Lactase#Industrial use

Lactase is an enzyme that some people are unable to produce in their small intestine.{{cite web|title=Lactose Intolerance|url=https://www.mayoclinic.org/diseases-conditions/lactose-intolerance/symptoms-causes/syc-20374232|website=Mayo Clinic|access-date=13 March 2018}} Technology to produce lactose-free milk, ice cream, and yogurt was developed by the USDA Agricultural Research Service in 1985.{{cite web|url=https://www.federallabs.org/successes/success-stories/lactose-free-milk-low-fat-cheese-and-more-dairy-breakthroughs|title=Lactose-Free Milk, Low-Fat Cheese, and More Dairy Breakthroughs|last=Porch|first=Kaitlyn|date=2018-04-12|website=www.federallabs.org|language=en|access-date=2018-10-26}}Lactase is added to milk, thereby hydrolyzing the lactose in the milk, leaving it slightly sweet but digestible by everyone.{{cite news|title=Asked: How do dairies make lactose-free milk?|url=https://www.azcentral.com/story/news/local/asked-answered/2014/09/03/asked-answered-lactose-free-milk/14821885/|access-date=13 March 2018|newspaper=USA Today|date=3 September 2014}} Without lactase, lactose-intolerant people pass the lactose undigested to the colon{{cite web|title=Lactose intolerance - Symptoms and causes|url=https://www.mayoclinic.org/diseases-conditions/lactose-intolerance/symptoms-causes/syc-20374232|access-date=2020-11-08|website=Mayo Clinic|language=en}} where bacteria break it down, creating carbon dioxide which leads to bloating and flatulence.

Lactase supplements can be used to treat lactose intolerance.{{cite web|title=Lactose Intolerance|url=https://www.niddk.nih.gov/health-information/health-topics/digestive-diseases/lactose-intolerance/Pages/facts.aspx|website=NIDDK|access-date=25 October 2016|date=June 2014}}

Lactase produced commercially can be extracted both from yeasts such as Kluyveromyces fragilis and Kluyveromyces lactis and from molds, such as Aspergillus niger and Aspergillus oryzae.{{cite journal|vauthors=Seyis I, Aksoz N |year=2004|title=Production of Lactase by Trichoderma sp. |journal=Food Technology and Biotechnology|volume=42|issue=2|pages=121–124|url=https://hrcak.srce.hr/file/163289}} Its primary commercial use in supplements is to break down lactose in milk to make it suitable for people with lactose intolerance.{{cite web |url=https://www.fda.gov/downloads/Food/IngredientsPackagingLabeling/GRAS/NoticeInventory/UCM400718 |archive-url=https://wayback.archive-it.org/7993/20171031042344/https://www.fda.gov/downloads/Food/IngredientsPackagingLabeling/GRAS/NoticeInventory/UCM400718.pdf#page=8 |url-status=dead |title=GRAS Notification for Acid Lactase from Aspergillus oryzae Expressed in Aspergillus niger |author=DSM Food Specialties |via=U.S. Food and Drug Administration |date=3 April 2014 |archive-date=31 October 2017 |page=1}}{{cite book |title=New Crops, New Uses, New Markets: 1992 Yearbook of Agriculture |last=Holsinger |first=Virginia H. |publisher=U.S. Department of Agriculture |year=1992 |pages=256–258 |chapter=Innovative Products for Food Industries: The Lactaid Story |chapter-url=https://naldc.nal.usda.gov/download/IND93048088/PDF |access-date=2022-01-11 |archive-date=2022-07-17 |archive-url=https://web.archive.org/web/20220717021856/https://naldc.nal.usda.gov/download/IND93048088/PDF |url-status=dead }} The U.S. Food and Drug Administration has not independently evaluated these products.{{cite web|url=https://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/ucm153949.htm|title=Agency Response Letter GRAS Notice No. GRN 000132|author=Tarantino, LM|date=12 December 2003|publisher=U.S. Food and Drug Administration|archive-url=https://web.archive.org/web/20110326081205/https://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/ucm153949.htm|archive-date=26 March 2011|url-status=dead}}

Lactase (or a similar form of β-galactosidase) is also used to screen for blue white colonies in the multiple cloning sites of various plasmid vectors in Escherichia coli or other bacteria.{{cite web |url=https://lactaseinbioinfo.weebly.com/introduction.html |title=Introduction |vauthors=Lau HM, Lee LS, Soh WC, Tue SW |publisher=Universiti Teknologi Malaysia |website=Lactase |date=March 2013 |access-date=16 November 2018}}{{cite web |url=http://www.fermentas.com/techinfo/nucleicacids/mappbluescriptiiskks.htm |archive-url=https://web.archive.org/web/20081019115101/http://www.fermentas.com/techinfo/nucleicacids/mappbluescriptiiskks.htm |url-status=dead |title=pBluescript II KS(+/−), pBluescript II SK(+/−): description & restriction map |publisher=Fermentas |archive-date=19 October 2008}}

