TOYOBO Ideas & Chemistry バイオ事業総括部

PREPARATION and SPECIFICATION

Appearance White amorphous powder, lyophilized
Activity GradeⅡ　1.0 U/mg-solid or more
　(containing approx. 70% of stabilizers)
Contaminant NADH oxidase ≤1.0×10-1%
Stabilizers Mg＋＋, Ca＋＋, bovine serum albumin, glycine, lysine

PROPERTIES

Stability Stable at −20℃ for at least one year（Fig.1）
Molecular weight approx. 150,000 (by gel filtration)
Isoelectric point 5.25 1)
Michaelis constants 8.0×10-5M (HCHO), 1.2×10-4M (NAD＋)
Structure 2 subunits (75,000) per enzyme molecule 1)
Inhibitors Chelating agents, Ni＋＋, Cd＋＋, Hg＋＋, PCMB, ionic detergents
Optimum pH 9.0（Fig.4）
Optimum temperature 40℃（Fig.5）
pH Stability pH 8.0−10.0 (30℃, 16hr)（Fig.6）
Thermal stability below 40℃ (pH 7.5, 30min)（Fig.7）
Substrate specificity(Table 1)
Effect of various chemicals (Table 2,3)

APPLICATIONS

This enzyme is useful for enzymatic determination of formaldehyde and of hydrogen peroxide when coupled with catalase.

ASSAY

Principle

Principle

The appearance of diformazan formed by the reduction of nitrotetrazorium blue (NTB) with phenazine methosulfate (PMS)(red) is measured at 570nm by spectrophotometry.

Unit definition

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

Method

Reagents

A. HCHO solution 10mM［0.08ml of 37% (W/V) HCHO/100ml of 50mM phosphate buffer, pH 7.5 contg. 0.5% Triton X-100］(Should be prepared fresh)
B. NAD＋ solution 0.4% (40mg NAD＋・3H2O/10ml of H2O)(Should be prepared fresh)
C. PMS-NTB solution 1.0mg phenazine methosulfate (PMS), 10mg nitrotetrazorium blue (NTB)/10ml of H2O (Store at 4℃ in brownish bottle)
D. HCl solution 0.3N
E. Enzyme diluent 50mM phosphate buffer, pH 7.5 contg. 0.2% BSA

Procedure

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

0.5ml Substrate solution (A)
0.1ml NAD＋ solution (B)
0.1ml PMS-NTB solution (C)
Concentration in assay mixture
Phosphate buffer 21 mM
HCHO 4.2 mM
NAD 0.46 mM
PMS 27 μM
NTB 0.10mM
Triton X-100 0.21 %

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

3.After exactly 15 minutes at 37℃, add 3.0ml of HCl solution (D) to stop the reaction and measure the optical density at 570nm against water (OD test).
At the same time, prepare the blank by first mixing the reaction mixture with 3.0ml of HCl solution after 15 min-incubation at 37℃, followed by the addition of the enzyme solution (OD blank).

*Dissolve the enzyme preparation in ice-cold enzyme diluent (E) and dilute to 0.01−0.02U/ml with the same buffer, immediately before assay.

Calculation

Activity can be calculated by using the following formula :

  • Volume activity (U/ml) =

  • ΔOD (OD test−OD blank)×Vt×df

    20.1×1.0×t×Vs

＝ ΔOD×0.0279×df

Weight activity (U/mg) ＝ (U/ml)×1/C

Vt : Total volume (4.2ml)
Vs : Sample volume (0.1ml)
20.1 : Half a millimolar extinction coefficient of diformazan (cm2/0.5micromole)
1.0 : Light path length (cm)
t : Reaction time (15 minutes)
df : Dilution factor
C : Enzyme concentration in dissolution (c mg/ml)

REFERENCES

1)M.Ando, T.Yoshimoto, S.Ogushi, K.Rikitake, S.Shibata and D.Tsuru; J.Biochem., 85, 1165 (1979).

2)Application
D.Tsuru; Creatinine.Rinsho Kensa (Supple), 22, 1331 (1978).

