Recombinant Chymosin

Cheeses represent a traditional way of preserving a perishable foodstuff, milk. Cheese is made by addition of milk to a starter culture of lactic acid bacteria whic acidify the milk to about pH 5.5. In addition, milk clotting enzymes called rennet are added, resulting in a coagulated protein gel trapping other proteins and fat. Traditionally the rennet used is a preparation of several enzymes isolated from calf stomach. Modern cheeses making, however, increasingly relies upon microbial sources of the most important enzyme, the protease chymosin. This is now produced by recombinant micro-organisms.

Milk Proteins
Milk consists of water, fat, protein, phosphate, lactose, citric acid and inorganics such as calcium phosphate. The protein component of milk can be divided into two groups, the casein fraction and the whey proteins:

The casein proteins are the ones that will form the curd during cheese making. Casein proteins tend not to have a particularly compact globular structure and they tend to be rather susceptible to proteolysis. As they are all phosphorylated, they bind the calcium content of the milk and exisit in the form of casein micelles.

Chymosin Reaction
The chymosin content of the rennin causes a specific and rapid cleaveage of the kappa-casein component of the casein micelles. This protein stabilises the micelles and after cleavage, the casein proteins precipitate under the influence of the calcium ions. Chymosin specifically recognises the sequence from His 98 to Lys 111 and cleaves the peptide bond between Phe 105 and Met 106 in the kappa-casein chain:

In addition to its role in milk clotting, chymosin is also involved in the proteolytic changes occurring during ripening.

Chymosin Structure
Bovine chymosin is an acid protease produced in the fourth stomach of the cow in the form of a precursor. After a series of proteolysis events, the mature chymosin has a molecular weight of 35600 Da and exisits in one of two allelic forms, A and B, differing in the nature of the amino acid at position 244 (Asp or Gly). The A form has a slightly higher specific activity for kappa-casein but is marginally less stable than the B form.

[Click on image to explore protein with Chime] Microbial alternatives to bovine chymosin are very desirable due to their lower price and more stable availability as the supply of calf-rennet is in decline. In addition, vegetarians find the use of bovine rennet unacceptible. Suitable candidates are the proteases from Mucor miehei, Mucor pusillis and Endothia parasitica. All of these are acid proteases of similar structure to the bovine enzyme and all have the neccessary narrow specificity for kappa-casein. The biggest problem with these proteases is that they are not suitable for long ripening cheeses as they have a different range of non-specific activities than chymosin and do not produce the correct flavours on prolonged ripening. Recombinant Chymosin Production The problems outlined above for chymosin and its microbial substitutes can be alleviated by cloning the bovine gene into a suitable production strain and producing the enzyme by fermentation. The most important decisions to be made were the choice of host/vector system. There are several possibilities for a production host:
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Eschrichia coli. E. coli is the favourite organism of molecular biologists and is most frequently used in gene cloning experiments. The problem with E. coli, however, is that recombinant proteins are frequently synthesised as intracellular inclusion bodies, increasing process costs cosiderably. Another issue with E. coli is that it is not generally recognised as safe for human consumption. Bacillus sp. Bacillus species are non-pathogenic and are used industrially to produce several enzymes used in food processing such as amylases. B.

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licheniformis could produce the chymosin, but the signal sequences did not allow the secretion of the enzyme into the medium. Lactococcus lactis. This host was chosen as it is already used in starter cultures, but production levels were found to be very low. Saccharomyces cerevisiae. Difficulties were experienced in achieving high secretion rates in yeast. Kluveromyces lactis. K. lactis is used for the production of dairy grade beta-galactosidase and its fermentation properties are well understood. It was found that the chtmosin could be produced in this host and good levels of secretion into the medium were achieved.

The chymosin gene was inserted into the K. lactis chromosome and the yeast is grown by fed-batch fermentation. After fermentation, the yeast is killed by addition of benzoic acid and the chymosin is isolated by filtration.

Rennet (rennin or rennilase) and cheese making: Back in the 1960's, the world faced a severe shortage of calf rennet. Rennet is a protease enzyme added to milk, along with certain bacteria, which coagulate milk proteins, producing the curds. This then separates from the liquid, whey. The semi-solid curds are then treated by adding salt and then matured in containers to make the cheddar-style cheeses. (Rennet is an enzyme found in the stomachs of young mammals, like calves, and is important in the digestion of milk proteins.) Over the past 30 years, alternatives to calf rennet have been sought. Alternative sources of Protease for coagulating milk: There are a number of alternatives available, which are not derived from animal sources directly. One form is comes from fungi, such as Rhizomucor miehei. Fungi produce natural proteolytic enzymes as part of their extracellular digestion process.

The other are from genetically modified microbes, such as E.coli bacteria, a fungus Aspergillus niger and food yeasts. Obtaining Chymosin from yeast cells: About 90% of hard cheeses like Cheddar are made now using chymosin from GM microbes. Advantages of using GM Chymosin: 1. 2. 3. Chymosin was the first enzyme to be approved for used in the food industry, so its effects are well known. Chymosin behaves exactly the same way as the animal equivalent, but due to its structure, it is much more predictable and there are fewer impurities. As it is made in yeast, it is fully accepted by vegetarians and some religious authorities. Chymosin and GMO's: Cheese produced using genetically engineered Chymosin is not regarded as a problem as it does not contain the organism, but the product (enzyme) of that GMO. The enzyme does not remain in the cheese either, but soon breaks down, as it is a relatively unstable protein.