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Comparative study of methods for the isolation and purification of bovine κ-casein and its hydrolysis by chymosin

Published online by Cambridge University Press:  01 June 2009

Kate P. Coolbear ,
David F. Elgar ,
Tim Coolbear  and
John S. Ayers
Kate P. Coolbear
Affiliation:
Department of Chemistry and Biochemistry, Massey University, Private Bag 11222, Palmerston North, New Zealand
David F. Elgar
Affiliation:
Department of Chemistry and Biochemistry, Massey University, Private Bag 11222, Palmerston North, New Zealand
Tim Coolbear
Affiliation:
New Zealand Dairy Research Institute, Private Bag 11029, Palmerston North, New Zealand
John S. Ayers
Affiliation:
Department of Chemistry and Biochemistry, Massey University, Private Bag 11222, Palmerston North, New Zealand
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Summary

κ-Casein was purified from a single batch of whole acid casein (κ-A variant) using different methods in order to compare their merits in producing a purified material with a carbohydrate and phosphate heterogeneity representative of the whole κ-casein complement in milk. Ion-exchange methods of purification gave products of higher purity than precipitation techniques involving final purification by ethanol fractionation, but all methods resulted in κ-caseins of apparently similar heterogeneity and chemical composition. The purified κ-caseins were hydrolysed with chymosin and the derived macropeptides isolated. These were all virtually identical as determined by reversed-phase chromatography and gel electrophoresis. Some observations on chymosin hydrolysis of κ-casein were made. In addition to formation of the major para-κ-casein (Glu1–Phe105) and macropeptide (Met106–Val169), chymosin hydrolysis at pH 6·6 also resulted in two minor para-κ-caseins with N-termini corresponding to Phe18 and Ser33 of κ-casein. At pH 5·5 and 4·5 para-κ-casein was rapidly hydrolysed into at least six fragments, one of which had an N-terminus corresponding to Trp76 of κ-casein. At pH 6·6, 5·5 and 4·5 the κ-casein macropeptide was stable to chymosin, but at pH 2·3 it was hydrolysed by chymosin into fragments with N-termini corresponding to Met106, He125, Ala138, Val139, Thr145 and Glu147 of κ-casein.

Type
Original Articles
Information
Journal of Dairy Research , Volume 63 , Issue 1 , February 1996 , pp. 61 - 71
Copyright
Copyright © Proprietors of Journal of Dairy Research 1996

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