A METHOD FOR DETERMINING THE RENNET ACTIVITY OF CHYMO-TRYPSIN

The rennet activity of chymo-trypsin (or pepsin) is conveniently measured by allowing a standard solution of milk to which chymotrypsin has been added to flow slowly through a graduated pipette and observing the rate and distance of flow of the milk before it clots. The time required for chymo-trypsin to clot milk may be calculated from these observations. The rennet activity is expressed as the reciprocal of the time in minutes required for 1 ml. of enzyme solution to clot 10 ml. of standard milk powder solution.     

Pepsin immobilized on inorganic supports for the continuous coagulation of skim milk.

The milk-clotting enzyme pepsin was immobilized onto beads of alumina, titania, glass, stainless steel, iron oxide, and Teflon for treating skim milk in a fluidized-bed reactor. Two covalent attachment procedures using silanized supports and glutaraldehyde and two adsorption procedures were evaluated. The three best catalysts were titania and glass, using the covalent attachment procedure, and alumina, using the adsorption procedure at pH 1.2. The pepsin adsorbed on alumina catalyst has commercial potential compared to the previously used glass catalyst. Attempts to increase the stability of pepsin adsorbed on alumina by cross-linking with glutaraldehyde were unsuccessful owing to the low pH necessary for optimum pepsin adsorption; Desorption of pepsin from alumina during reactor operation was determined. Regeneration of spent catalysts was only partially successful.     

Influence du degré de phosphorylation de la pepsine A bovine sur son activité enzymatique

Proteolytic and clotting activities of bovine pepsin A with respect to its degree of phosphorylation were studied on various substrates. The occurence of phosphate group(s) on bovine pepsin A more or less strongly affects its enzymic properties according to the substrate and its environment. This is particularly obvious as far as κ-casein is concerned. The specific flocculating activity of unphosphorylated (fA₀) as well as dephosphorylated (treated with potato) acid phosphatase) bovine pepsin A, determined on a 0.2% κ-casein solution, is significantly higher than that observed with phosphorylated pepsins, especially after κ-casein was treated with α-d.N-acetyl galactosaminyl oligosaccharidase, while specific milk clotting activity remains unchanged regardless to the level of phosphorylation of bovine pepsin A is.     

PEPSIN ACTIVITY UNITS AND METHODS FOR DETERMINING PEPTIC ACTIVITY

Experimental methods are described for determining the activity of pepsin preparations by means of changes in the viscosity of gelatin, casein, edestin, and powdered milk solutions, and by the rate of formation of non-protein nitrogen from casein and edestin solutions, or by the increase in formol titration of casein, edestin or gelatin. Activity units for pepsin are defined in terms of these measurements.     

Effect of dimethyl sulphoxide on the crystal structure of porcine pepsin

The structure of porcine pepsin crystallized in the presence of dimethyl sulphoxide has been analysed by X-ray crystallography to obtain insights into the structural events that occur at the onset of chemical denaturation of proteins. The results show that one dimethyl sulphoxide molecule occupies a site on the surface of pepsin interacting with two of its residues. An increase in the average temperature factor of pepsin in the presence of dimethyl sulphoxide has been observed indicating protein destabilization induced by the denaturant. Significant increase in the temperature factor and weakening of the electron density have been observed for the catalytic water molecule located between the active aspartates. The conformation of pepsin remains unchanged in the crystal structure. However, the enzyme assay and circular dichroism studies indicate that dimethyl sulphoxide causes a slight change in the secondary structure and complete loss of activity of pepsin in solution.     

Pepsin D: A minor component of commercial pepsin preparations

Methods are described for the isolation and purification of pepsin D, an enzyme which accounts for about 10% of the enzymic activity in commercial preparations of pepsin. Pepsin D is similar to pepsin in having a molecular weight of about 35000, the same C-terminal amino acid sequence, and an N-terminal isoleucine residue. It differs in having no phosphate residue. Pepsin D is similar to pepsin in its ability to digest haemoglobin, acetyl-l-phenylalanyl-l-di-iodotyrosine and gelatin but it is twice as active as pepsin in the clotting of milk. It has the same specificity as pepsin in its action on the B-chain of oxidized insulin. It is probable that the pepsin D in commercial preparations of pepsin arises from the activation of gastric pepsinogen D.     

