Lilla S, Caira S, Ferranti P, Addeo F
Istituto di Scienze dell'Alimentazione del C.N.R., Via Roma 52, I-83100 Avellino, Italy.
Rapid Commun Mass Spectrom. 2001;15(13):1101-12. doi: 10.1002/rcm.345.
The protein composition of natural rennet and of chromatographic and crystalline 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 was found to consist of six chymosin species, corresponding to chymosin A and B genetic variants, each of which comprised a mixture of two other forms differing at theN-terminal end, with one being three residues longer, and the other two residues shorter, than the mature chymosin. Two main tissue proteins were also identified as lysozyme (isozyme 2 plus a novel isozyme labelled 4) and bovine serum albumin. In addition to the proteins, chymosin fragments 247-323 and 288-323 were consistently present in natural rennet. Conversely, chromatographic and crystalline chymosin preparations lacked bovine serum albumin and/or lysozyme, although they contained the same six chymosin species as natural rennet. Since these tissue-specific contaminating proteins each possess specific functions in terms of stabilising enzyme solutions and protecting proteins from proteolytic enzymes, oxidising agents and bacterial proliferation, the rennet may be considered as a functional enzyme preparation that is effectively and naturally adapted to the purposes of cheesemaking. In practice, the highly complex protein composition inherent to natural rennet provided the possibility to differentiate the natural product from other bovine chymosin-based milk-clotting preparations examined in this work.
天然凝乳酶以及色谱法和结晶凝乳酶制剂的蛋白质组成已通过在线反相高效液相色谱/电喷雾电离质谱法(RP-HPLC/ESI-MS)和串联质谱法(MS/MS)得以确定。结果发现,天然凝乳酶由六种凝乳酶组成,分别对应凝乳酶A和B的基因变体,每种变体又包含另外两种在N端不同的形式的混合物,其中一种比成熟凝乳酶长三个残基,另一种短两个残基。还鉴定出两种主要的组织蛋白为溶菌酶(同工酶2加上一种标记为4的新型同工酶)和牛血清白蛋白。除了这些蛋白质外,凝乳酶片段247-323和288-323也始终存在于天然凝乳酶中。相反,色谱法和结晶凝乳酶制剂不含牛血清白蛋白和/或溶菌酶,尽管它们含有与天然凝乳酶相同的六种凝乳酶。由于这些组织特异性污染蛋白在稳定酶溶液以及保护蛋白质免受蛋白水解酶、氧化剂和细菌增殖影响方面各自具有特定功能,因此可以认为凝乳酶是一种有效地、自然地适用于奶酪制作目的的功能性酶制剂。实际上,天然凝乳酶固有的高度复杂的蛋白质组成使得区分天然产品与本研究中检测的其他基于牛凝乳酶的凝乳制剂成为可能。