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Beta-galactosidase (1tg7).png
β-galactosidase from Penicillum sp.
EC number
CAS number 9031-11-2
IntEnz IntEnz view
ExPASy NiceZyme view
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Gene Ontology AmiGO / EGO
galactosidase, beta 1
Symbol GLB1
Alt. symbols ELNR1
Entrez 2720
HUGO 4298
OMIM 230500
RefSeq NM_000404
UniProt P16278
Other data
Locus Chr. 3 p22.3

β-galactosidase, also called beta-gal or β-gal, is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins.[1]

Properties and functions[edit]

β-galactosidase is an exoglycosidase which hydrolyzes the β-glycosidic bond formed between a galactose and its organic moiety. It may also cleave fucosides and arabinosides but with much lower efficiency. It is an essential enzyme in the human body. Deficiencies in the protein can result in galactosialidosis or Morquio B syndrome. In E. coli, the gene of β-galactosidase, the lacZ gene, is present as part of the inducible system lac operon which is activated in the presence of lactose when glucose level is low.

It is commonly used in molecular biology as a reporter marker to monitor gene expression. It also exhibits a phenomenon called α-complementation which forms the basis for the blue/white screening of recombinant clones. This enzyme can be split in two peptides, LacZα and LacZΩ, neither of which is active by itself but when both are present together, spontaneously reassemble into a functional enzyme. This property is exploited in many cloning vectors where the presence of the lacZα gene in a plasmid can complement in trans another mutant gene encoding the LacZΩ in specific laboratory strains of E. coli. However, when DNA fragments are inserted in the vector, the production of LacZα is disrupted, the cells therefore show no β-galactosidase activity. The presence or absence of an active β-galactosidase may be detected by X-gal, which produces a characteristic blue dye when cleaved by β-galactosidase, thereby providing an easy means of distinguishing the presence or absence of cloned product in a plasmid.

In 1995, Dimri et al. proposed a new isoform for beta-galactosidase with optimum activity at pH 6.0 (Senescence Associated beta-gal or SA-beta-gal)[2] which would be specifically expressed in senescence (The irreversible growth arrest of cells). Specific quantitative assays were even developed for its detection.[3][4][5] However, it is now known that this is due to an overexpression and accumulation of the lysosomal endogenous beta-galactosidase,[6] and its expression is not required for senescence. Nevertheless, it remains the most widely used biomarker for senescent and aging cells, because it is reliable and easy to detect.


Ribbon diagram of the 3MUY structure.
Ribbon diagram of the 3MUY structure (tetramer), E. coli (lacZ) beta-galactosidase (R599A).[7]

The 1,024 amino acids of E. coli β-galactosidase were first sequenced in 1970,[8] and its structure determined twenty-four years later in 1994. The protein is a 464-kDa homotetramer with 2,2,2-point symmetry.[9] Each unit of β-galactosidase consists of five domains; domain 1 is a jelly-roll type barrel, domain 2 and 4 are fibronectin type III-like barrels, domain 5 a β-sandwich, while the central domain 3 is a TIM-type barrel.

The third domain contains the active site.[10] The active site is made up of elements from two subunits of the tetramer, and disassociation of the tetramer into dimers removes critical elements of the active site. The amino-terminal sequence of β-galactosidase, the α-peptide involved in α-complementation, participates in a subunit interface. Its residues 22-31 help to stabilize a four-helix bundle which forms the major part of that interface, and residue 13 and 15 also contributing to the activating interface. These structural features provide a rationale for the phenomenon of α-complementation, where the deletion of the amino-terminal segment results in the formation of an inactive dimer.


The active site of β-galactosidase catalyzes the hydrolysis of its disaccharide substrate via "shallow" and "deep" binding. Monovalent potassium ions (K+) as well as divalent magnesium ions (Mg2+) are required for the enzyme's optimal activity. The beta-linkage of the substrate is cleaved by a terminal carboxyl group on the side chain of a glutamic acid.

