TOYOBO Ideas & Chemistry Biotechnology Operating
Department

PREPARATION and SPECIFICATION

Appearance White amorphous powder, lyophilized
Activity GradeⅡ　20 U/mg-solid or more
Contaminants α-amylase ≤1.0×10-5 %
Stabilizers BSA

PROPERTIES

Stability Stable at −20 ℃ 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＋, Hg2＋, PCMB, MIA
Optimum pH 6.0−7.0(Fig.4)
Optimum temperature 60 ℃(Fig.5)
pH Stability pH 5.0−9.0(Fig.6)
Effect of various chemicals (Table 1)
Thermal stability below 60℃ (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℃

APPLICATIONS

This enzyme is useful for structural investigation of carbohydrates and for enzymatic determination of α-amylase when in combination hexokinase (HXK-311) and G-6-P dehydrogenase (G6D-311, G6D321) 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. 0.1M Phosphate buffer, pH 7.0 (at 25℃)
B. PNPG solution 20 mM (603mg P-Nitrophenyl-α-D-glucopyranoside /100 mL of H2O)(Stable for two weeks if stored at 0−5℃)
C. Na2CO3 solution 0.2 M (21.2g Na2CO3 /1,000 mL of H2O)
D. Enzyme diluent 0.2 M K-phosphate buffer, pH 7.0 containing 1mM of EDTA and 0.05 % of Tween 20

Procedure

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

1.0 mL 0.1 M phosphate buffer (A)
0.5 mL Substrate solution (B)
Concentration in assay mixture
Phosphate buffer 0.1 M
PNPG 5.0 mM
EDTA 0.25 mM
Tween 20 0.125 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 400nm 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 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 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×t×1.0×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) 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 (5 U/mL) was incubated with each chemical at 25 ℃ for 1 hr.]

  • Chemical Concn.(mM) Residual
    activity(%)
    None - 100
    Metal salt 2.0
    MgSO4 97
    CaCl2 71
    Ba(OAc)2 106
    FeCl2 50
    CoCl2 63
    MnCl2 69
    ZnCl2 104
    CdCl2 47
    NiCl2 110
    CuSO4 39
    Pb(OAc)2 75
    AgNO2 0.3
    HgCl2 1.2
    2-Mercaptoethanol 2.0 111
    PCMB 1.0 1.3
  • Chemical Concn.(mM) Residual
    activity(%)
    MIA 2.0 0.8
    NEM 2.0 120
    IAA 2.0 106
    Hydroxylamine 2.0 115
    EDTA 5.0 112
    o-Phenanthroline 2.0 114
    α,α′-Dipyridyl 1.0 122
    Borate 50 119
    NaF 2.0 118
    NaN3 2.0 123
    Triton X-100 0.10 % 123
    Brij 35 0.10 % 121
    Tween 20 0.10 % 124
    Span 20 0.10 % 43
    Na-cholate 0.10 % 102
    SDS 0.05 % 10
    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.

  • 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)

    Fig.3. Stability (Liquid form)

    in 50mM PIPES buffer solution, pH7.0 (contg. 0.5mM CaCl2, 0.1 % detergent) enzyme concn,:5U/mL

  • Fig.4. pH-Activity

    Fig.4. pH-Activity

    37℃, 15 min-reaction in 100mM buffer solution: ●, pH4.0-6.0 acetate ; O, pH6.0-8.0, phosphate; ■, pH8.0-9.0, borate

  • Fig.5. Thermal activity

    Fig.5. Thermal activity

    15 min-reaction in 100mM phosphate buffer, pH7.0

  • Fig.6. pH-Stability

    Fig.6. pH-Stability

    25℃, 20hr-treatment with 50mM buffer solution contg; 0.2 % of BSA: ●, pH4.0-6.0 acetate: ○, pH6.0-8.0, phosphate; ■, pH8.0-9.0, borate. enzyme concn. : 5U/mL

  • Fig.7. Thermal stability

    Fig.7. Thermal stability

    15min-treatment with 0.2M K-phosphate buffer, pH7.0 contg. 1mM EDTA and 0.05 % Tween20. enzyme concn.: 5U/mL

活性測定法（Japanese）

1. 原理

原理

p-Nitrophenolの生成量を400nmの吸光度変化で測定する。

2.定義

下記条件下で1分間に1マイクロモルのp-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, pH 7.0 (25℃)
  • 20mM PNPG水溶液〔603mgのP-ニトロフェニル-α-D-グルコピラノシドを100㎖の蒸留水に攪拌溶 解する〕(1～5℃保存で2週間は使用可能)
  • 0.2M Na2CO3溶液(21.2gの無水炭酸ナトリウムを蒸留水に溶解し1,000 mLとする)

酵素溶液：酵素標品を予め氷冷した1mM EDTA・2Naと0.05 %Tween20を含む0.2Mリン酸緩衝液,pH7.0で溶解し0.006〜0.022U/mLに希釈する。

4.手順

1.試験管に下記反応混液を調製し,37℃で約5分間予備加温する。

1.0 mL リン酸緩衝液, pH 7.0 (A)
0.5 mL 基質溶液 (B)

2.酵素溶液を0.5 mLを加え,反応を開始する。

3.37℃で正確に15分間反応させた後,Na2CO3溶液(C)2.0 mL加えて反応を停止させる。この液につき400nmにおける吸光度を測定する(OD test)。

4.盲検は反応混液①を37℃で15分間放置後,Na2CO3溶液(C) 2.0 mLを加えて混和し,次いで酵素溶液0.5 mLを加えて調整する。以下同様に吸光度を測定する(ODblank)。

5.計算式

  • U/mL =

  • ΔOD (OD test−OD blank)×4.0(mL)×希釈倍率

    18.1×1.0×15(分)×0.5(mL)

＝ ΔOD×0.0295×希釈倍率
U/mg = U/mL×1/C
18.1 : p-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)
C : 溶解時の酵素濃度(c mg/mL)