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| Appearance : | Yellowish amorphous powder, lyophilized | |
|---|---|---|
| Activity : | Grade Ⅲ 15 U/mg-solid or more (30 U/mg-protein or more) (containing approx. 30 % of stabilizers) | |
| Contaminants : | Catalase | ≤ 1.0 % |
| NADH oxidase | ≤ 1.0×10-3 % | |
| Stabilizers : | Mannitol, EDTA | |
| Appearance | Yellowish amorphous powder, lyophilized | |
|---|---|---|
| Activity | GradeⅢ 15 U/mg-solid or more (30 U/mg-protein or more) (containing approx. 30 % of stabilizers) | |
| Contaminants | Catalase | ≤ 1.0 % |
| NADH oxidase | ≤ 1.0×10-3 % | |
| Stabilizers | Mannitol, EDTA | |
| Stability | Stable at 20 ℃ for at least one year（Fig.1） |
|---|---|
| Molecular weight | approx. 98,000 |
| Isoelectric point | 4.6±0.1 |
| Michaelis constant | 2.5×10-3 M (N-Acetylneuraminic acid) |
| Structure | 3 subunits (approx. 35,000) per enzyme molecule |
| Inhibitors | p-Chloromercuribenzoate, SDS, Hg2＋, Ag＋ |
| Optimum pH | 7.58.0（Fig.3） |
| Optimum temperature | 70 ℃（Fig.4） |
| pH Stability | pH 6.09.0 (10 ℃, 25 hr)（Fig.5） |
| Thermal stability | below 65 ℃ (pH 7.5, 30 min)（Fig.6） |
| Effect of various chemicals | (Table 1) |
This enzyme is useful for enzymatic determination of N-acetylneuraminic acid and sialic acid in combination with related enzymes, in clinical analysis. 57）
In industrial use, this enzyme is useful for enzymatic synthesis of sialic acid. 89）
The elimination of NADH is measured at 340 nm by spectrophotometry.
One unit causes the oxidation of one micromole of NADH per minute under the conditions detailed below.
| A. NANA solution | 50 mM: Dissolve 309 mg of N-acetylneuraminic acid (MW ＝ 309) in approx. 15 mL of 50 mM potassium phosphate buffer, pH 7.5, and, after adjusting the pH to 7.5 with 1 N KOH, make up to 20 mL with the same buffer (stable for at least 1 week if stored at 0 5 ℃). |
|---|---|
| B. LDH solution | Approx. 50 U/mL: Dilute pig heart lactate dehydrogenase (Toyobo, grade II, ammonium sulfate suspension) to approx. 50 U/mL with ice-cold 50 mM potassium phosphate buffer, pH 7.5 (should be freshly prepared). |
| C. NADH solution | 1.0 mM: Dissolve 7.6 mg of NADH・Na2・3H2O (MW ＝ 763) in 10 mL of 50 mM potassium phosphate buffer, pH 7.5 (should be freshly prepared). |
| D. Buffer solution | 50 mM potassium phosphate buffer pH 7.5 |
| E. Enzyme diluent | 50 mM potassium phosphate buffer pH 7.5, containing 0.2 % BSA |
1.Prepare the following reaction mixture in a cuvette (d = 1.0 cm) and equilibrate at 37 ℃ for approximately 5 minutes.
| 1.0 mL | Substrate solution | (A) |
|---|---|---|
| 0.5 mL | LDH solution | (B) |
| 0.5 mL | NADH solution | (C) |
| 0.4 mL | Buffer solution | (D) |
| Concentration in assay mixture | |
|---|---|
| Potassium phosphate buffer | 50 mM |
| NANA | 20 mM |
| NADH | 0.2mM |
| LDH | Approx. 10 U/mL |
2.Add 0.1 mL of the enzyme solution* and mix by gentle inversion.