High Pressure Homogenization of Porcine Pepsin Protease: Effects on Enzyme Activity,Stability, Milk Coagulation Profile and Gel Development

This study investigated the effect of high pressure homogenization (HPH) (up to 190 MPa) on porcine pepsin (proteolytic and milk-clotting activities), and the consequences of using the processed enzyme in milk coagulation and gel formation (rheological profile, proteolysis, syneresis, and microstructure). Although the proteolytic activity (PA) was not altered immediately after the HPH process, it reduced during enzyme storage, with a 5% decrease after 60 days of storage for samples obtained with the enzyme processed at 50, 100 and 150 MPa. HPH increased the milk-clotting activity (MCA) of the enzyme processed at 150 MPa, being 15% higher than the MCA of non-processed samples after 60 days of storage. The enzyme processed at 150 MPa produced faster aggregation and a more consistent milk gel (G’ value 92% higher after 90 minutes) when compared with the non-processed enzyme. In addition, the gels produced with the enzyme processed at 150 MPa showed greater syneresis after 40 minutes of coagulation (forming a more compact protein network) and lower porosity (evidenced by confocal microscopy). These effects on the milk gel can be associated with the increment in MCA and reduction in PA caused by the effects of HPH on pepsin during storage. According to the results, HPH stands out as a process capable of changing the proteolytic characteristics of porcine pepsin, with improvements on the milk coagulation step and gel characteristics. Therefore, the porcine pepsin submitted to HPH process can be a suitable alternative for the production of cheese.     

Hydrolysis of a chitosan-induced milk aggregate by pepsin, trypsin and pancreatic lipase.

The addition of chitosan to whole milk results in dose dependent destabilization and coagulation of the casein micelles and milk fat. The present study evaluates how the presence of chitosan could affect the hydrolysis of this chitosan-induced aggregate by different gastrointestinal proteases (pepsin and trypsin) and by pancreatic lipase. The chitosan-milk aggregate was hydrolyzed by pepsin and trypsin, as evaluated by the UV absorbance of TCA-soluble peptides and by urea-PAGE. The kinetics and extent of hydrolysis were independent of the casein being soluble or aggregated. The release of soluble peptides from the aggregate was independent of the presence of chitosan. A progressive inhibition of pancreatic lipase was observed in proportion to the increase in molecular weight of the chitosan employed to induce the formation of the aggregate. Interestingly, the presence of chitosan not only affected the initial velocity of the reaction, but also reduced its extent. The results indicate that a milk aggregate induced by chitosan was very well digested by gastric and intestinal proteases independently of the molecular weight of the chitosan used, and that the aggregate could retain the lipid-lowering effect of chitosan.     

Purification and Some Properties of Crystalline Acid Protease from Acrocylindrium sp.

A crystalline acid protease produced by a strain of Acrocylindrium in a submerged culture was prepared by treatment with acetone (60%), salting out with ammonium sulfate (saturated) and, after chromatography on Duolite GS-101 column, dialysis against distilled water. This preparation was homogeneous on sedimentation analysis, starch-gel electrophoresis and gel filtration with Sephadex G-75. The optimum pH was 2.0 for milk casein digestion and the pH stability was for 2.0~5.0 at 30°C for one day. The crystalline enzyme was completely stable below 50°C, but lost the activity at 70°G in ten minutes. The acid protease was almost equal to pepsin on specific activity when milk casein solution (pH 2.0) was used as substrate.     

Effect of products of alphaS1-casein proteolysis on the activity of angiotensin-converting enzyme

The influence of alpha s1-casein proteolysis products of cow milk on the activity of angiotensin-converting enzyme (ACE) has been investigated. The peptide fraction has been obtained after incubation of alpha s1-casein with different strains Lactococcus lactis ssp. lactis and pepsin. The peptides with low molecular weight has been obtained with the help of a gel filtration. It is shown, that such peptides received with the help of some strains of Lactococcus lactis ssp. lactis and pepsin are capable to inhibit the activity of ACE. A conclusion about possibility of appearing antihypertensive peptides as a result of proteolitic processes in milk products has been made.     

