TOYOBO Ideas & Chemistry バイオ事業総括部

β-GALACTOSIDASE

PREPARATION and SPECIFICATION

Appearance White amorphous powder, lyophilized.
Activity GradeⅡ　500U/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＋＋

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 3.0×10-4M (o-Nitrophenyl-β-D-galactoside), 6.7×10-5M (pNitrophenyl-β-D-galactoside), 2.3×10-4sup>M (Phenyl-β-D-galactoside), 2.5×10-3M (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 280nm1cm (1%)＝20.9 1cm
Inhibitors p-Chloromercuribenzoate, lodoacetamide, heavy metal ions (Zn＋＋, Fe＋＋＋, Cd＋＋, Cu＋＋, Pb＋＋, Ag＋, Hg＋＋), 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℃, 20hr) （Fig.4）
Thermal stability below 50℃ (pH 7.3, 15min) （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 investigation of carbohydrates, the determination of lactose (foodstuff analysis) and as an enzyme label for enzyme immunoassay.

ASSAY

Principle

Principle

The appearance of o-nitrophenol is measured at 410nm by spectrophotometry.

Unit definition

One unit causes the formation of one micromole of ONP per minute under the conditions described below.

Method

Reagents

A. Phosphate buffer, pH 7.3 0.1M (Prepare by mixing 0.1M Na2HPO4 and 0.1M KH2PO4 to reach pH 7.3 at 37℃.)
B. Mercaptoethanol solution 3.36M［Dilute 4.0ml of 2-mercaptoethanol (14.2M) to 17ml with H2O.］ (Should be prepared fresh)
C. MgCl2 solution 30mM (Dissolve 610mg of MgCl2・6H2O in about 80ml of H2O and, after adjusting the pH to 7.3 with 1.0 N NaOH, fill up to 100ml with H2O.)
D. ONPG solution 34mM (205mg ONPG/20ml of Reagent A)(Stable for one week if stored at 0−5℃)
E. Enzyme diluent 50mM phosphate buffer, pH 7.3 contg. 1.0mM MgCl2 and 0.1% BSA

Procedure

1.Prepare the following reaction mixture in a cuvette (d＝1.0cm) and equilibrate at 37℃ for about 5 minutes.

2.5ml 0.1M Phosphate buffer,pH 7.3 (A)
0.1ml Mercaptoethanol solution (B)
0.1ml MgCl2 solution (C)
0.2ml ONPG solution (D)
Concentration in assay mixture
Phosphate buffer 92 mM
ONPG 2.3mM
Mercaptoethanol 0.11M
MgCl2 1.0mM

2.Add 0.1ml of the enzyme solution* and mix by gentle inversion

3.Record the increase in optical density at 410nm against water for 2 to 3 minutes in 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) by 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 0.17−0.85U/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 (3.0ml)
Vs : Sample volume (0.1ml)
3.5 : Millimolar extinction coefficient of ONP under the assay condition (cm2/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
    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)

    Fig.1. Stability (Powder form)

    (kept under dry conditions)

  • Fig.2. pH-Activity

    Fig.2. pH-Activity

    37℃,15 min-reaction in BrittonRobinson buffer

  • Fig.3. Temperature activity

    Fig.3. Temperature activity

    15min-reaction in 0.1M phospate buffer, pH7.3

  • Fig.4. pH-Stability

    Fig.4. pH-Stability

    25℃,20hr-treatment with BrittonRobinson buffer

  • Fig.5. Thermal stability

    Fig.5. Thermal stability

    15min-treatment with 50mM phosphate buffer,pH7.3 contg. 1.0mM MgCl2 enzyme concn.:80U/ml

活性測定法（Japanese）

1. 原理

原理

o-Nitrophenolの生成量を410nmの吸光度の変化で測定する。

2.定義

下記条件で1分間に1マイクロモルのo-Nitrophenolを生 成する酵素量を1単位 (U)とする。

3.試薬

  • 0.1Mリン酸緩衝液,pH7.3(37℃)(0.1M Na2 HPO4溶液と0.1M KH2PO4溶液を混合して37℃ でpHを7.3に調製する)
  • 3.36Mメルカプトエタノール溶液〔4.0mlの 2メル カプトエタノール(14.2M)を蒸留水で17mlに希釈 する〕(用時調製)
  • 30mM MgCl2溶液(610mgのMgCl2・6H2Oを約 80mlの蒸留水に溶解後,1.0N NaOHでpHを7.3に 調整し,蒸留水で100mlとする)
  • 34mM ONPG溶液(205mgのONPGを20mlの試 薬Aに攪拌溶解する)

酵素溶液：酵素標品を予め氷冷した1.0mM MgCl2, 0.1％BSAを含む50mMリン酸緩衝液， pH7.3で0.17〜0.85U/mlに希釈する。

4.手順

1.下記反応混液をキュベット(d＝1.0cm)に調製し,37℃ で約５分間予備加温する。

2.5ml 0.1Mリン酸緩衝液，pH7.3 (A)
0.1ml メルカプトエタノール溶液 (B)
0.1ml MgCl2溶液 (C)
0.2ml ONPG溶液 (D)

2.酵素溶液0.1mlを添加し,ゆるやかに混和後,水を対照 に37℃に制御させた分光光度計で410nmの吸光度 変化を2〜3分間記録し，その初期直線部分から1分 間当りの吸光度変化を求める(ΔODtest)。

3.盲検は酵素溶液の代りに酵素希釈液(1.0mMの MgCl2を含む50mMリン酸緩衝液，pH 7.3)を0.1mlを 加え,上記同様に操作を行って1分間当りの吸光度変化 を求める(ΔODblank)。

5.計算式

  • U/ml ＝

  • ΔOD/min (ΔOD test−ΔOD blank)×3.0(ml)×希釈倍率

    3.5×1.0×0.1(ml)

＝ ΔOD/min×8.57×希釈倍率
3.5 : o-Nitrophenolの上記測定条件下でのミリ モル分子吸光係数(cm2/micromole)
1.0 : 光路長(cm)