β-GALACTOSIDASE from Escherichia coli

PREPARATION and SPECIFICATION Appearance White amorphous powder, lyophilized. Activity GradeⅡ　500 U/mg-solid or more Contaminants α-galactosidase ＜ 1×10 -4  % α-glucosidase ＜ 1×10 -4  % β-glucosidase ＜ 2×10 -3  % α-mannosidase ＜ 1×10 -4  % β-mannosidase ＜ 1×10 -4  % proteinasee ＜10mAbs/mg-P Stabilizer Mg 2＋ PROPERTIES Stability Stable at −20 ℃ for at least one year （Fig.1） Molecular weight 540,000  1,2) Isoelectric point  3) 4.6 Michaelis constants 3.0×10 -4  M (o-Nitrophenyl-β-D-galactoside), 6.7×10 -5  M (pNitrophenyl-β-D-galactoside), 2.3×10 -4  M (Phenyl-β-D-galactoside), 2.5×10 -3  M (Lactose) Structure  4〜8) The enzyme is composed of four identical subunits having a molecular weight of ca.135,000. The amino acid analysis indicates approximately 1,170 residues per subunit. E  280 nm 1 cm  (1%)＝20.9 1 cm Inhibitors p-Chloromercuribenzoate, lodoacetamide, heavy metal ions (Zn 2＋ , Fe 3＋ , Cd 2＋ , Cu 2＋ , Pb 2＋ , Ag ＋ , Hg 2＋ ), lonic detergents (SDS, DAC, etc.) Optimum pH 7.0−7.5 （Fig.2） Optimum temperature 50−55 ℃ （Fig.3） pH Stability pH 6.5−8.5 (25 ℃, 20 hr) （Fig.4） Thermal stability below 50 ℃ (pH 7.3, 15 min) （Fig.5） Substrate specificty This enzyme specifically hydrolyzes β-D-galactosyl linkage (Table 1). Effect of various chemicals (Table 2) APPLICATIONS This enzyme is useful for structural analysis of carbohydrates, measurement of lactose (foodstuff analysis) and as an enzyme label for enzyme immunoassays. ASSAY Principle The formation of o-nitrophenol is measured at 410 nm by spectrophotometry. Unit definition One unit causes the formation of one micromole of ONP per minute under the conditions detailed below. Method Reagents A. Phosphate buffer, pH 7.3 0.1 M: Prepare by mixing 0.1 M Na 2 HPO 4  and 0.1 M KH 2 PO 4  to pH 7.3 at 37 ℃.) B. Mercaptoethanol solution 3.36 M: Dilute 4.0 mL of 2-mercaptoethanol (14.2 M) to 17 mL with H 2 O (should be freshly prepared). C. MgCl 2  solution 30 mM: Dissolve 610mg of MgCl 2 ・6H 2 O in approx. 80 mL of H 2 O and, after adjusting the pH to 7.3 with 1.0 NaOH, fill up to 100 mL with H 2 O. D. ONPG solution 34 mM: 205 mg of ONPG / 20 mL of reagent A (stable for 1 week if stored at 0−5 ℃). E. Enzyme diluent 50mM phosphate buffer, pH 7.3, containing 1.0mM MgCl 2  and 0.1 % BSA Procedure 1.Prepare the following reaction mixture in a cuvette (d ＝ 1.0cm) and equilibrate at 37℃ for approximately 5 minutes. 2.5 mL 0.1M Phosphate buffer, pH 7.3 (A) 0.1 mL Mercaptoethanol solution (B) 0.1 mL MgCl2 solution (C) 0.2 mL ONPG solution (D) Concentration in assay mixture Phosphate buffer 92 mM ONPG 2.3 mM Mercaptoethanol 0.11 M MgCl 2 1.0 mM 2.Add 0.1 mL of the enzyme solution* and mix by gentle inversion 3.Record the increase in optical density at 410 nm against water for 2 to 3 minutes with a spectrophotometer thermostated at 37 ℃, and calculate ΔOD per minute from the initial linear portion of the curve (ΔOD test). At the same time, measure the blank rate (ΔOD blank) using the same method as the test except that the enzyme diluent (E) is added instead of the enzyme solution. * Dilute the enzyme preparation to 0.17−0.85 U/mL with ice-cold enzyme diluent (E). Calculation Activity can be calculated by using the following formula : Volume activity (U/mL) = ΔOD/min (ΔOD test−ΔOD blank)×Vt×df 3.5×1.0×Vs ＝ ΔOD/min×8.57×df Vt : Total volume (3.0 mL) Vs : Sample volume (0.1 mL) 3.5 : Millimolar extinction coefficient of ONP under the assay condition (cm 2 /micromole) 1.0 : Light path length (cm) df : Dilution factor REFERENCES 1)G.R.Graben, E.Steers, Jr.and C.B.Anfinsen; J.Biol.Chem., 240, 2468 (1965). 