View source for Chymosin

{{cs1 config|name-list-style=vanc}}
{{Short description|Class of enzymes}}
{{distinguish|text=[[renin]], an enzyme which takes part in regulation of arterial blood pressure}}
{{infobox enzyme
| Name       = Chymosin
| EC_number  = 3.4.23.4
| CAS_number = 9001-98-3
| GO_code    = 0030602
| image      = CHYMOSIN COMPLEX WITH THE INHIBITOR CP-113972.jpg
| width      =
| caption    = Crystal structure of bovine chymosin complex with the inhibitor CP-113972.<ref name="pmid9862200">{{PDB|1CZI}}; {{cite journal | vauthors = Groves MR, Dhanaraj V, Badasso M, Nugent P, Pitts JE, Hoover DJ, Blundell TL | title = A 2.3 A resolution structure of chymosin complexed with a reduced bond inhibitor shows that the active site beta-hairpin flap is rearranged when compared with the native crystal structure | journal = Protein Engineering | volume = 11 | issue = 10 | pages = 833–40 | date = October 1998 | pmid = 9862200 | doi = 10.1093/protein/11.10.833 | url = https://academic.oup.com/peds/article-pdf/11/10/833/18542197/110833.pdf | doi-access = free }}</ref> 
}}
'''Chymosin''' {{IPAc-en|ˈ|k|aɪ|m|ə|s|ᵻ|n}} or '''rennin''' {{IPAc-en|ˈ|r|ɛ|n|ᵻ|n}} is a [[protease]] found in [[rennet]]. It is an [[Aspartic protease|aspartic endopeptidase]] belonging to MEROPS A1 family. It is produced by newborn [[ruminant]] animals in the lining of the [[abomasum]] 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]].

Historically, chymosin was obtained by extracting it from the stomachs of slaughtered calves. Today, most commercial chymosin used in cheese production is produced [[Recombinant DNA|recombinantly]] in {{nobr|''[[Escherichia coli]]''}}, [[Aspergillus awamori|''Aspergillus niger'' var. ''awamori'']], and {{nobr|''[[Kluyveromyces lactis]]''}}.{{citation needed|date=April 2024}}

==Occurrence==
Chymosin is found in a wide range of [[tetrapods]],<ref name=lineage/> although it is best known to be produced by [[ruminant]] animals in the lining of the [[abomasum]]. Chymosin is produced by [[gastric chief cell]]s in newborn mammals<ref name="pmid11534329">{{cite journal | vauthors = Kitamura N, Tanimoto A, Hondo E, Andrén A, Cottrell DF, Sasaki M, Yamada J | title = Immunohistochemical study of the ontogeny of prochymosin--and pepsinogen-producing cells in the abomasum of sheep | journal = Anatomia, Histologia, Embryologia | volume = 30 | issue = 4 | pages = 231–5 | date = August 2001 | pmid = 11534329 | doi = 10.1046/j.1439-0264.2001.00326.x | s2cid = 7552821 }}</ref> 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,<ref name= OMIM>Staff,  Online Mendelian Inheritance in Man (OMIM) Database.  Last updated February 21, 1997 [http://www.omim.org/entry/118943 Chymosin pseudogene; CYMP prochymosin, included, in the OMIM]</ref> and [[chicken|chick]]s.<ref name=lineage/>

One study reported finding a chymosin-like enzyme in some human infants,<ref>{{cite journal | vauthors = Henschel MJ, Newport MJ, Parmar V | title = Gastric proteases in the human infant | journal = Biology of the Neonate | volume = 52 | issue = 5 | pages = 268–72 | year = 1987 | pmid = 3118972 | doi = 10.1159/000242719 }}</ref> but others have failed to replicate this finding.<ref>{{cite book | vauthors = Szecsi PB, Harboe M | veditors = Rawlings ND, Salvesen G |year=2013|title=Handbook of Proteolytic Enzymes|chapter=Chapter 5: Chymosin|chapter-url=https://www.researchgate.net/publication/278718218|language=en|volume=1|pages=37–42|doi=10.1016/B978-0-12-382219-2.00005-3}}</ref> Humans have a [[pseudogene]] for chymosin that does not generate a protein, found on [[Chromosome 1 (human)|chromosome 1]].<ref name= OMIM/><ref>{{cite book | vauthors = Fox PF | title = Cheese: Chemistry, Physics and Microbiology | date = 28 February 1999 | publisher = Springer | isbn = 9780834213388 | url = https://books.google.com/books?id=U_mj5DANAeoC&q=chymosin+gene+human&pg=PA62 }}</ref>  Humans have other proteins to digest milk, such as [[pepsin]] and [[lipase]].<ref>{{cite book | vauthors = Sanderson IR, Walker WA |title=Development of the gastrointestinal tract |date=1999 |publisher=B.C. Decker |location=Hamilton, Ontario |isbn=978-1-55009-081-9 | url = https://books.google.com/books?id=YhgKZ_dvda0C }}</ref>{{rp|262}}

