α-GLUCOSIDASE(MALTASE) from Microorganism

AGH-211

PREPARATION and SPECIFICATION
Appearance:  White amorphous powder, lyophilized
Activity:  GradeⅡ 20U/mg-solid or more
Contaminants:  α-amylase≤1.0×10⁻⁵%
Stabilizers:  BSA
PROPERTIES
Stability:  Stable at -20°C for at least One year (Fig.1)
Molecular weight:  approx. 65,000 (Gel-filtration and SDS-PAGE)
Isoelectric point :  5.2
Michaelis constant :  6.3×10⁻⁴M (p-Nitrophenyl-α-D-glucopyranoside)
Inhibitors :  Ag⁺, Hg⁺⁺, PCMB, MIA
Optimum pH :  6.0-7.0(Fig.4)
Optimum temperature:  60°C(Fig.5)
pH Stability:  pH 5.0-9.0(Fig.6)
Thermal stability:  below 60°C (pH 7.0, 15min)(Fig.7)
Substrate* Relative hydrolysis rate** Substrate* Relative hydrolysis rate**
PNPG 100.0 Maltose
 271.0
PNPG₂ 205.0 Maltotriose 203.0
PNPG₃ 284.0 Maltotetraose 168.0
PNPG₅ 164.0 Maltopentaose 100.0
*: Substrate concn. 2.2mM
** : Glucose-forming activity, pH 6.8 at 37°

Effect of various chemicals:   (Table 1)

APPLICATIONS

This enzyme is useful for structural investigations of carbohydrates and for the enzymatic determination of α-amylase when coupled with hexokinase (HXK-311) and G-6-P dehydrogenase (G6D-311, G6D-321) in clinical analysis.

ASSAY

Principle:

α-glucosidase

p-Nitrophenyl-α-D-glucopyranoside (PNPG)                                         ► p-Nitrophenol (PNP)+α-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. 0.1M Phosphate buffer, pH 7.0 (at 25°C)
B. PNPG solution:  20mM (603mg P-Nitrophenyl-α-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:  0.2M K-phosphate buffer, pH 7.0 containing 1mM of EDTA and 0.05% of Tween 20

Procedure

Concentration in assay mixture
Phosphate buffer 0.1 M
PNPG 5.0 mM
EDTA 0.25 mM
Tween 20 0.125mg/ml

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

1.0 ml 0.1 M phosphate buffer (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 first mixing the reaction mixture with 2.0ml of Na₂CO₃ solution (C)
after 15 min-incubation at 37°C, followed by the addition of the enzyme solution (OD blank).

* Dissolve the enzyme preparation in ice-cold enzyme diluent (D) and dilute to 0.006-0.022U/ml with the
same buffer, 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× t ×1.0×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. Y.Suzuki, M.Shinji and N.Eto; Biochim.Biophys.Acta., 787, 281 (1984).
  2. Y.Takii, K.Daimon and Y.Suzuki; Appl.Microbiol.Biotechnol., 38, 243 (1992).
  3. Y.Takii, K.Takahashi, K.Yamamoto, Y.Sogabe and Y.Suzuki; Appl.Microbiol.Biotechnol., 44, 629 (1996).
Table 1. Effect of Various Chemicals on α-Glucosidase
[The enzyme dissolved in 10mM phosphate buffer, pH 7.0 contg. 0.2% of BSA (5U/ml) was incubated with
each chemical at 25°C for 1hr.]
Chemical Concn.(mM) Residual
activity(%)		 Chemical Concn.(mM) Residual
activity(%)
None − 100 MIA 2.0 0.8
Metal salt 2.0   NEM 2.0 120
MgSO₄
   97
 IAA 2.0 106
CaCl₂   71 Hydroxylamine 2.0 115
Ba(OAc)₂   106 EDTA 5.0 112
FeCl₂ 50 o-Phenanthroline 2.0 114
CoCl₂   63 α,α′-Dipyridy 1.0 122
MnCl₂   69 Borate 50 119
ZnCl₂   104
NaF 2.0 118
CdCl₂   47 NaN₃ 2.0 123
NiCl₂   110 Triton X-100 0.10% 123
CuSO₄   39 Brij 35 0.10% 121
Pb(OAc)₂   75
 Tween 20 0.10% 124
AgNO₃   0.3 Span 20 0.10% 43
HgCl₂   1.2 Na-cholate 0.10% 102
2-Mercaptoethanol 2.0 111
 SDS 0.05% 10
PCMB 1.0 1.3 DAC 0.05% 124

Ac, CH₃CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; NEM, N-Ethylmaleimide; IAA, Iodoacetamide; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride.

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