In E. coli, Glu-461 was thought to be the nucleophile in the substitution reaction.[11] However, it is now known that Glu-461 is an acid catalyst. Instead, Glu-537 is the actual nucleophile,[12] binding to a galactosyl intermediate.

In humans, the nucleophile of the hydrolysis reaction is Glu-268.[13]

β-galactosidase reaction


The β-galactosidase assay is used frequently in genetics, molecular biology, and other life sciences. An active enzyme may be detected using X-gal, which forms an intense blue product after cleavage by β-galactosidase, and is easy to identify and quantify. It is used for example in blue white screen.[14] Its production may be induced by a non-hydrolyzable analog of allolactose, IPTG, which binds and releases the lac repressor from the lac operator, thereby allowing the initiation of transcription to proceed.

Since it is highly expressed and accumulated in lysosomes in senescent cells, it is used as a senescence biomarker both in vivo and in vitro in qualitative and quantitative [3] assays, despite its limitations.

In studies of leukaemia chromosomal translocations, Dobson and colleagues used a fusion protein of LacZ in mice,[15] exploiting β-galactosidase's tendency to oligomerise to suggest a potential role for oligomericity in MLL fusion protein function.[16]

Evolved beta-galactosidase[edit]

Some species of bacteria, including E. coli, have additional β-galactosidase genes. A second gene, called evolved β-galactosidase (ebgA) gene was discovered when strains with the lacZ gene deleted (but still containing the gene for galactoside permease, lacY), were plated on medium containing lactose (or other 3-galactosides) as sole carbon source. After a time, certain colonies began to grow. However, the EbgA protein is an ineffective lactase and does not allow growth on lactose. Two classes of single point mutations dramatically improve the activity of ebg enzyme toward lactose.[17][18] and, as a result, the mutant enzyme is able to replace the lacZ β-galactosidase.[19] EbgA and LacZ are 50% identical on the DNA level and 33% identical on the amino acid level.[20] The active ebg enzyme is an aggregate of ebgA -gene and ebgC-gene products in a 1:1 ratio with the active form of ebg enzymes being an α4 β4 hetero-octamer.[21]