The temperature optimum for human lactase is about 37 °C{{cite journal | vauthors = Hermida C, Corrales G, Cañada FJ, Aragón JJ, Fernández-Mayoralas A | title = Optimizing the enzymatic synthesis of β-D-galactopyranosyl-D-xyloses for their use in the evaluation of lactase activity in vivo | journal = Bioorganic & Medicinal Chemistry | volume = 15 | issue = 14 | pages = 4836–40 | date = Jul 2007 | pmid = 17512743 | doi = 10.1016/j.bmc.2007.04.067 | hdl = 10261/81580 }} and the pH optimum is 6.{{cite journal | vauthors = Skovbjerg H, Sjöström H, Norén O | title = Purification and characterisation of amphiphilic lactase/phlorizin hydrolase from human small intestine | journal = European Journal of Biochemistry | volume = 114 | issue = 3 | pages = 653–61 | date = Mar 1981 | pmid = 6786877 | doi = 10.1111/j.1432-1033.1981.tb05193.x | doi-access = }}

In metabolism, the β-glycosidic bond in D-lactose is hydrolyzed to form D-galactose and D-glucose, which can be absorbed through the intestinal walls and into the bloodstream. The overall reaction that lactase catalyzes is as follows:

: C₁₂H₂₂O₁₁ + H₂O → C₆H₁₂O₆ + C₆H₁₂O₆ + heat.

:lactose + H₂O → β-D-galactose + D-glucose

The catalytic mechanism of D-lactose hydrolysis retains the substrate anomeric configuration in the products.{{cite journal | vauthors = Sinnott M | title = Catalytic mechanisms of enzymic glycosyl transfer | journal = Chem. Rev. | volume = 90 | issue = 7 | pages = 1171–1202 |date=November 1990 | doi = 10.1021/cr00105a006}} While the details of the mechanism are uncertain, the stereochemical retention is achieved via a double displacement reaction. Studies of E. coli lactase have proposed that hydrolysis is initiated when a glutamate nucleophile on the enzyme attacks from the axial side of the galactosyl carbon in the β-glycosidic bond.{{cite journal | vauthors = Juers DH, Heightman TD, Vasella A, McCarter JD, Mackenzie L, Withers SG, Matthews BW | title = A structural view of the action of Escherichia coli (lacZ) β-galactosidase | journal = Biochemistry | volume = 40 | issue = 49 | pages = 14781–94 | date = Dec 2001 | pmid = 11732897 | doi = 10.1021/bi011727i }} The removal of the D-glucose leaving group may be facilitated by Mg-dependent acid catalysis. The enzyme is liberated from the α-galactosyl moiety upon equatorial nucleophilic attack by water, which produces D-galactose.

Substrate modification studies have demonstrated that the 3′-OH and 2′-OH moieties on the galactopyranose ring are essential for enzymatic recognition and hydrolysis.{{cite journal | vauthors = Fernandez P, Cañada FJ, Jiménez-Barbero J, Martín-Lomas M | title = Substrate specificity of small-intestinal lactase: study of the steric effects and hydrogen bonds involved in enzyme-substrate interaction | journal = Carbohydrate Research | volume = 271 | issue = 1 | pages = 31–42 | date = Jul 1995 | pmid = 7648581 | doi = 10.1016/0008-6215(95)00034-Q }} The 3′-hydroxy group is involved in initial binding to the substrate while the 2′- group is not necessary for recognition but needed in subsequent steps. This is demonstrated by the fact that a 2-deoxy analog is an effective competitive inhibitor (K_i = 10mM). Elimination of specific hydroxyl groups on the glucopyranose moiety does not eliminate catalysis.

File:LactaseMechanism2.png

Lactase also catalyzes the conversion of phlorizin to phloretin and glucose.

