Lactase

The animal (including human) versions

As mentioned above the human lactase is a protein with two β-galactosidase domains. The lactase domain functions as a lactase while the phlorizin hydrolase domain catalyzes the conversion of phlorizin to phloretin and glucose.

Structure and biosynthesis

Preprolactase, the primary translation product, has a single polypeptide primary structure consisting of 1927 amino acids. 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. 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.

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.

Genetic expression and regulation

In humans, lactase is encoded by a single genetic locus on chromosome 2. 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. 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.

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. 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. Both mutations, C→T at position -13910 and G→ A at position -22018, have been independently linked to lactase persistence.

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. Developmentally regulated DNA-binding proteins may down-regulate transcription or destabilize mRNA transcripts, causing decreased LPH expression after weaning.

Mechanism

The catalytic mechanism of D-lactose hydrolysis retains the substrate anomeric configuration in the products. 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. 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 recognition and hydrolysis by the mammalian lactase. 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 (Ki = 10mM). Elimination of specific hydroxyl groups on the glucopyranose moiety does not eliminate catalysis.

The temperature optimum for human lactase is about 37 °C and the pH optimum is 6.

Non-animal lactases and their uses

UsesLactase produced commercially is extracted both from yeasts such as Kluyveromyces fragilis and Kluyveromyces lactis and from molds, such as Aspergillus niger and Aspergillus oryzae.

As mentioned earlier, lactase is an enzyme that some people are unable to produce in their small intestine. Without lactase, lactose-intolerant people pass the lactose undigested to the colon where bacteria break it down, creating carbon dioxide which leads to bloating and flatulence. The commercial forms of lactase can break down lactose when they are either added to food or put in the human digestive tract.

Added to food

Lactase is added to dairy, thereby hydrolyzing the lactose in it, leaving it slightly sweet but digestible by everyone.

Technology to produce lactose-free milk, ice cream, and yogurt was developed by the USDA Agricultural Research Service in 1985. Lactase from select species of mold are considered GRAS by the US FDA; as a result, they are allowed to be added to food in limited quantities as a processing aid. This is one of the primary commercial uses of lactase.

Ingested

Lactase supplements can be used to treat lactose intolerance. The U.S. Food and Drug Administration has not independently evaluated these products, but there is consensus that they do work so long as the stated potency matches what is claimed by the label.

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.

Biotechnology

Lactase (technically, 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. Besides the fungal types of β-galactosidase mentioned above, biotechnology also makes use of the E. coli lacZ β-galactosidase found in the lac operon.

Notes

References

References

1. (Sep 1988). "Complete primary structure of human and rabbit lactase-phlorizin hydrolase: implications for biosynthesis, membrane anchoring and evolution of the enzyme". The EMBO Journal.
2. (Jul 1993). "Regional localization of the lactase-phlorizin hydrolase gene, LCT, to chromosome 2q21". Annals of Human Genetics.
3. (2009). "Molecular genetics of human lactase deficiencies". Annals of Medicine.
4. (Jan 1987). "Biosynthesis and maturation of lactase-phlorizin hydrolase in the human small intestinal epithelial cells". The Biochemical Journal.
5. (Oct 1994). "The pro region of human intestinal lactase-phlorizin hydrolase". The Journal of Biological Chemistry.
6. (Sep 1998). "Intestinal lactase-phlorizin hydrolase (LPH): the two catalytic sites; the role of the pancreas in pro-LPH maturation". FEBS Letters.
7. (Mar 1997). "Regulation of lactase-phlorizin hydrolase gene expression by the caudal-related homoeodomain protein Cdx-2". The Biochemical Journal.
8. Reference, Genetics Home. "LCT gene".
9. "Lactose intolerance: MedlinePlus Genetics".
10. (Jun 2004). "Genetic signatures of strong recent positive selection at the lactase gene". American Journal of Human Genetics.
11. (May 2003). "Transcriptional regulation of the lactase-phlorizin hydrolase gene by polymorphisms associated with adult-type hypolactasia". Gut.
12. (May 2005). "Adult-type hypolactasia and regulation of lactase expression". Biochimica et Biophysica Acta (BBA) - General Subjects.
13. (Jun 1998). "The genetically programmed down-regulation of lactase in children". Gastroenterology.
14. (November 1990). "Catalytic mechanisms of enzymic glycosyl transfer". Chem. Rev..
15. (Dec 2001). "A structural view of the action of ''Escherichia coli'' (lacZ) β-galactosidase". Biochemistry.
16. (Jul 1995). "Substrate specificity of small-intestinal lactase: study of the steric effects and hydrogen bonds involved in enzyme-substrate interaction". Carbohydrate Research.
17. (Jul 2007). "Optimizing the enzymatic synthesis of β-D-galactopyranosyl-D-xyloses for their use in the evaluation of lactase activity ''in vivo''". Bioorganic & Medicinal Chemistry.
18. (Mar 1981). "Purification and characterisation of amphiphilic lactase/phlorizin hydrolase from human small intestine". European Journal of Biochemistry.
19. (2004). "Production of Lactase by ''Trichoderma'' sp.". Food Technology and Biotechnology.
20. "Lactose Intolerance".
21. "Lactose intolerance - Symptoms and causes".
22. (3 September 2014). "Asked: How do dairies make lactose-free milk?". USA Today.
23. Porch, Kaitlyn. (2018-04-12). "Lactose-Free Milk, Low-Fat Cheese, and More Dairy Breakthroughs".
24. DSM Food Specialties. (3 April 2014). "GRAS Notification for Acid Lactase from ''Aspergillus oryzae'' Expressed in ''Aspergillus niger''".
25. Tarantino, LM. (12 December 2003). "Agency Response Letter GRAS Notice No. GRN 000132". U.S. Food and Drug Administration.
26. Holsinger, Virginia H.. (1992). "New Crops, New Uses, New Markets: 1992 Yearbook of Agriculture". U.S. Department of Agriculture.
27. (June 2014). "Lactose Intolerance".
28. (March 2013). "Introduction". Universiti Teknologi Malaysia.
29. "pBluescript II KS(+/−), pBluescript II SK(+/−): description & restriction map". Fermentas.