  1. ^ Dorland's Illustrated Medical Dictionary. Retrieved 2006-10-22. 
  2. ^ Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O (September 1995). "A biomarker that identifies senescent human cells in culture and in aging skin in vivo". Proc. Natl. Acad. Sci. U.S.A. 92 (20): 9363–7. doi:10.1073/pnas.92.20.9363. PMC 40985. PMID 7568133. 
  3. ^ a b Bassaneze V, Miyakawa AA, Krieger JE (January 2008). "A quantitative chemiluminescent method for studying replicative and stress-induced premature senescence in cell cultures". Anal. Biochem. 372 (2): 198–203. doi:10.1016/j.ab.2007.08.016. PMID 17920029. 
  4. ^ Gary RK, Kindell SM (August 2005). "Quantitative assay of senescence-associated beta-galactosidase activity in mammalian cell extracts". Anal. Biochem. 343 (2): 329–34. doi:10.1016/j.ab.2005.06.003. PMID 16004951. 
  5. ^ Itahana K, Campisi J, Dimri GP (2007). "Methods to detect biomarkers of cellular senescence: the senescence-associated beta-galactosidase assay". Methods Mol. Biol. Methods in Molecular Biology 371: 21–31. doi:10.1007/978-1-59745-361-5_3. ISBN 978-1-58829-658-0. PMID 17634571. 
  6. ^ Lee BY, Han JA, Im JS, Morrone A, Johung K, Goodwin EC, Kleijer WJ, DiMaio D, Hwang ES (April 2006). "Senescence-associated beta-galactosidase is lysosomal beta-galactosidase". Aging Cell 5 (2): 187–95. doi:10.1111/j.1474-9726.2006.00199.x. PMID 16626397. 
  7. ^ Dugdale, M.L. et al. (2010). "Importance of Arg-599 of b-galactosidase (Escherichia coli) as an anchor for the open conformations of Phe-601 and the active-site loop". Biochem.Cell Biol. (88): 969–979. doi:10.2210/pdb3muy/pdb.  rendered with PyMOL
  8. ^ Fowler AV, Zabin I (October 1970). "The amino acid sequence of beta galactosidase. I. Isolation and composition of tryptic peptides". J. Biol. Chem. 245 (19): 5032–41. PMID 4918568. 
  9. ^ Jacobson, R. H.; Zhang, X. -J.; Dubose, R. F.; Matthews, B. W. (1994). "Three-dimensional structure of β-galactosidase from E. Coli". Nature 369 (6483): 761–766. doi:10.1038/369761a0. PMID 8008071.  edit
  10. ^ Matthews BW (June 2005). "The structure of E. coli beta-galactosidase". C. R. Biol. 328 (6): 549–56. doi:10.1016/j.crvi.2005.03.006. PMID 15950161. 
  11. ^ Gebler JC, Aebersold R, Withers SG (June 1992). "Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) beta-galactosidase from Escherichia coli". J. Biol. Chem. 267 (16): 11126–30. PMID 1350782. 
  12. ^ Yuan J, Martinez-Bilbao M, Huber RE (April 1994). "Substitutions for Glu-537 of beta-galactosidase from Escherichia coli cause large decreases in catalytic activity". Biochem. J. 299 (Pt 2): 527–31. PMC 1138303. PMID 7909660. 
  13. ^ McCarter JD, Burgoyne DL, Miao S, Zhang S, Callahan JW, Withers SG (January 1997). "Identification of Glu-268 as the catalytic nucleophile of human lysosomal beta-galactosidase precursor by mass spectrometry". J. Biol. Chem. 272 (1): 396–400. doi:10.1074/jbc.272.1.396. PMID 8995274. 
  14. ^ Beta-Galactosidase Assay (A better Miller) - OpenWetWare
  15. ^ Dobson, C. L., Warren, A. J., Pannell, R., Forster, A. & Rabbitts, T. H. Tumorigenesis in mice with a fusion of the leukaemia oncogene Mll and the bacterial lacZ gene. EMBO J. 19, 843–851 (2000).
  16. ^ Krivtsov, A. V., & Armstrong, S. A. (2007). MLL translocations, histone modifications and leukaemia stem-cell development. Nature Reviews Cancer, 7(11), 823-833.
  17. ^ Hall, B. G. (1977). "Number of mutations required to evolve a new lactase function in Escherichia coli". Journal of bacteriology 129 (1): 540–3. PMC 234956. PMID 318653.  edit
  18. ^ Hall, B. G. (1981). "Changes in the substrate specificities of an enzyme during directed evolution of new functions". Biochemistry 20 (14): 4042–9. doi:10.1021/bi00517a015. PMID 6793063.  edit
  19. ^ Hall, B. G. (1976). "Experimental evolution of a new enzymatic function. Kinetic analysis of the ancestral (ebg) and evolved (ebg) enzymes". Journal of molecular biology 107 (1): 71–84. PMID 794482.  edit
  20. ^ Stokes, H. W.; Betts, P. W.; Hall, B. G. (1985). "Sequence of the ebgA gene of Escherichia coli: Comparison with the lacZ gene". Molecular biology and evolution 2 (6): 469–77. PMID 3939707.  edit
  21. ^ Elliott, A. C.; k, S; Sinnott, M. L.; Smith, P. J.; Bommuswamy, J; Guo, Z; Hall, B. G.; Zhang, Y (1992). "The catalytic consequences of experimental evolution. Studies on the subunit structure of the second (ebg) beta-galactosidase of Escherichia coli, and on catalysis by ebgab, an experimental evolvant containing two amino acid substitutions". The Biochemical journal. 282 ( Pt 1): 155–64. PMC 1130902. PMID 1540130.  edit

External links[edit]