Features of the acid protease partition in aqueous two-phase systems of polyethylene glycol-phosphate: chymosin and pepsin

The partitioning of chymosin (from Aspergilus niger) and pepsin (from bovine stomach) was carried out in aqueous-two phase systems formed by polyethyleneglycol-potassium phosphate. The effects of polymer concentration, molecular mass and temperature were analysed. The partition was assayed at pH 7.0 in systems of polyethyleneglycol of molecular mass: 1450, 3350, 6000 and 8000. Both proteins showed high affinity for the polyethyleneglycol rich phase. The increase of polyethyleneglycol concentration favoured the protein transfer to the top phase, suggesting an important protein-polymer interaction. Polyethyleneglycol proved to have a stabilizing effect on the chymosin and pepsin, increasing its protein secondary structure. This finding agreed with the enhancement of the milk clotting activity by the polyethyleneglycol. The method appears to be suitable as a first step for the purification of these proteins from their natural sources.     

ISOLATION,CRYSTALLIZATION, AND PROPERTIES OF PEPSIN INHIBITOR

A method has been described for the isolation and crystallization of swine pepsin inhibitor from swine pepsinogen. Solubility experiments and fractional recrystallization show no drift in specific activity. The reversible combination of pepsin with the inhibitor was found to obey the mass law. The inhibitor is quite specific, failing to act on other proteolytic and milk clotting enzymes. The inhibitor is destroyed by pepsin at pH 3.5. Chemical and physical studies indicate that the inhibitor is a polypeptide of approximately 5,000 molecular weight with an isoelectric point at pH 3.7. It contains arginine, tyrosine, but no tryptophane and has basic groups in its structure.     

Cooperativity between pepsin and crystallization of calcium carbonate in distilled water

Cooperativity between pepsin and crystallization of calcium carbonate in distilled water was studied. The results show that vaterite was formed under the influence of pepsin and the crystalline product was a composite of vaterite and pepsin. The component of this material was similar to that of nacre. At the same time, the crystallization of calcium carbonate had also an important effect on the secondary structure of the pepsin. The secondary structure of the pepsin was characterized through FT-IR technology. The result indicated that the pure pepsin is composed of 24.38% alpha-helices, 29.91% beta-sheets, 39.32% beta-turns and 6.49% random structures and the pepsin in the CaCO(3)-pepsin solution is composed of 2.09% alpha-helices, 93.304% beta-sheets, 4.592% beta-turns and 0.006% random structure. During the crystallization of the calcium carbonate from the pepsin solution, the secondary structure of the pepsin transformed. These results showed that there was cooperativity between the crystallization of vaterite and the pepsin. The cooperative mechanism is discussed.     

Continuous coagulation of milk using immobilized enzymes in a fluidized-bed reactor

Milk-clotting enzymes such as pepsin, chymosin, chymotrypsin, and M. miehei proteases were immobilized on porous, alkylamine glass and incorporated into a fluidized-bed continuous coagulation scheme. Only pepsin and calf rennet retained sufficient activity towards skim milk to warrant further studies. Comparison of kinetic data with fixed-bed reactors revealed the overall superior performance of fluidized beds; higher clotting activities were possible while avoiding plugging problems and high pressure drops common to fixed-bed reactors. Film diffusion and catalyst back-mixing appear to be significant factors in the overall kinetics. All enzymes lost activity on exposure to skim milk. The inactivation rates were lower at high substrate pH and insignificantly affected by reactor temperature. Nitrogen and sialic acid accumulation on the porous glass paralleled the loss in activity in the initial stages. Attempts to regenerate the immobilized enzymes were partially successful.     

ABSORPTION OF PEPSIN BY CRYSTALLINE PROTEINS

Crystalline proteins, such as edestin or melon globulin, remove pepsin from solution. The pepsin protein is taken up as such and the quantity of protein taken up by the foreign protein is just equivalent to the peptic activity found in the complex. The formation of the complex depends on the pH and is at a maximum at pH 4.0. An insoluble complex is formed and precipitates when pepsin and edestin solutions are mixed and the maximum precipitation is also at pH 4.0. The composition of the precipitate varies with the relative quantity of pepsin and edestin. It contains a maximum quantity of pepsin when the ratio of pepsin to edestin is about 2 to 1. This complex may consist of 75 per cent pepsin and have three-quarters of the activity of crystalline pepsin itself. The pepsin may be extracted from the complex by washing with cold N/4 sulfuric acid. If the complex is dissolved in acid solution at about pH 2.0 the foreign protein is rapidly digested and the pepsin protein is left and may be isolated. The pepsin protein may be identified by its tyrosine plus tryptophane content, basic nitrogen content, crystalline form and specific activity.     