2)C.C.Contaxis and F.J.Reithel; Biochem,J., 124, 623 (1971). 3)K.Wallenfels and R.Weil; The Enzymes,Vol. 7, p.617 (P.D.Boyer ed.), Academic Press. New York−London (1972). 4)A.Ulmann, M.E.Goldberg, D.Perrin and J.Monod; Biochemistry, 7, 261 (1968). 5)A.V.Fowler and I.Zabin; J.Biol.Chem., 245, 5032 (1970). 6)A.V.Fowler and I.Zabin; J.Biol.Chem., 247, 5425, 5432 (1972). 7)F.Melchers and W.Messer; Eur.J.Biochem., 34, 228 (1973). 8)K.E.Langley, A.V.Fowler and I.Zabin; J.Biol.Chem., 250, 2587 (1975). Table 1. Substrate Specificity of β-Galactosidase Substrate (2.3mM) Relative activity(%) Vmax ** (Relative value) o-Nitrophenyl-β-D-galactopyranoside 100 100 p-Nitrophenyl-β-D-galactopyranoside 14.7 13.4 Phenyl-β-D-galactopyranoside* 1.1 1.3 Lactose* 2.1 3.9 p-Nitrophenyl-α-D-galactopyranoside 0 0 p-Nitrophenyl-α-D-glucopyranoside 0 0 p-Nitrophenyl-β-D-glucopyranoside 0 0 Substrate (2.3mM) Relative activity(%) Vmax ** (Relative value) p-Nitrophenyl-α-D-mannopyranoside 0 0 p-Nitrophenyl-β-D-mannopyranoside 0 0 p-Nitrophenyl-α-L-fucopyranoside 0 0 p-Nitrophenyl-β-L-fucopyranoside 0 0 p-Nitrophenyl-α-D-xylopyranoside 0 0 p-Nitrophenyl-β-D-xylopyranoside 0 0 * Liberation of galactose was measured using galactose dehydrogenase as a coupling enzyme. **Vmax was obtained from Lineweaver-Burk plots (Vmax with o-Nitrophenyl-β-D-galactopyranoside was 1,000 micromoles of hydrolyzed substrate per min per mg-protein). Table 2. Effect of Various Chemicals on β-Galactosidase ［This enzyme dissolved in 50mM PIPES buffer, pH 7.0(10U/mL) was incubated with each chemical at 30℃ for 30minutes. The residual activity was assayed according to the routine method described above.］ Chemical Concn.(mM) Residual activity(%) None - 100 Metal salt 2.0 MgCl2 99 CaCl2 102 Ba(OAc)2 80 FeCl3 59 CoCl2 83 MnCl2 100 ZnSO4 6.2 Cd(OAc)2 4.7 NiCl2 77 CuSO4 0.9 Pb(OAc)2 1.3 AgNO3 0 HgCl2 2.0 Mercaptoethanol 2.0 99 Cysteine 2.0 102 PCMB 2.0 0.3 Chemical Concn.(mM) Residual activity(%) MIA 2.0 86 NEM 2.0 95 IAA 2.0 1.4 Hydroxylamine 2.0 78 EDTA 5.0 103 o-Phenanthroline 2.0 99 α,α′-Dipyridyl 2.0 103 Borate 50 98 NaF 2.0 99 NaN 3 20 98 Triton X-100 0.1 % 101 Brij 35 0.1 % 103 Tween 20 0.1 % 103 Span 20 0.1 % 107 Na-cholate 0.1 % 109 SDS 0.05 % 75 DAC 0.05 % 0 Ac, CH 3 CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; NEM, N-Ethylmaleimide; IAA, lodoacetamide; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride. Fig.1. Stability (Powder form) (kept under dry conditions) Fig.2. pH-Activity 37℃,15 min-reaction in BrittonRobinson buffer Fig.3. Temperature activity 15min-reaction in 0.1M phospate buffer, pH7.3 Fig.4. pH-Stability 25℃,20hr-treatment with BrittonRobinson buffer Fig.5. Thermal stability 15min-treatment with 50mM phosphate buffer,pH7.3 contg. 1.0mM MgCl 2  enzyme concn.:80U/mL