Chymosin or rennin is a protease found in

rennet Rennet () is a complex set of enzymes produced in the stomachs of ruminant mammals. Chymosin, its key component, is a protease enzyme that curdles the casein in milk. In addition to chymosin, rennet contains other enzymes, such as pepsin and ...

. It is an aspartic endopeptidase belonging to MEROPS A1 family. It is produced by newborn ruminant animals in the lining of the

abomasum The abomasum, also known as the maw,The Cham ...

to curdle the milk they ingest, allowing a longer residence in the bowels and better absorption. It is widely used in the production of

cheese Cheese is a dairy product produced in wide ranges of flavors, textures, and forms by coagulation of the milk protein casein. It comprises proteins and fat from milk, usually the milk of cows, buffalo, goats, or sheep. During production, ...

. Bovine chymosin is now produced recombinantly in , '' Aspergillus niger var awamori'', and as alternative resource.

Occurrence

The chymosin is found in a wide range of tetrapods, although it is best known to be produced by ruminant animals in the lining of the

abomasum The abomasum, also known as the maw,The Cham ...

. Chymosin is produced by

gastric chief cell A gastric chief cell (or peptic cell, or gastric zymogenic cell) is a type of gastric gland cell that releases pepsinogen and gastric lipase. It is the cell responsible for secretion of chymosin in ruminant animals. The cell stains basophilic u ...

s in newborn mammals to curdle the milk they ingest, allowing a longer residence in the bowels and better absorption. Non-ruminant species that produce chymosin include pigs, cats, seals,Staff, Online Mendelian Inheritance in Man (OMIM) Database. Last updated February 21, 199
Chymosin pseudogene; CYMP prochymosin, included, in the OMIM
/ref> and chicks. One study reported finding a chymosin-like enzyme in some human infants, but others have failed to replicate this finding. Humans have a pseudogene for chymosin that does not generate a protein, found on

chromosome 1 Chromosome 1 is the designation for the largest human chromosome. Humans have two copies of chromosome 1, as they do with all of the autosomes, which are the non-sex chromosomes. Chromosome 1 spans about 249 million nucleotide base pairs, which ...

. Humans have other proteins to digest milk, such as

pepsin Pepsin is an endopeptidase that breaks down proteins into smaller peptides. It is produced in the gastric chief cells of the stomach lining and is one of the main digestive enzymes in the digestive systems of humans and many other animals, ...

and lipase. In addition to the primate lineage leading up to humans, some other mammals have also lost the chymosin gene.

Enzymatic reaction

Chymosin is used to bring about the extensive precipitation and

curd Curd is obtained by coagulating milk in a sequential process called curdling. It can be a final dairy product or the first stage in cheesemaking. The coagulation can be caused by adding rennet or any edible acidic substance such as lemon ...

formation in

-making. The native substrate of chymosin is K-casein which is specifically cleaved at the peptide bond between amino acid residues 105 and 106, phenylalanine and methionine. The resultant product is

calcium phosphocaseinate Calcium is a chemical element with the symbol Ca and atomic number 20. As an alkaline earth metal, calcium is a reactive metal that forms a dark oxide-nitride layer when exposed to air. Its physical and chemical properties are most similar to ...

. When the specific linkage between the

hydrophobic In chemistry, hydrophobicity is the physical property of a molecule that is seemingly repelled from a mass of water (known as a hydrophobe). In contrast, hydrophiles are attracted to water. Hydrophobic molecules tend to be nonpolar and, t ...

(para-casein) and

hydrophilic A hydrophile is a molecule or other molecular entity that is attracted to water molecules and tends to be dissolved by water.Liddell, H.G. & Scott, R. (1940). ''A Greek-English Lexicon'' Oxford: Clarendon Press. In contrast, hydrophobes are no ...

(acidic

glycopeptide Glycopeptides are peptides that contain carbohydrate moieties (glycans) covalently attached to the side chains of the amino acid residues that constitute the peptide. Over the past few decades it has been recognised that glycans on cell surfac ...

) groups of

casein Casein ( , from Latin ''caseus'' "cheese") is a family of related phosphoproteins ( αS1, aS2, β, κ) that are commonly found in mammalian milk, comprising about 80% of the proteins in cow's milk and between 20% and 60% of the proteins in human ...

is broken, the hydrophobic groups unite and form a 3D network that traps the aqueous phase of the milk. Charge interactions between

histidine Histidine (symbol His or H) is an essential amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated –NH3+ form under biological conditions), a carboxylic acid group (which is in the d ...

s on the kappa-casein and glutamates and

aspartate Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. Like all other amino acids, it contains an amino group and a carboxylic acid. Its α-amino group is in the pro ...

s of chymosin initiate enzyme binding to the substrate. When chymosin is not binding substrate, a beta-hairpin, sometimes referred to as "the flap," can hydrogen bond with the active site, therefore covering it and not allowing further binding of substrate.

Examples

Listed below are the ruminant ''Cym'' gene and corresponding human pseudogene:

Recombinant chymosin

Because of the imperfections and scarcity of microbial and animal rennets, producers sought replacements. With the development of genetic engineering, it became possible to extract rennet-producing genes from animal stomach and insert them into certain bacteria, fungi or

yeasts Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to constitut ...

to make them produce chymosin during fermentation. The genetically modified microorganism is killed after fermentation and chymosin is isolated from the fermentation broth, so that the fermentation-produced chymosin (FPC) used by cheese producers does not contain any GM component or ingredient. FPC contains the identical chymosin as the animal source, but produced in a more efficient way. FPC products have been on the market since 1990 and are considered the ideal milk-clotting enzyme. FPC was the first artificially produced enzyme to be registered and allowed by the US Food and Drug Administration. In 1999, about 60% of US

hard cheese There are many different types of cheese. Cheeses can be grouped or classified according to criteria such as length of fermentation, texture, methods of production, fat content, animal milk, and country or region of origin. The method most com ...

was made with FPC and it has up to 80% of the global market share for rennet. By 2008, approximately 80% to 90% of commercially made cheeses in the US and Britain were made using FPC. The most widely used fermentation-produced chymosin is produced either using the fungus ''

Aspergillus niger ''Aspergillus niger'' is a mold classified within the ''Nigri'' section of the ''Aspergillus'' genus. The ''Aspergillus'' genus consists of common molds found throughout the environment within soil and water, on vegetation, in fecal matter, on de ...

'' or using ''

Kluyveromyces lactis ''Kluyveromyces lactis'' is a '' Kluyveromyces'' yeast commonly used for genetic studies and industrial applications. Its name comes from the ability to assimilate lactose and convert it into lactic acid. ''Kluyveromyces lactis'' (formerly ' ...

''. FPC contains only chymosin B,Bovine chymosins A and B differ by one amino acid residue. This is probably an alleic variant, according to Uniprot:P00794. The two isoforms have identical catalytic activity, so any improvement in the product is due to the elimination of other impurities. achieving a higher degree of purity compared with animal rennet. FPC can deliver several benefits to the cheese producer compared with animal or microbial rennet, such as higher production yield, better curd texture and reduced bitterness.

References

External links

* The

MEROPS MEROPS is an online database for peptidases (also known as proteases, proteinases and proteolytic enzymes) and their inhibitors. The classification scheme for peptidases was published by Rawlings & Barrett in 1993, and that for protein inhibitors ...