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

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
  NaN3	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, CH₃CO; 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 MgCl₂ enzyme concn.:80U/mL