Mass spectrometric characterisation of proteins in rennet and in chymosin-based milk-clotting preparations

The protein composition of natural rennet and of chromatographic and crysta lline chymosin preparations has been defined by on-line reverse-phase high performance liquid chromatography/electrospray ionisation mass spectrometry (RP-HPLC/ESI-MS) and by tandem mass spectrometry (MS/MS). Natural rennet w as found to consist of six chymosin species, corresponding to chymosin A an d B genetic variants, each of which comprised a mixture of two other forms differing at the N-terminal end, with one being three residues longer, and the other two residues shorter, than the mature chymosin. Two main tissue p roteins were also identified as lysozyme (isozyme 2 plus a novel isozyme la belled 4) and bovine serum albumin. In addition to the proteins, chymosin f ragments 247-323 and 288-323 were consistently present in natural rennet. C onversely, chromatographic and crystalline chymosin preparations lacked bov ine serum albumin and/or lysozyme, although they contained the same six chy mosin species as natural rennet. Since these tissue-specific contaminating proteins each possess specific functions in terms of stabilising enzyme sol utions and protecting proteins from proteolytic enzymes, oxidising agents a nd bacterial proliferation, the rennet may be considered as a functional en zyme preparation that is effectively and naturally adapted to the purposes of cheesemaking. In practice, the highly complex protein composition inhere nt to natural rennet provided the possibility to differentiate the natural product from other bovine chymosin-based milk-clotting preparations examine d in this work. Copyright (C) 2001 John Wiley & Sons, Ltd.