Lactase

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 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. 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. 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. 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 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). and the pH optimum is 6. == Non-animal lactases and their uses ==

Lactase 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. == See also ==