In addition to the primate lineage leading up to humans, some other mammals have also lost the chymosin gene.<ref name=lineage>{{cite journal | vauthors = Lopes-Marques M, Ruivo R, Fonseca E, Teixeira A, Castro LF | title = Unusual loss of chymosin in mammalian lineages parallels neo-natal immune transfer strategies | journal = Molecular Phylogenetics and Evolution | volume = 116 | pages = 78–86 | date = November 2017 | pmid = 28851538 | doi = 10.1016/j.ympev.2017.08.014 | bibcode = 2017MolPE.116...78L }}</ref>

==Enzymatic reaction==
Chymosin is used to bring about the extensive [[Precipitation (chemistry)|precipitation]] and [[curd]] formation in [[cheese]]-making. The native substrate of chymosin is [[K-casein]] which is specifically [[Bond cleavage|cleaved]] at the [[peptide bond]] between amino acid residues 105 and 106, [[phenylalanine]] and [[methionine]].<ref name="Gilliland">{{cite book | vauthors = Gilliland GL, Oliva MT, Dill J | title = Structure and Function of the Aspartic Proteinases | chapter = Functional Implications of the Three-Dimensional Structure of Bovine Chymosin | series = Advances in Experimental Medicine and Biology | volume = 306 | pages = 23–37 | year = 1991 | pmid = 1812710 | doi = 10.1007/978-1-4684-6012-4_3 | isbn = 978-1-4684-6014-8 }}</ref> The resultant product is [[calcium phosphocaseinate]].{{Citation needed|date=August 2010}} When the specific linkage between the [[hydrophobic]] (para-casein) and [[hydrophilic]] (acidic [[glycopeptide]]) groups of [[casein]] is broken, the hydrophobic groups unite and form a [[Three-dimensional space|3D]] network that traps the aqueous phase of the milk.

Charge interactions between [[histidine]]s on the kappa-casein and [[glutamate]]s and [[aspartate]]s of chymosin initiate enzyme binding to the substrate.<ref name="Gilliland"/>  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.<ref name="pmid9862200"/>

==Examples==
Listed below are the ruminant ''Cym'' gene and corresponding human pseudogene:
{|
|{{infobox nonhuman protein
|Name=Chymosin [Precursor]
|caption=X-ray analysis of calf chymosin <ref name="Newman">{{PDB|4CMS}}; {{cite journal | vauthors = Newman M, Safro M, Frazao C, Khan G, Zdanov A, Tickle IJ, Blundell TL, Andreeva N | display-authors = 6 | title = X-ray analyses of aspartic proteinases. IV. Structure and refinement at 2.2 A resolution of bovine chymosin | journal = Journal of Molecular Biology | volume = 221 | issue = 4 | pages = 1295–309 | date = October 1991 | pmid = 1942052 | doi = 10.1016/0022-2836(91)90934-X }}</ref>
|image=X-RAY ANALYSES OF ASPARTIC PROTEINASES IV. STRUCTURE AND REFINEMENT AT 2.2 ANGSTROMS RESOLUTION OF BOVINE CHYMOSIN.jpg
|width=
|Organism=Bos taurus
|Symbol=Cym
|AltSymbols=CPC
|EntrezGene=529879
|UniProt=P00794
|PDB=4CMS
|ECnumber=
|Chromosome=
|Arm=
|Band=
|LocusSupplementaryData=
}}
|{{infobox protein
|Name=chymosin pseudogene (human)
|caption=
|image=
|width=
|HGNCid=2588
|Symbol=CYMP
|AltSymbols=
|EntrezGene=643160
|OMIM=118943
|RefSeq= NR_003599
|UniProt=
|PDB=
|ECnumber=
|Chromosome=1
|Arm= p
|Band= 13.3
|LocusSupplementaryData=
}}
|}

