptible to nucleophilic attack by the catalytic apparatus without the need of a conformational change in the enzyme.

Structure and Proposed Amino-Acid Sequence of a Pepsin from Atlantic Cod (Gadus morhua)

The crystal structure of a pepsin from the gastric mucosa of Atlantic cod has been determined to 2.16 A resolution. Data were collected on orthorhombic crystals with cell dimensions a = 35.98, b = 75.40 and c = 108.10 A, on a FAST area-detector system. The phase problem was solved by the molecular-replacement method using porcine pepsin (PDB entry 5PEP) as a search model. The structure has been refined to a crystallographic R factor of 20.8% using all reflections between 8.0 and 2.16 A, without prior knowledge of the primary sequence. The resulting crystal structure is very similar to the porcine enzyme, consisting of two domains with predominantly beta-sheet structure in the same sequential positions as the enzyme from pig. In the course of the model building, 122 residues were substituted and two residues deleted from the starting model to give a polypeptide chain of 324 amino acids and a sequence identity of 57.7% with the pig pepsin. No carbohydrate residues were located. Sequence alignment with available aspartic proteinases, indicates that the fish enzyme seems to be more related to mammalian gastric pepsins than to the mammalian gastricsins and chymosins, lysosomal cathepsin D's and a pepsin from tuna fish. The amino-acid composition of the cod enzyme, however, is more in accordance with the cathepsin D's.

The primary structure and enzymic properties of porcine prochymosin and chymosin

Preliminary investigations by N-terminal sequence analysis showed that pig and calf chymosin possessed 80% amino acid sequence identity but showed considerable differences in their enzymatic properties. A comparison of their structures may therefore contribute to an understanding of the significance of the amino acid residues responsible for the differences in these properties. Pig chymosis was extracted from the stomachs of pigs of less than 3 weeks of age, and was purified by ion exchange chromatography. Half of the primary structure was determined by amino acid sequencing and the complete structure was deduced from a cloned chymosin cDNA. Results showed that the zymogen showed 81% sequence identity with calf prochymosin and 57% identity with pig pepsinogen A. The size of the propart and location of the residue which becomes the N-terminus in the active molecule were the same in the prochymosins. The maximum general proteolytic activity at pH 3.5 of pig chymosin was 2–3% of that of the activity of pig pepsin A at pH 2, whereas the milk clotting activity relative to the general proteolytic activity of pig chymosin was much higher than that of calf chymosin. Agar gel electrophoresis at pH 5.3 of stomach extracts of individual pigs showed the existence of two predominant genetic variants of zymogen and enzyme. The two variants could not be distinguished by amino acid composition or N-terminal sequencing, and no differences in the enzymatic properties of the genetic variants were observed. It was concluded that of the residues that participate in the substrate binding, calf and pig chymosin differ in the following positions (pig pepsin numbering, subsites in parentheses): Ser 12 Thr (S 4), Leu 30 Val (S 1/S 3), His 74 Gln (S 2′), Val 111 Ile (S 1/S 3), Lys 220 Met (S 4). With regard to the low general proteolytic activity of pig chymosin, the substitution Asp 303 Val relative to calf chymosin may contribute to an explanation of this.

Engineering the substrate specificity of rhizopuspepsin: The role of Asp 77 of fungal aspartic proteinases in facilitating the cleavage of oligopeptide substrates with lysine in P1

