Cheese is made using rennet, which are milk clotting enzymes. Rennet traps proteins and fat in the cheese making process. An important enzyme within this collection is a protease called chymosin. This protease coagualates the milk, an important part of the cheese making process. These collection of enzymes were initially isolated from calf intestines. They can also be isolated from other sources such as pigs and fungi. The rarity and imperfections of this enzyme isolated from calf intestines led to an alternative choice to create chymosin by recombinant methods.
How it WorksEditChymosin is a proteolitic enzyme or protease. In milk there are two different catagories that the components can be divided into. They are caseins and whey proteins. The casein components exist as micelles in the milk mixture. chymosin causes a rapid cleavage of the kappa-casein component of the casein micelles. Specifically, chymosin recognizes the amino acids in positions 105 and 106, Phe and Met, respectively. The kappa-casein protein helps to keep the other milk proteins soluble by helping stabilize other casein proteins that have formed micelles. Chymosin cleaves kappa-casein, removing its ability to stabilize micelle structures leaving the casein proteins to precipitate and tangle into a solid mass called a curd.
Chymosin StructureEditChymosin consists of 323 amino acids containing intramolecular disulfide linkages. There are 2 types of chymosin, A and B. The difference is in one amino acid at position 286 where A has an aspartic acid while B has a glycine instead.
Constructing Recombinant ChymosinEdit
Since calf rennet is increasing in rarity because of the decline in calves, an alternative method of producing chymosin for cheese production was needed. Creating the chymosin is a fairly straightforward process.A host must be first chosen for this process. There are many hosts such as E.coli K12 or Kluyveromyces lactis. Any of these hosts can work and the process is the same. First, the copy of the chymosin gene is isolated from calf intestinal cells. This isolated gene is then inserted into a plasmid, a circular piece of DNA. The plasmid is then inserted into a host (transformation) and cultured. The host cell will start transcribing and translating the plasmid and thus the chymosin gene on the plasmid will produce chymosin. The chymosin is then purified through techniques such as column filtration to remove everything except chymosin. It is advantagous to produce chymosin through recombinant techniques because of the purity of the product. Using this method, only chymosin is isolated instead of other proteins associated with rennet in the calf's intestine.
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