Open Collections

Chemical modification of carboxyl groups in porcine pepsin

University of British Columbia

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.

Text

2010-03-12

For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

http://hdl.handle.net/2429/21817

Food Science

University of British Columbia

Graduate