Rhizopuspepsin and other fungal aspartic proteinases are distinct from the mammalian enzymes in that they are able to cleave substrates with lysine in the P1 position. Sequence and structural comparisons suggest that two aspartic acid residues, Asp 30 and Asp 77 (pig pepsin numbering), may be responsible for generating this unique specificity. Asp 30 and Asp 77 were changed to the corresponding residues in porcine pepsin, Ile 30 and Thr 77, to create single and double mutants. The zymogen forms of the wild-type and mutant enzymes were overexpressed in Escherichia coli as inclusion bodies. Following solubilization, denaturation, refolding, activation, and purification to homogeneity, structural and kinetic comparisons were made. The mutant enzymes exhibited a high degree of structural similarity to the wild-type recombinant protein and a native isozyme. The catalytic activities of the recombinant proteins were analyzed with chromogenic substrates containing lysine in the P1, P2, or P3 positions. Mutation of Asp 77 resulted in a loss of 7 kcal mol-1 of transition-state stabilization energy in the hydrolysis of the substrate containing lysine in P1. An inhibitor containing the positively charged P1-lysine side chain inhibited only the enzymes containing Asp 77. Inhibition of the Asp 77 mutants of rhizopuspepsin and several mammalian enzymes was restored upon acetylation of the lysine side chain. These results suggest that an exploitation of the specific electrostatic interaction of Asp 77 in the active site of fungal enzymes may lead to the design of compounds that preferentially inhibit a variety of related Candida proteinases in immunocompromised patients.

Production of acute gastric erosions in the cat by individual human pepsins.

1. Differing amounts of human pepsins 1, 3 (pepsin A) and 5 (pepsin C or gastriscin) in aqueous HCl/NaCl at pH 1.3 were placed in Perspex rings on the exposed luminal surface of the perfused cat stomach to test for mucolytic and erosive activity in vivo, with the acid medium in control rings. 2. After incubation at 37 degrees C for 60 or 120 min, the test and control solutions were replaced by the same volume of each test or control solution containing 18 mg of aspirin and incubated for a further 120 min. 3. The number of bleeding points or erosions was counted at 15 min intervals. None was observed with human pepsins 1, 3 and 5 or with pig pepsin A or in the control rings. 4. With aspirin, erosions developed in all ringed areas. Their rate of development with time for pepsin 1 and pig pepsin did not differ from controls. Significantly more erosions developed with human pepsins 3 (5 mg per ring, P < 0.001) and 5 (0.32 mg per ring, P < 0.05). 5. Human pepsins 3 and 5, applied at pH 1.3 to the mucosal surface of the perfused cat stomach, therefore cause erosions when administered with aspirin, but do not produce erosions when given alone. In man, pepsins secreted in increased amount or concentration may therefore be unlikely to cause significant mucosal erosion unless human gastric mucosa is less resistant to pepsins than cat mucosa. Pepsins may perhaps facilitate the action of a second factor, such as aspirin, or infection with Helicobacter pylori.

Secondary Substrate Binding in Aspartic Proteinases: Contributions of Subsites S3 and S′2 to kcat

The kinetic parameters kcat and Km were determined at 25°C and pH 5.5 for endothiapepsin and rhizopuspepsin acting on the series of substrates Ac-Alam-Lys-Nph-Alan-amide where Nph is p-nitrophenylalanine, and m and n equal 0-4. Kinetic parameters were also determined at 25°C and pH 3.5 for pig pepsin acting on the series of substrates Ac-Alam-Phe-Nph-Argn-Alan-amide, where m equals 0 to 2 and n equals 0 or 1, and another series based on -Ile-Glu-Phe-Nph-Arg-, which is the core of a series of peptides designed by Dunn et al. (1986, Biochem. J. 237, 899—906). Km values were found to be largely independent of increases in the chain length of the substrates whereas kcat values showed large increases with increasing chain length. With endothiapepsin and rhizopuspepsin the largest increases (between 13-fold and over 150-fold) were obtained when alanine residues were added in positions P3 and P′2 and thus are similar to those observed previously with penicillopepsin. Additions of alanines to positions P2, P′3, and P′4 gave much smaller increases. In the case of pig pepsin the results were not as clearcut. Whereas the largest increases were seen for positions P3 and P′2 only with some of the peptides, the increases were strongly dependent on the length of the peptides. With some of the longer peptides the increases were comparable to those seen for positions P2 and P′3. Thus, whereas the addition of two alanines in position P3 to the dipeptide Ac-Phe-Nph-amide caused a large proportional increase in kcat, the increase was much smaller when the addition was made to the homologous tetrapeptide and even smaller when made to the pentapeptide Ac-Ala-Phe-Nph-Arg-Ala-amide. Additions of arginine in position P′2 gave large increases in kcat for all three peptides of the series.

