Variants of chymosin with improved milk-clotting properties
AssigneeChr. Hansen A/S
InventorsJohannes Maarten Van Den Brink, Jesper Langholm Jensen, Jonas Jacobsen, Martin Lund, Iben Jeppesen, Christian Jaeckel
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Abstract
Variants of chymosin with improved milk-clotting properties are disclosed.
Claims18
An isolated chymosin polypeptide variant obtained by a method comprising:
- producing and isolating a chymosin polypeptide variant of a parent polypeptide having chymosin activity, wherein the amino acid sequence of the variant differs from the amino acid sequence of the parent by having an alteration comprising a substitution at at least one amino acid position of the parent sequence corresponding to any of positions 77, 154, 212, 224, 246, and 367, wherein the corresponding position in the parent sequence is determined by alignment of the parent sequence with SEQ ID NO:1 (bovine chymosin), thereby obtaining the isolated chymosin polypeptide variant, wherein the variant has chymosin activity;
- wherein:
- the amino acid sequence of the parent polypeptide has at least 65% sequence identity with the amino acid sequence from position 59 to position 381 of SEQ ID NO:1 (the mature bovine chymosin polypeptide);
- and
- the variant has fewer than 30 amino acid alterations in the region from amino acid position 59 to amino acid position 381 as compared to the mature polypeptide from amino acid position 59 to amino acid position 381 of SEQ ID NO:1 or as compared to the mature polypeptide from amino acid position 59 to amino acid position 381 of SEQ ID NO:2, as determined by an alignment of the amino acid sequence of the variant with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, respectively;
- with the proviso that the variant is NOT a specific variant having any of the following sets of substitutions based on the numbering of SEQ ID NO:1:
- Q246E+G309D+S329P+D337E;
- R125Q+G128N+H204R+Q246E+S284T;
- Y185F+R213Q+Q246E;
- D216S+L224V+V263I+F281V+G309D;
- Y79S+L224V+L311I;
- and
- R119S+L224V+T297S.
An isolated chymosin polypeptide variant of a parent polypeptide having chymosin activity, wherein the amino acid sequence of the variant differs from the amino acid sequence of the parent by having an alteration comprising a substitution at at least one amino acid position of the parent sequence corresponding to any of positions 77, 154, 212, 224, 246, and 367, wherein the corresponding position in the parent sequence is determined by alignment of the parent sequence with SEQ ID NO:1 (bovine chymosin),
- wherein the variant has chymosin activity;
- and
- wherein:
- (i) the amino acid sequence of the parent polypeptide has at least 90% sequence identity with the amino acid sequence from position 59 to position 381 of SEQ ID NO:1 (the mature bovine chymosin polypeptide);
- and
- (ii) the amino acid sequence of the variant has less than 100% sequence identity with the amino acid sequence from position 59 to position 381 of SEQ ID NO:1;
- and
- (ii) the variant has fewer than 30 amino acid alterations in the region from amino acid position 59 to amino acid position 381 as compared to the mature polypeptide from amino acid position 59 to amino acid position 381 of SEQ ID NO:1, as determined by an alignment of the amino acid sequence of the variant with the amino acid sequence of SEQ ID NO:1;
- with the proviso that the variant is NOT a specific variant having any of the following sets of substitutions based on the numbering of SEQ ID NO:1:
- Q246E+G309D+S329P+D337E;
- R125Q+G128N+H204R+Q246E+S284T;
- Y185F+R213Q+Q246E;
- D216S+L224V+V263I+F281V+G309D;
- Y79S+L224V+L311I;
- and
- R119S+L224V+T297S;
- wherein the variant has a chymosin activity giving a higher clotting activity to proteolytic activity (C/P) ratio as compared to the C/P ratio of bovine chymosin comprising the amino acid sequence from position 59 to position 381 of SEQ ID NO:1.
An isolated chymosin polypeptide variant of a parent polypeptide having chymosin activity, wherein the amino acid sequence of the variant differs from the amino acid sequence of the parent by having an alteration comprising a substitution at at least one amino acid position of the parent sequence corresponding to any of positions 77, 154, 212, 224, 246, and 367, wherein the corresponding position in the parent sequence is determined by alignment of the parent sequence with SEQ ID NO:1 (bovine chymosin),
- wherein the variant has chymosin activity;
- and wherein:
- (i) the amino acid sequence of the parent polypeptide has at least 90% sequence identity with the amino acid sequence from position 59 to position 381 of SEQ ID NO:2;
- and
- (ii) the amino acid sequence of the variant has less than 100% sequence identity with the amino acid sequence from position 59 to position 381 of SEQ ID NO:2;
- and
- (iii) the variant has fewer than 30 amino acid alterations in the region from amino acid position 59 to amino acid position 381 as compared to the mature polypeptide from amino acid position 59 to amino acid position 381 of SEQ ID NO:2, as determined by an alignment of the amino acid sequence of the variant with the amino acid sequence of SEQ ID NO:2;
- with the proviso that the isolated chymosin polypeptide variant is NOT a specific variant having any of the following sets of substitutions based on the numbering of SEQ ID NO:1:
- Q246E+G309D+S329P+D337E;
- R125Q+G128N+H204R+Q246E+S284T;
- Y185F+R213Q+Q246E;
- D216S+L224V+V263I+F281V+G309D;
- Y79S+L224V+L311I;
- and
- R119S+L224V+T297S;
- wherein the variant has a chymosin activity giving a higher clotting activity to proteolytic activity (C/P) ratio as compared to the C/P ratio of camel chymosin comprising the amino acid sequence from position 59 to position 381 of SEQ ID NO:2.
Dependencies are inferred from the claim text.