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Carboxyl groups in porcine pepsin were chemically modified "by the carbodiimide reaction using waterrsoluble l-ethyl-3-(3-dimethylaminopropyl) carbodiimide and amino acid esters as nucleophiles. The modification resulted in profound changes in the activities, specificity and some physico-chemical properties of the enzyme. These include (1) significant decrease in milk clotting activity without changes in proteolytic activity against hemoglobin; (2) decrease in peptidase activity against N-acetyl-L-phenylalanyl-diiodo-L-tyrosine; (3) increase in clotting activity against k-casein but decrease in clotting activity against K-α sl⁻casein mixture; (4) shift -in proteolytic pH profile with pH optimum increased from 2.0 to about 3.5; (5) decrease in relative electrophoretic mobility and a slight decrease in isoelectric point; (6) increase in Km without much change in kcat; and (7) increase in stability at pH above 6.0. Results suggest that the drop in milk clotting activity was due to a change in the charge distribution on the enzyme affecting enzyme-micelle interaction. The presence of dipeptide substrates interfered with the carboxyl modification suggestive of the proximity of the modified groups to the enzyme active site. The modified enzyme remained reactive to site-specific inactivators but at rates slower than the native enzyme. The modification was not specific, causing similar changes in pepsinogen and chymosin. The modified and native pepsins had similar caseinolytic properties and produced comparable rates of syneresis and curd tension development on curdled milk. The increase in pH stability suggested that the modified enzyme may be a better calf rennet substitute than native pepsin for cheese-making. / Land and Food Systems, Faculty of / Graduate

Several studies have demonstrated that certain genetic variants of beta-casein are closely related to milk production, milk composition and technological properties of milk such as coagulation properties during cheese making, calcium precipitation and water binding properties. The objective of current study is to investigate the effect of the genetic variants on hydrolysis of beta-casein by chymosin and pepsin. On the basis of a preliminary analysis, a total of 50 milk samples which provided representatives of different available genetic variants of beta-casein were collected from different herds in Quebec. Casein was prepared from milk samples by the acid precipitation and the genetic variants of beta-casein were identified by both alkaline and acid urea-PAGE. An anion exchange chromatography was employed for the separation of beta-casein from whole casein. An initial hydrolysis of beta-casein of different phenotypes by chymosin and pepsin were achieved under the optimized hydrolytic conditions. Hydrolysates were periodically removed from the reaction mixture and they were analyzed by both RP-HPLC and SDS-PAGE in order to study the hydrolytic pattern of each beta-casein variant with the increasing hydrolytic time. (Abstract shortened by UMI.)

Caseins are present in milk in the form of large spherical complexes called micelles that are stabilized by kappa-casein found on the surface. This stabilizing effect is lost when milk clotting enzymes hydrolyze kappa-casein thereby initiating the coagulation process. The objective of this study was to analyze the effect of kappa-casein polymorphism upon hydrolysis by proteases. The A and B forms are the most common genetic variants of kappa-casein in Canadian Holstein dairy herds, representing three phenotypes AA, AB and BB. Whole casein from Holstein milk samples was fractionated into its four major components by ion-exchange chromatography using Express Ion Exchanger Q Anion Exchanger and Macro-Prep High Q Anion Exchange Support columns. The kappa-casein fraction was isolated, dialyzed, and assessed for its phenotype and purity by PAGE. The pure forms of the three different phenotypes of kappa-casein (AA, AB, BB) at a final concentration of 0.5% were then hydrolyzed by calf chymosin (1:500) and porcine pepsin (1:1000) at a pH of 5.8 at 37°C. Aliquots were collected at 0, 5, 15, 30, 60 and 90 min and the reaction stopped by using 24% NH4OH. The rate of hydrolysis of the three phenotypes was analyzed by comparing the disappearance of the substrate with time from the RP-HPLC chromatograms using Waters MAXIMA software. SDS-PAGE was used to calculate the approximate molecular weight of the hydrolytic products. Analysis of the hydrolysate profiles indicated that there was significant difference (P < 0.05) in the rate of hydrolysis between the phenotypes. Under the present conditions at a pH of 5.8, the AA phenotype showed a significantly slower rate of hydrolysis by both chymosin and pepsin, than the other two phenotypes. There was no significant difference in the rate of hydrolysis between the phenotypes AB and BB during chymosin hydrolysis. The BB phenotype is hydrolyzed more extensively and the AB phenotype is intermediate between the two variants in the p

Thesis (doctoral)--Universität Leipzig, 1903. / Includes bibliographical references.

IN ENGLISH Human gastric juice contains mainly aspartate proteinases: pepsin A and pepsin C. Both pepsins are produced by gastric mucosa as inactive pepsinogens and they are activated to the corresponding pepsins in the acidic environment of the gastric lumen. The levels of pepsinogens in serum reflect the morphological and functional status of gastric mucosa. A subject of this thesis is a part of a long-term investigation that focuses on the elaboration of methods for separation gastric aspartate proteainases that would be suitable for diagnostic purposes. The preparation of new type ligands was a concrete subject of PhD. thesis that after their immobilization they can enable the separation of aspartate proteinases. Four heptapeptides containing D-leucinyl residue were synthetized (Val-D-Leu-Pro-Phe-Phe-Val- D-Leu, Val-D-Leu-Pro-Tyr-Phe-Val-D-Leu, Val-D-Leu-Pro-Tyr-Tyr-Val-D-Leu and Val-D- Leu-Pro-Phe-Tyr-Val-D-Leu. The prepared heptapeptides immobilized on agarose magnetic particles were used for the study of their interaction with porcine pepsin A and rat pepsin C. While porcine pepsin A was adsorbed to all heptapeptides immobilized to magnetic particles, rat pepsin C was not retarded. Similar results were obtained using heptapeptides immobilized to Sepharose. The situation was more complicated...

IN ENGLISH Human gastric juice contains mainly aspartate proteinases: pepsin A and pepsin C. Both pepsins are produced by gastric mucosa as inactive pepsinogens and they are activated to the corresponding pepsins in the acidic environment of the gastric lumen. The levels of pepsinogens in serum reflect the morphological and functional status of gastric mucosa. A subject of this thesis is a part of a long-term investigation that focuses on the elaboration of methods for separation gastric aspartate proteainases that would be suitable for diagnostic purposes. The preparation of new type ligands was a concrete subject of PhD. thesis that after their immobilization they can enable the separation of aspartate proteinases. Four heptapeptides containing D-leucinyl residue were synthetized (Val-D-Leu-Pro-Phe-Phe-Val- D-Leu, Val-D-Leu-Pro-Tyr-Phe-Val-D-Leu, Val-D-Leu-Pro-Tyr-Tyr-Val-D-Leu and Val-D- Leu-Pro-Phe-Tyr-Val-D-Leu. The prepared heptapeptides immobilized on agarose magnetic particles were used for the study of their interaction with porcine pepsin A and rat pepsin C. While porcine pepsin A was adsorbed to all heptapeptides immobilized to magnetic particles, rat pepsin C was not retarded. Similar results were obtained using heptapeptides immobilized to Sepharose. The situation was more complicated...

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