Secondary enzyme-substrate interactions: kinetic evidence for ionic interactions between substrate side chains and the pepsin active site.

The possibility that pig pepsin has a cation binding specificity in its secondary binding subsites has been examined by the pepsin-catalyzed hydrolysis of a series of synthetic octa- to undecapeptide substrates. These chromophoric substrates are cleaved by pepsin in the phenylalanyl-p-nitrophenylalanyl (Phe-Nph) bond. Lys and Arg residues were placed into seven different positions in the substrates, and their effect on kcat and Km was examined between pH 2.8 and pH 5.8 (I = 0.1 M, 37 degrees C). Kinetic evidence indicates the existence in the enzyme binding subsites S4, S3, S2, S3', S4', and S5' of a group(s) which become(s) negatively charged at higher pH. For most substrates, the magnitude as well as the pH dependence of kcat was unaffected by the presence of Lys or Arg in these peptides. In contrast, changes up to 5 orders of magnitude were observed for Km, depending on the number of basic residues and on their positions in the sequence. Km for a group of substrates at pH greater than 5.5 was lower than 50 nM. Values for kcat/Km for some substrates exceed the level of 10(8) M-1 s-1. Therefore, the free energy derived from ionic interactions in secondary binding sites influences mostly the binding step on the reaction pathway. This result is in contrast to the previous observations that the length and the hydrophobic character of the substrate residues in some positions influence kcat with little effect on Km toward shorter substrates of pepsin [Fruton, J. (1976) Adv. Enzymol. Relat. Areas Mol. Biol. 44, 1-36].

Degradation of human epidermal keratin by fish pepsin

Stomach extract of Atlantic herring Clupea harengus, Atlantic salmon Salmo salar, cod Gadus morhua, redfish Sebastes marinus, and plaice Pleuronectes platessa, degraded human epidermal keratin effectively in vitro. The keratin-degrading activity of all extracts showed a pH optimum around 3.3-3.4, and sheets of plantar callus were degraded with about the same efficacy as keratin. Pepstatin sensitivity, heat lability, and the acidic pH optimum demonstrated that the keratin-degrading activity was pepsin. The keratin-degrading activity of cod stomach extract had a temperature optimum of around 42 degrees C at optimal pH, and showed a similar pH dependency with collagen as with keratin as substrate. The keratin-degrading activity of pepsin I and pepsin II purified from cod showed a pH optimum of 3.7 and 3.1, respectively, similar to that obtained with hemoglobin as substrate. Pig pepsin showed a pH optimum of about 2 with keratin, hemoglobin, and collagen as substrates. The present investigation demonstrates that fish pepsin is effective in degrading human epidermal keratin in vitro, and in a contemporary study the same was shown with fish trypsin. This may suggest a possible mechanism for the development of irritative hand eczema caused by exposure to fish and acidified fish material.

A simple assay for determining keratin and collagen degradation in vitro

A simple assay measuring degradation of human epidermal keratin and bovine tendon collagen is presented. Insoluble protein substrate (30 mg) was incubated with 1 ml buffer and enzyme sample for 1 h at 37 degrees C, following addition of 1 ml distilled water and removal of the remaining substrate by filtration/centrifugation. The protein content was determined in the filtrate/supernatant by the Lowry method. Keratin was prepared as follows: Freeze-drying, homogenization in a mortar or beetling mill, extraction in 0.9% (w/v) NaCl followed by water and 100% ethanol, drying at 37 degrees C. The assay was tested with pig pepsin, bovine trypsin, and crude extract of fish stomach, demonstrating that these preparations are effective in degrading human epidermal keratin.

