ß-GLUCOSIDASE from Sweet almond

BGH-101

PREPARATION and SPECIFICATION
Appearance:  Light yellow amorphous powder, lyophilized
Activity:  GradeⅠ 15U/mg-solid or more
(containing approx. 30% of BSA)
Contaminant:  α-Amylase ≤5.0×10⁻⁴%
Stabilizers:  BSA, glutathione (reduced)
PROPERTIES
Stability:  Stable at -20°C for at least One year(Fig.1)
Molecular weight:  approx. 110,000
Isoelectric point :  7.3 ¹ ⁾
Michaelis constants :  2.8×10⁻³M (p-Nitrophenyl-ß-D-glucopyranoside), 3.3×10⁻³M
(2,4-Dichlorophenyl-ß-D-glucopyranoside)
Structure :  2 subunits per enzyme molecule
Optimum pH :  5.5(Fig.4)
Optimum temperature:  50-55°C(Fig.5)
pH Stability:  pH 6.0-9.0 (25°C, 64hr)(Fig.6)
Thermal stability:  below 50°C (pH 7.3, 1hr)(Fig.7)
Effect of various chemicals:   (Table 1)

APPLICATIONS

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

ASSAY

Principle:

ß-glucosidase

p-Nitrophenyl-ß-D-glucopyranoside (PNPG)                                     ► p-Nitrophenol (PNP)+D-Glucose

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

Unit definition:

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

Method:

Reagents
A. Acetate buffer, pH 5.0 (at 25°C):  0.1M
B. PNPG solution:  20mM (603mg p-nitrophenyl-ß-D-glucopyranoside/100ml of H₂O)(Stable for two weeks if stored at 0-5°C)
C. Na₂CO₃ solution:  0.2M (21.2g Na₂CO₃ /1,000ml of H₂O)
D. Enzyme diluent:  10mM phosphate buffer, pH 7.0 containing 0.2% of BSA.

Procedure

Concentration in assay mixture
Acetate buffer 50 mM
PNPG 5.0 mM
BSA 0.05mg/ml

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

1.0 ml 0.1M Acetate buffer, pH 5.0 (A)
0.5 ml Substrate solution (B)

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

3. After exactly 15 minutes at 37°C, add 2.0ml of Na₂CO₃ solution (C) to stop the reaction and measure the
optical density at 400nm against water (OD test).
At the same time, prepare the blank by mixing the reaction mixture with 2.0ml of Na₂CO₃ solution (C)
after incubation for 15 minutes at 37°C, followed by addition of the enzyme solution (OD blank).

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

Calculation

Activity can be calculated by using the following formula :

ΔOD (OD test−OD blank ) ×Vt × df

Volume activity (U/ml) =                                                               =ΔOD×0.0295×df

18.1×1.0 × t ×Vs


Weight activity (U/mg)=(U/ml)×1/C

Vt
: Total volume (4.0ml)
Vs
: Sample volume (0.5ml)
18.1
: Millimolar extinction coefficient of p-nitrophenol under the assay condition (㎠/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 of ß-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 ß-glucosidase
[Residual activity after 1 hr-treatment at 30°C.]
Chemical Concn.(mM) Residual
activity(%)		 Chemical Concn.(mM) Residual
activity(%)
None − 100 MnCl₂   94.3
Metal salt 0.5   BaCl₂   93.9
CaCl₂
   92.7
FeCl₃   99.8
FeSO₄   94.1 o-Phenanthroline 0.5 94.3
CoCl₂   95.5 α,α′-Dipyridy 0.5 94.3
ZnCl₂ 95.0 Borate 25 94.1
CuSO₄   94.5 PCMB 0.05 94.5
HgCl₂   99.8 MIA 0.5 89.3
CrCl₂   93.9
NaF 0.5 96.6
MgSO₄   96.8 NaN₃ 10 98.9
SnCl₂   93.6 EDTA 5.0 96.1
CdCl₂   93.0 Triton X-100 0.5% 102.3
AgNO₃   92.7
Na-cholate 0.5% 99.5
NiCl₂   95.5      
　

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

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