Commercial lactase is used as a medication for lactose intolerance. Since it is an enzyme, its function can be inhibited by the acidity of the stomach. However, it is packaged in an acid-proof tablet, allowing the enzyme to pass through the stomach intact and remain in the small intestine. In the small intestine it can act on ingested lactose molecules, allowing the body to absorb the digested sugar which would otherwise cause cramping and diarrhea. Since the enzyme is not absorbed, it is excreted.{{cn|date=May 2024}}

Preprolactase, the primary translation product, has a single polypeptide primary structure consisting of 1927 amino acids.{{cite journal | vauthors = Mantei N, Villa M, Enzler T, Wacker H, Boll W, James P, Hunziker W, Semenza G | title = Complete primary structure of human and rabbit lactase-phlorizin hydrolase: implications for biosynthesis, membrane anchoring and evolution of the enzyme | journal = The EMBO Journal | volume = 7 | issue = 9 | pages = 2705–13 | date = Sep 1988 | pmid = 2460343 | pmc = 457059 | doi = 10.1002/j.1460-2075.1988.tb03124.x}} It can be divided into five domains: (i) a 19-amino-acid cleaved signal sequence; (ii) a large prosequence domain that is not present in mature lactase; (iii) the mature lactase segment; (iv) a membrane-spanning hydrophobic anchor; and (v) a short hydrophilic carboxyl terminus. The signal sequence is cleaved in the endoplasmic reticulum, and the resulting 215-kDa pro-LPH is sent to the Golgi apparatus, where it is heavily glycosylated and proteolytically processed to its mature form.{{cite journal | vauthors = Naim HY, Sterchi EE, Lentze MJ | title = Biosynthesis and maturation of lactase-phlorizin hydrolase in the human small intestinal epithelial cells | journal = The Biochemical Journal | volume = 241 | issue = 2 | pages = 427–34 | date = Jan 1987 | pmid = 3109375 | pmc = 1147578 | doi=10.1042/bj2410427}} The prodomain has been shown to act as an intramolecular chaperone in the ER, preventing trypsin cleavage and allowing LPH to adopt the necessary 3-D structure to be transported to the Golgi apparatus.{{cite journal | vauthors = Naim HY, Jacob R, Naim H, Sambrook JF, Gething MJ | title = The pro region of human intestinal lactase-phlorizin hydrolase | journal = The Journal of Biological Chemistry | volume = 269 | issue = 43 | pages = 26933–43 | date = Oct 1994 | doi = 10.1016/S0021-9258(18)47109-8 | pmid = 7523415 | doi-access = free }}

Image:Lactase Processing.png

Mature human lactase consists of a single 160-kDa polypeptide chain that localizes to the brush border membrane of intestinal epithelial cells. It is oriented with the N-terminus outside the cell and the C-terminus in the cytosol. LPH contains two catalytic glutamic acid sites. In the human enzyme, the lactase activity has been connected to Glu-1749, while Glu-1273 is the site of phlorizin hydrolase function.{{cite journal | vauthors = Zecca L, Mesonero JE, Stutz A, Poirée JC, Giudicelli J, Cursio R, Gloor SM, Semenza G | title = Intestinal lactase-phlorizin hydrolase (LPH): the two catalytic sites; the role of the pancreas in pro-LPH maturation | journal = FEBS Letters | volume = 435 | issue = 2–3 | pages = 225–8 | date = Sep 1998 | pmid = 9762914 | doi = 10.1016/S0014-5793(98)01076-X | s2cid = 33421778 | doi-access = free | bibcode = 1998FEBSL.435..225Z }}

In humans, lactase is encoded by a single genetic locus on chromosome 2.{{cite journal | vauthors = Troelsen JT, Mitchelmore C, Spodsberg N, Jensen AM, Norén O, Sjöström H | title = Regulation of lactase-phlorizin hydrolase gene expression by the caudal-related homoeodomain protein Cdx-2 | journal = The Biochemical Journal | volume = 322 | issue = Pt. 3 | pages = 833–8 | date = Mar 1997 | pmid = 9148757 | pmc = 1218263 | doi = 10.1042/bj3220833 }} It is expressed exclusively by mammalian small intestine enterocytes and in very low levels in the colon during fetal development. Humans are born with high levels of lactase expression. In most of the world's population, lactase transcription is down-regulated after weaning, resulting in diminished lactase expression in the small intestine, which causes the common symptoms of adult-type hypolactasia, or lactose intolerance.{{cite web|url=https://ghr.nlm.nih.gov/gene/LCT|title=LCT gene|first=Genetics Home|last=Reference|website=Genetics Home Reference|access-date=3 April 2018}} The LCT gene provides the instructions for making lactase. Lactose intolerance in infants (congenital lactase deficiency) is caused by mutations in the LCT gene. Mutations are believed to interfere with the function of lactase, causing affected infants to have a severely impaired ability to digest lactose in breast milk or formula.{{cite web |title=Lactose intolerance: MedlinePlus Genetics |url=https://medlineplus.gov/genetics/condition/lactose-intolerance/ |access-date=2022-03-22 |website=medlineplus.gov |language=en}}