THE INFLUENCE OF HYDROGEN ION CONCENTRATION ON THE INACTIVATION OF PEPSIN SOLUTIONS

1. Pepsin in solution at 38°C. is most stable at a hydrogen ion concentration of about 10^–5 (pH 5.0). 2. Increasing the hydrogen ion concentration above pH 5.0 causes a slow increase in the rate of destruction of pepsin. 3. Decreasing the hydrogen ion concentration below pH 5.0 causes a very rapid increase in the rate of destruction of the enzyme. 4. Neither the purity of the enzyme solution nor the anion of the acid used has any marked effect on the rate of destruction or on the zone of hydrogen ion concentration in which the enzyme is most stable. 5. The existence of an optimum range of hydrogen ion concentration for the digestion of proteins by pepsin cannot be explained by the destruction of the enzyme by either too weak or too strong acid.     

THE COMBINATION OF ENZYME AND SUBSTRATE : I. A METHOD FOR THE QUANTITATIVE DETERMINATION OF PEPSIN. II. THE EFFECT OF THE HYDROGEN ION CONCENTRATION.

1. A quantitative method for the determination of pepsin is described depending on the change in conductivity of a digesting egg albumin solution. 2. The combination of pepsin with an insoluble substrate has been followed by this method. 3. The amount of pepsin removed from solution by a given weight of substrate is independent of the size of the particles of the substrate. 4. There is an optimum zone of hydrogen ion concentration for the combination of enzyme and substrate corresponding to the optimum for digestion. 5. It is suggested that the pepsin combines largely or entirely with the ionized protein.     

CRYSTALLINE TRYPSIN : II. GENERAL PROPERTIES

A method is described for isolating a crystalline protein of high tryptic activity from beef pancreas. The protein has constant proteolytic activity and optical activity under various conditions and no indication of further fractionation could be obtained. The loss in activity corresponds to the decrease in native protein when the protein is denatured by heat, digested by pepsin, or hydrolyzed in dilute alkali. The enzyme digests casein, gelatin, edestin, and denatured hemoglobin, but not native hemoglobin. It accelerates the coagulation of blood but has little effect on the clotting of milk. It digests peptone prepared by the action of pepsin on casein, edestin or gelatin. The extent of the digestion of gelatin caused by this enzyme is the same as that caused by crystalline pepsin and is approximately equivalent to tripling the number of carboxyl groups present in the solution. The activity of the preparation is not increased by enterokinase. The molecular weight by osmotic pressure measure is about 34,000. The diffusion coefficient in ½ saturated magnesium sulfate at 6°C. is 0.020 ±0.001 cm.² per day, corresponding to a molecular radius of 2.6 x 10^–7 cm. The isoelectric point is probably between pH 7.0 and pH 8.0. The optimum pH for the digestion of casein is from 8.0–9.0. The optimum stability is at pH 1.8.     

Isolation and Identification of Three Novel Antioxidant Peptides from the Bactrian Camel Milk Hydrolysates

The aim of this study was isolation and purification of antioxidant peptides from Bactrian camel milk (BCM) hydrolysate. Trypsin, pepsin, alcalase, and pap     

Improved pepsin inhibitor derived from activation peptide 1-16 of porcine pepsinogen.

The peptide Leu-Val-Lys-Val-Pro-Leu-Val-Arg-Lys-Lys-Ser-Leu-Arg-Gln-Asn-Leu, a known pepsin inhibitor, is derived from the first 16 amino acids of porcine pepsinogen. It was prepared from the activation mixture and was modified by guanidination of its three lysine residues to form homoarginine residues. The modified peptide is a better pepsin inhibitor than the native peptide; for 50% inhibition of the milk clotting action of pepsin at pH 5.3, the molar ratio of peptide to pepsin required is 9 for the native inhibitor and only 2 for the guanidinated inhibitor. The dissociation constants (k1) of the inhibitor-pepsin complexes are 7 X 10(-8) and 1.4 X 10(-8) M for the native and guanidinated peptides, respectively. The guanidinated peptide is more resistant to digestion by pepsin at pH 3.5. The native and modified peptides partially protect pepsin from inactivation at pH 7. Stepwise removal of the amino-terminal Leu-Val-Har residues from the guanidinated inhibitor by Edman degradation decreases the pepsin-inhibiting activity only slightly at the first step, but markedly at the second and third steps. Thus, all of the amino-terminal sequence except the leucine residue is necessary for full activity.