Table 1. Substrate Specificity of Formaldehyde dehydrogenase

  • Substrate(final 50mM) Relative activity(%)
    HCHO 100
    CH3CHO 47
    CH3・CH2CHO 5
    (CH3)2CH・CHO 0
    CH3(CH2)2CHO 3
    HO・CHO 19
    CH3・CO・CHO 34
  • Substrate(final 50mM) Relative activity(%)
    CH3OH 0
    CH3CH2OH 0
    CH2OH・CH2OH 0
    CH2(OH)・CH(OH)CH2OH 0
    HCOOH 0
    CH3CH2COOH 0
    COOH・COOH 0

Table 2. Effect of Various Chemicals on Formaldehyde dehydrogenase

(Residual activity after 30 min-treatment at 30℃)

  • Chemical Concn.(mM) Residual activity(%)
    NiCl2 1.0 3
    Zn(OAc)2 1.0 31
    MnCl2 1.0 29
    CoSO4 1.0 96
    FeCl3 1.0 95
    FeSO4 1.0 89
    BaCl2 1.0 110
    Ca(OAc)2 1.0 122
  • Chemical Concn.(mM) Residual activity(%)
    MgCl2 1.0 124
    Pb(OAc)2 1.0 103
    HgCl2 1.0 0.4
    Cd(OAc)2 1.0 1.0
    EDTA 10 53
    NaCl 50 100
    KBr 50 93
    KI 50 51
Ac, CH3CO; EDTA, Ethylenediaminetetraacetate

Table 3. Effect of Various Detergents on Formaldehyde dehydrogenase

(Residual activity after 30 min-treatment at 30℃)

  • Detergent Concn. Residual activity(%)
    Emulgen 0.1 101
    Brij 35 0.1 101
    Brij 58 0.1 102
    Span 20 0.1 102
    Span 80 0.1 99
    Tween 20 0.1 100
  • Detergent Concn. Residual activity(%)
    Triton X-100 0.1% 105
    Na-cholate 0.1 100
    DAC 0.1 9
    SDS 10 mM 38
    NLS 10 mM 76
DAC, Dimethylbenzylalkylammonium chloride; SDS, Sodium dodecyl sulfate; NLS, N-Lauroyl sarcosine-Na salt

  • 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)

    enzyme concetration: 10U/ml buffer composition:50mM K-phosphate buffer, pH7.5

  • Fig.4. pH-Activity

    Fig.4. pH-Activity

    37℃,15min-reaction in 50mM buffer solution: pH6-7.6,phosphate; pH7.2-8.8, Tris-HCl; pH8.7-11 Na2CO3;pH11.5-12.5;NaOH-KCI
    ○,activity for formaldehyde;
    ●,activity for n-butanol

  • Fig.5. Temperature activity

    Fig.5. Temperature activity

    15min-reaction in 50mM phosphate buffer, pH7.5

  • Fig.6. pH-Stability

    Fig.6. pH-Stability

    30℃,16hr-treatment with 50mM buffer solution: pH5.8, acetate;pH6.8-8.0,phosphate pH8.0-10.0 borate

  • Fig.7. Thermal stability

    Fig.7. Thermal stability

    30min-treatment with 50mM phosphate buffer,pH7.5

活性測定法（Japanese）

1. 原理

原理

生成したNADHによりPMS (phenazine methosulfate)を介してNTB (nitrotetrazorium blue)を還元し，生成したdiformazanの570nmにおける吸光度を測定する。

2.定義

下記条件下で1分間あたり1/2マイクロモルのdiformazanを生成する酵素量を1単位 (U)とする。

3.試薬

  • 10mM HCHO溶液〔0.08mlの37%(W/V)HCHO溶液を0.5%トリトンX-100を含む50mMリン酸緩衝液， pH7.5で100mlとする〕(用時調製)
  • 0.4%NAD＋水溶液(40mgのNAD・3H2Oを10mlの蒸留水に溶解する)(用時調製)
  • PMS-NTB水溶液(1.0mgPMS及び10mgNTBを10mlの蒸留水に溶解する)(褐色瓶中で4℃保存)
  • 0.3N HCl溶液

酵素溶液：酵素標品を予め氷冷した0.2%BSAを含む50mMリン酸緩衝液,pH7.5で溶解し,分析直前に同緩衝液で0.01～0.02U/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。

0.5ml 基質溶液 (A)
0.1ml NAD＋溶液 (B)
0.1ml PMS-NTB水溶液 (C)

2.酵素溶液0.5ml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，HCl溶液(D)3.0mlを加えて反応を停止させる。この液につき570nmにおける吸光度を測定する(ODtest)。

4.盲検は反応混液1を37℃で15分間放置後,HCl溶液(D)3.0mlを加えて混和し,次いで酵素溶液0.5mlを加えて調製する。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)×4.2(㎖)×希釈倍率

    20.1×1.0×15(分)×0.5(ml)

＝ ΔOD×0.0279×希釈倍率
U/mg = U/ml×1/C
20.1 : diformazanの1/2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)