Journal of Dairy Research (1991), 58, 421-429
Printed in Great Britain
421
Enzymic modification of asl-casein with peptidylarginine deiminase: preparation of less acid-coagulable and less calcium-sensitive casein BY NORIHIRO AZUMA, KAZUTOSHI NARA AND CHOEMON KANNO Department of Animal Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya 321, Japan (Received 23 October 1990 and accepted for publication 18 June 1991) SUMMARY. Enzymic modification with peptidylarginine deiminase (EC 3.5.3.15) enabled five out of six arginyl residues in asl-casein to be converted to citrullyl residues, only the N-terminal arginyl residue remaining unaffected. An increase in the net negative charge was confirmed by PAGE. The isoelectric point was decreased from 4-46 for the intact asl-casein to 4-30 for the deiminated type, while simultaneously lowering the acid-precipitation starting point from pH 5-17 to pH 4-62. The deiminated asl-casein self-associated less in the absence of Ca and was less Ca-sensitive than the native type, although its Ca-binding ability was slightly enhanced. In the presence of 25 mM-CaCl2 and K-casein, Ca-induced precipitation of asl-casein did not occur, the solution of the mixture remaining transparent. Deimination of asl-casein resulted in altering its characteristics, possibly by interfering with interactions through hydrophobicity and/or hydrogen bonding. The positive charge of the arginyl residues might play an important role in casein micelle formation.
Caseins are recognized to be digestible and well-balanced amino acid resources. Their coagulation characteristics in the presence of Ca and their precipitation under acidic conditions limit their utilization as a food ingredient, especially for soft drinks. To increase the solubility of caseins, many investigators have attempted to modify them chemically or enzymically. Hoagland (1966) demonstrated that succinylation of the e-amino group of the lysyl residues of /?-casein made the protein insensitive to Ca2+ and that the succinylated /?-casein did not form a fast sedimenting peak. Creamer et al. (1971) also prepared acid-soluble casein by succinylation which was soluble down to pH 4-4 where normal casein is insoluble. It was shown by Evans et al. (1971) that acetylated or propionylated /?-casein associated less than the native type because of its enhanced negative charge. Home (1983) reported the effect of modifying lysyl residues by reacting with dansyl chloride and with fluorescamine on the Ca-induced precipitation of asl-casein. For a given Ca input level, the coagulation time for modified caseins increased with the degree of modification. Although chemically modified proteins are not allowed to be used as food proteins, it is possible that the use of enzymically modified proteins may be permitted. Home & Moir (1984) further examined the mechanism for the Ca-induced aggregation of
Printed in Great Britain
421
Enzymic modification of asl-casein with peptidylarginine deiminase: preparation of less acid-coagulable and less calcium-sensitive casein BY NORIHIRO AZUMA, KAZUTOSHI NARA AND CHOEMON KANNO Department of Animal Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya 321, Japan (Received 23 October 1990 and accepted for publication 18 June 1991) SUMMARY. Enzymic modification with peptidylarginine deiminase (EC 3.5.3.15) enabled five out of six arginyl residues in asl-casein to be converted to citrullyl residues, only the N-terminal arginyl residue remaining unaffected. An increase in the net negative charge was confirmed by PAGE. The isoelectric point was decreased from 4-46 for the intact asl-casein to 4-30 for the deiminated type, while simultaneously lowering the acid-precipitation starting point from pH 5-17 to pH 4-62. The deiminated asl-casein self-associated less in the absence of Ca and was less Ca-sensitive than the native type, although its Ca-binding ability was slightly enhanced. In the presence of 25 mM-CaCl2 and K-casein, Ca-induced precipitation of asl-casein did not occur, the solution of the mixture remaining transparent. Deimination of asl-casein resulted in altering its characteristics, possibly by interfering with interactions through hydrophobicity and/or hydrogen bonding. The positive charge of the arginyl residues might play an important role in casein micelle formation.
Caseins are recognized to be digestible and well-balanced amino acid resources. Their coagulation characteristics in the presence of Ca and their precipitation under acidic conditions limit their utilization as a food ingredient, especially for soft drinks. To increase the solubility of caseins, many investigators have attempted to modify them chemically or enzymically. Hoagland (1966) demonstrated that succinylation of the e-amino group of the lysyl residues of /?-casein made the protein insensitive to Ca2+ and that the succinylated /?-casein did not form a fast sedimenting peak. Creamer et al. (1971) also prepared acid-soluble casein by succinylation which was soluble down to pH 4-4 where normal casein is insoluble. It was shown by Evans et al. (1971) that acetylated or propionylated /?-casein associated less than the native type because of its enhanced negative charge. Home (1983) reported the effect of modifying lysyl residues by reacting with dansyl chloride and with fluorescamine on the Ca-induced precipitation of asl-casein. For a given Ca input level, the coagulation time for modified caseins increased with the degree of modification. Although chemically modified proteins are not allowed to be used as food proteins, it is possible that the use of enzymically modified proteins may be permitted. Home & Moir (1984) further examined the mechanism for the Ca-induced aggregation of