==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]] to make them produce chymosin during fermentation.<ref name="pmid6304731">{{cite journal | vauthors = Emtage JS, Angal S, Doel MT, Harris TJ, Jenkins B, Lilley G, Lowe PA | title = Synthesis of calf prochymosin (prorennin) in Escherichia coli | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 80 | issue = 12 | pages = 3671–5 | date = June 1983 | pmid = 6304731 | pmc = 394112 | doi = 10.1073/pnas.80.12.3671 | bibcode = 1983PNAS...80.3671E | doi-access = free }}</ref><ref name="pmid6283469">{{cite journal | vauthors = Harris TJ, Lowe PA, Lyons A, Thomas PG, Eaton MA, Millican TA, Patel TP, Bose CC, Carey NH, Doel MT | display-authors = 6 | title = Molecular cloning and nucleotide sequence of cDNA coding for calf preprochymosin | journal = Nucleic Acids Research | volume = 10 | issue = 7 | pages = 2177–87 | date = April 1982 | pmid = 6283469 | pmc = 320601 | doi = 10.1093/nar/10.7.2177 }}</ref>  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.<ref name="GMO Database"/> 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.<ref name="Law 2010 100–101">{{cite book|author = Law BA | title = Technology of Cheesemaking|year=2010|publisher= Wiley-Blackwell | location = UK | isbn = 978-1-4051-8298-0 | pages = 100–101 |url = http://eu.wiley.com/WileyCDA/WileyTitle/productCd-1405182989.html}}</ref>

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]] was made with FPC<ref name="USDA">{{cite web |url=https://fpc.state.gov/6176.htm|title=Food Biotechnology in the United States: Science, Regulation, and Issues|publisher=U.S. Department of State|access-date=2006-08-14}}</ref> and it has up to 80% of the global market share for rennet.<ref name="pmid16537950">{{cite journal | vauthors = Johnson ME, Lucey JA | title = Major technological advances and trends in cheese | journal = Journal of Dairy Science | volume = 89 | issue = 4 | pages = 1174–8 | date = April 2006 | pmid = 16537950 | doi = 10.3168/jds.S0022-0302(06)72186-5 | doi-access = free }}</ref>

By 2008, approximately 80% to 90% of commercially made cheeses in the US and Britain were made using FPC.<ref name="GMO Database">{{cite web|url=http://www.gmo-compass.org/eng/database/enzymes/83.chymosin.html|publisher=GMO Compass|title=Chymosin|access-date=2011-03-03|url-status=dead|archive-url=https://web.archive.org/web/20150326181805/http://www.gmo-compass.org/eng/database/enzymes/83.chymosin.html|archive-date=2015-03-26}}</ref> The most widely used fermentation-produced chymosin is produced either using the fungus ''[[Aspergillus niger]]'' or using ''[[Kluyveromyces lactis]]''.

FPC contains only chymosin B,<ref>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.</ref> 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.<ref name="Law 2010 100–101"/>

== References ==
{{reflist}}

== Further reading ==
{{refbegin}}
* {{cite journal | vauthors = Foltmann B | title = A review on prorennin and rennin | journal = Comptes-Rendus des Travaux du Laboratoire Carlsberg | volume = 35 | issue = 8 | pages = 143–231 | year = 1966 | pmid = 5330666 }}
* {{cite journal | vauthors = Visser S, Slangen CJ, van Rooijen PJ | title = Peptide substrates for chymosin (rennin). Interaction sites in kappa-casein-related sequences located outside the (103-108)-hexapeptide region that fits into the enzyme's active-site cleft | journal = The Biochemical Journal | volume = 244 | issue = 3 | pages = 553–8 | date = June 1987 | pmid = 3128264 | pmc = 1148031 | doi = 10.1042/bj2440553 }}
{{refend}}

== External links ==
* The [[MEROPS]] online database for peptidases and their inhibitors: [https://www.ebi.ac.uk/merops/cgi-bin/pepsum?id=A01.006 A01.006]

{{Aspartic acid proteases}}
{{Enzymes}}
{{Portal bar|Biology|border=no}}

[[Category:EC 3.4.23]]