Process for separating milk clotting enzymes, and stable rennet

Details Process for separating milk clotting enzymes, and stable rennet Process for separating milk clotting enzymes, and stable rennet

1996-12-12

1999-03-30

Tsang, Cecilia J.

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

514 21, 530350, 530412, 530414, 530416, 530418, 424 9466, A61K 3843, C07K 114

Patent

active

058889663

DESCRIPTION:

BRIEF SUMMARY
FIELD OF INVENTION

The present invention relates to the manufacturing of milk clotting enzymes, in particular to the manufacturing of rennet compositions containing a high proportion of chymosin and to rennet compositions in which the milk clotting enzyme has been stabilized.


TECHNICAL BACKGROUND AND PRIOR ART

Milk clotting enzymes are widely used in the cheese industry to provide a curd of the major milk proteins. Commercially available milk clotting enzymes include native enzymes derived from microbial or animal tissue sources, or the enzymes may be provided as gene products of recombinant cells expressing a milk clotting enzyme of animal or microbial origin.
Native milk clotting enzymes of animal origin are isolated by extraction from animal tissues containing one or several of these enzymes. Thus, animal milk clotting enzymes include several enzymes of the group of aspartic endopeptidases having molecular weights which are in the range of about 35,000 to 42,000 daltons (group 3.4.23 according to the Enzyme Nomenclature, 1992 of the International Union of Biochemistry and Molecular Biology, IUBMB) such as pepsin A (3.4.23.1) and gastricsin (3.4.23.3) which is excreted into the gastric juice of vertebrates including ruminants, pigs and humans, and chymosin (3.4.23.4) which is a predominantly neonatal gastric enzyme with high milk clotting activity, excreted in mammals. The molecular weights of animal milk clotting enzymes are in the range of about 35,000 to about 42,000 daltons. Thus, chymosin has a calculated molecular weight of 35,652 daltons.
The primary industrial source of native animal milk clotting enzymes are stomachs of calves and adult cattle in which essentially all of the in-vivo milk clotting activity is associated with the presence in the gastric juice of chymosin and pepsin A. However, when produced in the stomach tissue cells, these enzymes occur as enzymatically inactive pre-enzymes which are designated pre-prochymosin and pre-pepsinogen A, respectively. When chymosin is excreted, an N-terminal fragment is cleaved off to give prochymosin including a pro-fragment. Prochymosin is an essentially inactive form of the enzyme which, however, under acidic conditions becomes activated to the active chymosin molecule by removal of the pro-fragment. This activation takes place in-vivo in the gastric lumen under appropriate pH conditions. Pepsinogen A is activated into the active enzyme by partial hydrolysis under acidic conditions.
Pseudochymosin is the designation of a chymosin species where only part of the pro-fragment (amino acid residues 1-27) is removed. Pseudochymosin will e.g. occur in an extract which has been exposed to a low pH, such as a pH of 2. Pseudochymosin has enzyme activity and is stable at low pH but is processed to chymosin at higher pH. The isoelectric point of pseudochymosin is not known, but it is assumed to be higher than 4.9.
Recently, animal chymosin that is produced in recombinant microorganisms including filamentous fungi has been introduced into the industrial market. Such recombinantly manufactured milk clotting enzyme products are also referred to as fermentation produced chymosin or rennet.
In addition to the above milk clotting enzymes of animal origin several natively produced microbial enzymes are used in the dairy industry. Such enzymes are referred to as microbial milk clotting enzymes or microbial coagulants in the following. Examples of such enzymes include Rhizomucor (previously Mucor) miehei proteases including destabilized, i.e. oxidized Rhizomucor miehei protease, Mucor pusillus protease and Endothia parasitica protease.
Preparations or compositions containing native milk clotting enzymes of animal origin are prepared industrially by extraction from stomach tissues, in particular from ruminants including calves and adult cattle. Enzyme-containing crude extracts contain chymosin species including precursors, and pepsin species in ratios which depend primarily on the age of the animal. Thus, the distribution between chymosin and pepsin in stomachs from

REFERENCES:
patent: 3763010 (1973-10-01), Schleich
patent: 3950221 (1976-04-01), Kokusho et al.
patent: 4721673 (1988-01-01), Uren et al.
patent: 5151358 (1992-09-01), Heinsohn et al.
Subramanian, S., "Separation of chymosin and pepsin in calf rennet by dye-ligand affinity chromatography", Preparative Biochemistry, vol. 17, No. 3, pp. 297-312 (1987).

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