Some population segments exhibit lactase persistence resulting from a mutation that is postulated to have occurred 5,000–10,000 years ago, coinciding with the rise of cattle domestication.{{cite journal | vauthors = Bersaglieri T, Sabeti PC, Patterson N, Vanderploeg T, Schaffner SF, Drake JA, Rhodes M, Reich DE, Hirschhorn JN | title = Genetic signatures of strong recent positive selection at the lactase gene | journal = American Journal of Human Genetics | volume = 74 | issue = 6 | pages = 1111–20 | date = Jun 2004 | pmid = 15114531 | pmc = 1182075 | doi = 10.1086/421051 }} This mutation has allowed almost half of the world's population to metabolize lactose without symptoms. Studies have linked the occurrence of lactase persistence to two different single-nucleotide polymorphisms about 14 and 22 kilobases upstream of the 5'-end of the LPH gene.{{cite journal | vauthors = Kuokkanen M, Enattah NS, Oksanen A, Savilahti E, Orpana A, Järvelä I | title = Transcriptional regulation of the lactase-phlorizin hydrolase gene by polymorphisms associated with adult-type hypolactasia | journal = Gut | volume = 52 | issue = 5 | pages = 647–52 | date = May 2003 | pmid = 12692047 | pmc = 1773659 | doi = 10.1136/gut.52.5.647 }} Both mutations, C→T at position -13910 and G→ A at position -22018, have been independently linked to lactase persistence.{{cite journal | vauthors = Troelsen JT | title = Adult-type hypolactasia and regulation of lactase expression | journal = Biochimica et Biophysica Acta (BBA) - General Subjects | volume = 1723 | issue = 1–3 | pages = 19–32 | date = May 2005 | pmid = 15777735 | doi = 10.1016/j.bbagen.2005.02.003 }}

The lactase promoter is 150 base pairs long and is located upstream of the site of transcription initiation. The sequence is highly conserved in mammals, suggesting that critical cis-transcriptional regulators are located nearby. Cdx-2, HNF-1α, and GATA have been identified as transcription factors. Studies of hypolactasia onset have demonstrated that despite polymorphisms, little difference exists in lactase expression in infants, showing that the mutations become increasingly relevant during development.{{cite journal | vauthors = Wang Y, Harvey CB, Hollox EJ, Phillips AD, Poulter M, Clay P, Walker-Smith JA, Swallow DM | title = The genetically programmed down-regulation of lactase in children | journal = Gastroenterology | volume = 114 | issue = 6 | pages = 1230–6 | date = Jun 1998 | pmid = 9609760 | doi = 10.1016/S0016-5085(98)70429-9 | doi-access = free }} Developmentally regulated DNA-binding proteins may down-regulate transcription or destabilize mRNA transcripts, causing decreased LPH expression after weaning.

{{Portal|Biology}}

• MCM6
• Lactase persistence

{{Reflist|35em}}

{{Commons category|Lactase}}

• {{usurped|1=[https://archive.today/20130128074840/http://macromoleculeinsights.com/lactase.php The Lactase Protein]}}
• E. coli β-galactosidase: {{PDB|1JYY}}
• [http://www.ebi.ac.uk/ego/DisplayGoTerm?id=GO:0000016 Gene Ontology for Lactase]
• [http://www.hhmi.org/biointeractive/making-fittest-got-lactase-co-evolution-genes-and-culture Making of the Fittest: Got Lactase? The Co-evolution of Genes and Culture]
• [https://www.ncbi.nlm.nih.gov/pubmed/23252911 Lactase persistence shows indication of association with Obesity]
• [https://www.ebi.ac.uk/pdbe/pdbe-kb/proteins/P16278 PDBe-KB] provides an overview of all the structure information available in the PDB for Human Beta-galactosidase
• [https://www.ebi.ac.uk/pdbe/pdbe-kb/proteins/P16278 PDBe-KB] provides an overview of all the structure information available in the PDB for Escherichia coli Beta-galactosidase

{{Fructose and galactose metabolism}}

{{Sugar hydrolases}}

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Category:Food additives

Category:Antiflatulents

Category:EC 3.2.1

Category:Genes on human chromosome 2

Category:Enzymes of known structure

Category:Enzymes used as drugs

Category:Lactose