Variants of chymosin with improved milk-clotting properties
A technology of rennet, bovine rennet, applied in the field of rennet variants
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[0281] 2. The method for preparing an isolated rennet polypeptide variant according to claim 1, wherein said isolated chymosin polypeptide variant has:
[0282] - a chymosin activity producing a C / P ratio higher than that of bovine chymosin comprising the mature polypeptide of SEQ ID NO: 1; and
[0283] - a chymosin activity producing a C / P ratio higher than that of camelid chymosin comprising the mature polypeptide of SEQ ID NO:2.
[0284] 3. The method for preparing an isolated chymosin polypeptide variant as claimed in any one of the preceding claims, wherein said alteration comprises positions corresponding to Substitution, deletion or insertion of at least one amino acid position at any one of positions 311, 352 and 353.
[0285] 4. The method for preparing an isolated chymosin polypeptide variant as claimed in any one of the preceding claims, wherein said alteration comprises positions corresponding to , 298, 300, 307, 309, 311, 325, 350, 352 and 353 at least one amino...
Embodiment 1
[0404] Example 1: Alignment and numbering of chymosin protein sequences and variant sequences
[0405] Using EBI (EBI, tools, multiple sequence alignment, CLUSTALW", http: / / www.ebi.ac.uk / Tools / msa / clustalw2 / ) and presented in Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007). Bioinformatics 23 (21 ), 2947-2948 to align chymosin protein sequences using the ClustalW algorithm described in 2947-2948.
[0406] ClustalW2 settings for multiple sequence alignment are Protein weight Matrix=BLOSUM, GAP open=10, GAP EXTENSION=0,05, GAP DISTANCES=8, No End Gaps, ITERATION=none, NUMITER=1, CLUSTERING=NJ.
[0407] Bovine chymosin B prochymosin precursor was used as a reference sequence (Genbank accession number P00794 - disclosed herein as SEQ ID NO: 1), where the N-terminal methionine has number 1 (MRCL...) and The C-terminal isoleucine (...LAKAI in the protein sequence) has n...
Embodiment 2
[0408] Example 2: Design of chymosin variants
[0409] Design of chymosin variants using different strategies.
[0410] When referring to camelid chymosin, the text refers to camelid chymosin comprising the polypeptide of SEQ ID NO:2.
[0411] The camelid chymosin of SEQ ID NO: 2 can be considered as the relevant parent polypeptide having chymosin activity for use in the preparation of its camelid chymosin variant.
[0412] When referring to bovine chymosin, the text refers to bovine chymosin comprising the polypeptide of SEQ ID NO:1.
[0413] The bovine chymosin of SEQ ID NO: 1 may be considered as the relevant parent polypeptide having chymosin activity for use in the preparation of bovine chymosin variants thereof.
[0414] Camelid chymosin variants were designed based on the alignment of a large set of publicly known aspartic protease sequences with 25% or more identity to bovine chymosin B.
[0415] Typically changes are introduced into hypervariable regions without ...
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