The pH dependence of the hydrolysis of chromogenic substrates of the type, Lys-Pro-Xaa-Yaa-Phe-(NO 2)Phe-Arg-Leu, by selected aspartic proteinases: evidence for specific interactions in subsites S 3 and S 2

Variation in the kinetic parameters, k cat and K m, with pH has been used to obtain evidence for significant acid-dissociation processes in the hydrolysis of octapeptide substrates by three aspartic proteinases. These substrates are all cleaved at the peptide bond between a Phe (P1) and a p-nitroPhe (P1′) residue resulting in a shift in absorbance at 300 nm that facilitates kinetic measurements. The substrates differ in the amino-acid residues present in the P3 and P2 positions. Porcine pepsin, calf chymosin, and the aspartic proteinase from Endothia parasitica all show pH dependencies that imply that favorable or unfavorable interactions can occur with the S3 or S2 areas of the enzyme-active site. Examination of the crystallographically determined structure of the E. parasitica proteinase and consideration of the amino-acid sequence differences between the three enzymes suggests that the origin of the pH effects arises from favorable interactions between Glu-13 (COO −) of pig pepsin and Thr (OH) or His (ImH +) in P3 of a substrate. Similarly, Lys-220 (NH + 3) of chymosin and a Glu (COO −) in P2 of a substrate may produce a favorable interaction and Asp-77 (COO −) of E. parasitica proteinase and a Glu (COO −) in P2 of a substrate may produce an unfavorable interaction. These results lead to possible explanations for subtle specificity differences within a family of homologous enzymes, and suggest loci for study by site-directed mutagenesis.

Mucus degradation by pepsin: comparison of mucolytic activity of human pepsin 1 and pepsin 3: implications in peptic ulceration.

The ability to digest mucus, mucolytic activity of isolated pepsins and samples of human gastric juice has been assayed by measuring the fall in viscosity when incubated with purified pig gastric mucus glycoprotein. Pure human pepsin 1, the peptic ulcer associated pepsin, digested gastric mucus glycoprotein at a faster rate than did pure human pepsin 3 (the principal human pepsin), or the equivalent pig pepsin (pepsin A). At pH 2.0 pepsin 1 had twice the mucolytic activity of pepsin 3. Above pH 3.8 this difference became more marked and whereas pepsin 1 caused substantial mucolysis up to and including pH 5.1, pepsin 3 had minimal activity. At pH 4.0 pepsin 1 had six times the mucolytic activity of pepsin 3. Gastric juices from patients with duodenal ulcer each exhibited substantial mucolytic activity between pH 2 to 5, similar to that of pepsin 1. In contrast, gastric juice from non-symptomatic volunteers exhibited little mucolytic activity above pH 4. Analysis of the mucus glycoprotein by gel filtration showed that an increase in lower molecular weight, pepsin degraded, glycoprotein was associated with the fall in mucus viscosity for all enzyme preparations. These results showed that pepsin 1 can digest the mucus more effectively than pepsin 3 and at higher pH values. The raised concentrations of pepsin 1 in the juice of peptic ulcer patients may thus promote the ulcerative process by increased erosion of the mucus barrier under conditions likely to pertain in the duodenal bulb as well as the stomach.

Assessment of the suitability for Cheddar cheesemaking of purified and commercial chicken pepsin preparations

SummaryCommercial and pure chicken pepsin preparations were compared with rennet as cheesemaking coagulants. From the milk clotting and caseinolytic activities and enzymic stabilities, predictions were made of the relative activities of the chicken pepsins and rennet during cheese ripening. By this means mixtures of chicken and pig pepsins for cheesemaking trials were formulated. These and mixtures of chicken pepsins with rennet were approximately additive in coagulating activity provided they were used immediately. The amounts of chicken pepsins used in laboratory scale Cheddar cheesemaking trials were minimized by acidification and calcification of the milk. The nature of the coagulant did not affect the cheesemaking conditions, yields or cheese composition. Cheeses made with chicken pepsins alone showed faster proteolysis, more intense flavour, off-flavours and bitterness and were softer than those made with rennet. Cheeses made with chicken pepsin/rennet mixtures were intermediate and those made with chicken pepsin/pig pepsin mixtures were similar to those made with rennet. It was concluded that neither of the chicken pepsin preparations was suitable for Cheddar cheesemaking and that the predictive tests had led to correct assessments.