Validation of recombinant and bovine chymosin by mass spectrometry.
Mass spectrometry has been used to map chymosin from a fermentative source. The copresence of the two known genetic variants A (Asp(244)) and B (Gly(244)) was ascertained in bovine chymosin. By contrast, either the A or the B genetic variant occurred in the three commercial samples of recombinant calf chymosin (RCC). Specific biomarker proteins were searched to identify the enzyme source, in both bovine chymosin and RCC samples. Analyzing the derived tryptic peptides, evidence was provided that RCC and bovine chymosin are mainly formed by (1-323), (3-323), and (40p-323) (suffix "p" denotes residues in the pro-segment region of chymosin), whereas the minor components, (4-323), (5-323), and (6-323), were only detected in bovine chymosin. Additionally, the three commercial RCC samples contained the protein species (1-323), (38p-323), (39p-323), and (40p-323) and the shorter form (3-323). Differentiation of the natural and bioengineered enzyme is based upon the detection of these unique minor components by mass spectrometry.
Lilla
Sergio
S
Dipartimento di Scienza degli Alimenti, Università degli Studi di Napoli Federico II, Portici, Italy. lilla@unina.it
Mogna
Giovanni
G
Addeo
Francesco
F
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D012085:Chymosin / Q000737:chemistry / Q000145:classification* / Q000235:genetics; D014644:Genetic Variation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis / Q000145:classification*; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D014357:Trypsin; D012085:Chymosin