β-GLUCOSIDASE from Sweet almond

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Appearance :Light yellow amorphous powder, lyophilized
Activity :GradeⅠ 15 U/mg-solid or more
(containing approx. 30 % of BSA)
Stability :Stable at −20 ℃ for at least one year
Molecular weight :approx. 110,000
Isoelectric point :7.3 1)
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PREPARATION and SPECIFICATION

AppearanceLight yellow amorphous powder, lyophilized
ActivityGradeⅠ 15 U/mg-solid or more
(containing approx. 30 % of BSA)
Contaminantα-Amylase≤ 5.0×10-4 %
StabilizersBSA, glutathione (reduced)

PROPERTIES

StabilityStable at −20 ℃ for at least one year(Fig.1)
Molecular weightapprox. 110,000
Isoelectric point7.3 1)
Michaelis constants2.8×10-3 M (p-Nitrophenyl-β-D-glucopyranoside), 3.3×10-3 M (2,4-Dichlorophenyl-β-D-glucopyranoside)
Structure2 subunits per enzyme molecule
Optimum pH5.5(Fig.4)
Optimum temperature50−55 ℃(Fig.5)
pH StabilitypH 6.0−9.0 (25 ℃, 64 hr)(Fig.6)
Thermal stabilitybelow 50 ℃ (pH 7.3, 1 hr)(Fig.7)
Effect of various chemicals(Table 1)

APPLICATIONS

This enzyme is useful for structural investigation of carbohydrates and for enzymatic determination of α-amylase in combination with α-glucosidase (AGH-211) in clinical analysis.

ASSAY

Principle

Principle

The formation of p-nitrophenol is measured at 400 nm by spectrophotometry.

Unit definition

One unit causes the formation of one micromole of PNP per minute under the conditions detailed below.

Method

Reagents

A. Acetate buffer, pH 5.0 (at 25 ℃)0.1 M
B. PNPG solution20 mM (603 mg p-nitrophenyl-β-D-glucopyranoside/100 mL of H2O)(Stable for two weeks if stored at 0−5 ℃)
C. Na2CO3 solution0.2 M (21.2 g Na2CO3 /1,000 mL of H2O)
D. Enzyme diluent10 mM phosphate buffer, pH 7.0 containing 0.2 % of BSA.

Procedure

1.Prepare the following reaction mixture in a test tube and equilibrate at 37 ℃ for approximately 5 minutes.

1.0 mL0.1 M Acetate buffer, pH 5.0(A)
0.5 mLSubstrate solution(B)
Concentration in assay mixture
Acetate buffer50 mM
PNPG5.0 mM
BSA0.05 mg/mL

2.Add 0.5 mL of the enzyme solution* and mix.

3.After exactly 15 minutes at 37 ℃, add 2.0 mL of Na2CO3 solution (C) to stop the reaction and measure the optical density at 400 nm against water (OD test).

At the same time, prepare the blank by mixing the reaction mixture with 2.0 mL of Na2CO3 solution (C) after incubation for 15 minutes at 37 ℃, followed by addition of the enzyme solution (OD blank).

*Dissolve the enzyme preparation in ice-cold 50 mM Tris-HCl buffer pH 7.8 (approx. 1 mg/mL) and dilute to 0.006−0.022 U/mL with the enzyme diluent (D), immediately before the assay.

Calculation

Activity can be calculated by using the following formula :

  • Volume activity (U/mL) =

  • ΔOD (OD test−OD blank)×Vt×df


    18.1×1.0×t×Vs

  • = ΔOD×0.0295×df

Weight activity (U/mg) = (U/mL)×1/C

Vt: Total volume (4.0 mL)
Vs: Sample volume (0.5 mL)
18.1: Millimolar extinction coefficient of p-nitrophenol under the assay condition (cm2/micromole)
1.0: Light path length (cm)
t: Reaction time (15 minutes)
df: Dilution factor
C: Enzyme concentration in dissolution (c mg/mL)

REFERENCES

1)A.K.Grover, D.D.Macmurchie and R.J.Cushley; Biochim.Biophys.Acta, 482, 98 (1977).

(Characteristics ofβ-Glucosidase from almond)

2)R.Heyworth and P.G.Walker; Biochem.J., 83, 331 (1962).

3)J.H.Hash and K.W.King; J.Biol.Chem., 232, 395 (1958)

Table 1. Effect of Various Chemicals on β-Glucosidase

[Residual activity after 1 hr-treatment at 30 ℃.]

  • ChemicalConcn.(mM)Residual
    activity(%)
    None100
    Metal salt0.5
    CaCl292.7
    FeSO494.1
    CoCl295.5
    ZnCl295.0
    CuSO494.5
    HgCl299.8
    CrCl293.9
    MgSO496.8
    SnCl293.6
    CdCl293.0
    AgNO392.7
    NiCl295.5
  • ChemicalConcn.(mM)Residual
    activity(%)
    MnCl294.3
    BaCl293.9
    FeCl399.8
    o-Phenanthroline0.594.3
    α,α′-Dipyridyl0.594.3
    Borate2594.1
    PCMB0.0594.5
    MIA0.589.3
    NaF0.596.6
    NaN31098.9
    EDTA5.096.1
    Triton X-1000.5 %102.3
    Na-cholate0.5 %99.5

PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate.

  • Fig.1. Stability (Powder form)

    Fig.1. Stability (Powder form)

    (kept under dry conditions)

  • Fig.2. Stability (Powder form)

    Fig.2. Stability (Powder form)

    (kept under dry conditions)

  • Fig.3. Stability (Liquid form at 25℃)

    Fig.3. Stability (Liquid form at 25 ℃)

    (enzyme concentration: 1.0 mg/mL buffer composition: 50 mM Tris-HCI buffer, pH 7.8)

  • Fig.4. pH-Activity

    Fig.4. pH-Activity

    (37 ℃.15 min-reaction in 50 mM acetate buffer.)

  • Fig.5.Temperature activity

    Fig.5.Temperature activity

    (15 min-reaction in 50 mM acetate buffer, pH 5.0)

  • Fig.6. pH-Stability

    Fig.6. pH-Stability

    (25 ℃, 64 hr-treatment with 50 mM buffer solution:pH 3.5-6.0, acetate; pH 6.5-9.0, Tris-HCI)

  • Fig.7. Thermal stability

    Fig.7. Thermal stability

    (1 hr-treatment with 50 mM Tris-HCI buffer,pH 7.3.)

2. MSDS

3. Tech Data Sheets/Manuals

Size

1 MG, 10 MG, 5 MG

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