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You are here:Home » Molecular Biology » MB-Enzymes, Glycosidase » -Galactosidase (E. coli)

-Galactosidase (E. coli)


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Beta-galactosidases are widespread in microorganisms, animals and plants. That from the Escherichia coli strain K12 has been particularly studied at Anfinsen's laboratory in connection with genetic experiments on gene regulation of protein synthesis.
Catalog #G1041-05
Beta-galactosidase is tetrameric, being composed of four identical subunits of 135kD, each with an active site which may be independently active (Melcher and Messe 1973). The enzyme is readily fragmented into small peptides (Marinkovic et al. 1975). The amino acid analysis indicates approximately 1170 residues per subunit (Fowler and Zabin 1970). See also Langley et al. (1975) and Naider et al. (1972). Kaneshiro et al. (1975) report on an active dimer.
Contaxis and Reithel (1974) report a small molecule with high 260nm absorbance which is bound to the enzyme in crude extracts as a stabilizer. On adding it to the pure enzyme, activity and heat stability are enhanced.
Bronskil and Wong (1971) suggest that the ribosomal enzyme may be an intermediate stage in the assembly of the quarternary structure.
ActivityThe reaction probably involves a galactosyl-enzyme intermediate ( van de Groen 1973). See also Hill and Huber (1971) and Sinnott and Souchard (1973a and b). Hartl and Hall (1974) report a second beta-galactosidase without lactase activity in Vivo but with high ONPG (o-nitrophenyl, beta-D-galactopyranoside) activity. Monovalent cations have a stimulatory effect on the enzyme (Becker and Evans 1969). The alcohols, methanol, ethanol, i-propanol, and n-propanol, at 5% concentration, all increase the rate of o-nitrophenyl, beta]D-galactopyranoside cleavage (Shifrin and Hunn 1969). The enzyme is protected against heat-inactivation by 5-phosphorylribose 1-pyrophosphate in the presence of beta-mercaptoethanol (Moses and Sharp 1970).
Specific Activity 300 units per mg protein
Unit Definition1 unit hydrolyzes 1umole o-nitrophenyl-beta-D-galactopyranoside (ONPG) per min. at 25°C, pH 7.5 (Craven et al., 1965).
Quality ControlSDS-PAGE
Molecular Weight540kD ( Steers et al. 1965; Cohen and Mire 1971; (Contaxis and Reithel 1971).
Optimum pH6-8. See also Tenu et al. (1971 and 1972).
Extinction coefficient 20.9.
SpecificityA review of substrate requirements is offered by Wallenfels and Malhotra (1960).
InhibitorsIn the absence of Mg2+ ions, beta-mercaptoethanol causes dissociation (Shifrin et al. 1970. See also Case et al. (1973) and Loontiens et al. (1970).
Reagents0.3M Sodium phosphate buffer, pH 7.5, 0.003M magnesium chloride
Substrate diluent: 0.01M Tris acetate pH 7.5, 0.01M magnesium chloride
Enzyme diluent: 0.01 M Tris HCl, pH 7.5, 0.01M magnesium chloride, 0.01M mercaptoethanol, 0.01M sodium chloride, 0.10M Sodium/potassium phosphate buffer, pH 7.0, 1.0M Mercaptoethanol, 0.014M o-Nitrophenyl-beta-D-galactopyranoside (ONPG) in substrate diluent. Prepare fresh daily.
EnzymePrepare a one mg/ml stock solution in 0.10M sodium/potassium phosphate buffer pH 7.0. Immediately prior to use, dilute further to 0.02-0.04% in enzyme diluent. The protein concentration of the chromatographically purified enzyme may be determined as follows:
mg Protein/ml = A280 x 0.478
Note This enzyme is not stable when diluted. All dilutions should be made as quickly as possible and used immediately.
Storage and StabilityMay be stored at 4°C. For long-term storage, aliquot and store at 4°C. Do not freeze. Aliquots are stable for 6 months. For maximum recovery of product, centrifuge the original vial prior to removing the cap. Further dilutions can be made in assay buffer.
Molecular Weight540kD
SourceE. coli
PurityPurified by chromatography.
FormSupplied as a suspension in 1.6M ammonium sulfate.
Important NoteThis product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.
Alternate namesEC=; E. coli

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