3.Record the decrease in optical density at 340 nm against water for 3 to 4 minutes with a spectrophotometer thermostated at 37 ℃, and calculate the ΔOD per minute from the initial linear portion of the curve (ΔOD test). At the same time, measure the blank rate (ΔOD blank) using the same method in the test except that the enzyme diluent (E) is added instead of the enzyme solution.
*Dissolve the enzyme preparation in ice-cold enzyme diluent (E) and dilute to 0.10.3 U/mL with the same buffer, immediately before the assay.
Activity can be calculated by using the following formula :
Volume activity (U/mL) =
ΔOD/min (ΔOD testΔOD blank)×Vt×df
6.22×1.0×Vs
＝ ΔOD/min×4.02×df
Weight activity (U/mg) ＝ (U/mL)×1/C
| Vt | : Total volume (2.5 mL) |
| Vs | : Sample vol ume (0.1 mL) |
| 6.22 | : Millimolar extinction coefficient of NADH (cm2/micromole) |
| 1.0 | : Light path length (cm) |
| df | : Dilution factor |
| C | : Enzyme concentration in dissolution (c mg/mL) |
1)D.G.Comb and S.Roseman; J.Biol.Chem., 235, 2529 (1960).
2)D.G.Comb and S.Roseman; Meth.Enzymol., 5, 391 (1960).
3)S.B.Arden, W.Chang and L.Barksdale; J.Bacteriol., 112, 1260 (1972).
4)Y.Uchida, Y.Tsukada and T.Sugimori; Agric.Biol.Chem., 49, 181 (1985).
5)P.Burunetti, A.Swanson and S.Roseman; Meth.Enzymol., 6, 465 (1963).
6)K.Taniuchi, Y.Miyamoto, Y.Uchida, K.Chifu, M.Mukai, N.Yamaguchi, Y.Tsukada, T.Sugimori, K.Doi and S.Baba; J.Med.Technol. (Japanese), 7, 403 (1979).
7)K.Sugahara, K.Sugimoto, O.Nomura and T.Usui; Clin.Chim.Acta, 108, 493 (1980).
8)Mahn-Joo Kim,William J.Hennen,H.Marcel Sweers and Chi-Huey Wong; J.Am.Chem.Soc.,110, 6481 (1988).
9)Ethan S.Simon,Mark D.Bednarski,and George M.Whitesides; J.Am.Chem.Soc.,110, 7159 (1988).
［The enzyme dissolved in 0.1 M Tris-HCI buffer, pH 7.5 (5 U/mL) was incubated at 30 ℃ for 1hr.］
| Chemical | Concn.(mM) | Residual activity(%) |
|---|---|---|
| None | – | 100 |
| Metal salt | 2.0 | |
| MgCl2 | 107 | |
| CaCl2 | 87 | |
| Ba(OAc)2 | 95 | |
| FeCl3 | 89 | |
| CoCl2 | 93 | |
| MnCl2 | 98 | |
| ZnSO4 | 92 | |
| NiCl2 | 99 | |
| CuSO4 | 64 | |
| Pb(OAc)2 | 87 | |
| AgNO3 | 0 | |
| HgCl2 | 0 |
| Chemical | Concn.(mM) | Residual activity(%) |
|---|---|---|
| PCMB | 2.0 | 0 |
| NEM | 2.0 | 103 |
| NaF | 2.0 | 100 |
| NaN3 | 20 | 100 |
| EDTA | 5.0 | 95 |
| o-Phenanthroline | 2.0 | 100 |
| α,α′-Dipyridyl | 2.0 | 101 |
| Borate | 50 | 86 |
| Triton X-100 | 0.10 % | 109 |
| Na-cholate | 0.10 % | 95 |
| SDS | 0.10 % | 0 |
| Tween 40 | 0.10 % | 96 |
| Span 85 | 0.10 % | 93 |
Fig.1. Stability (Powder form)
(kept under dry conditions)
Fig.2. Stability (Powder form)
(kept under dry conditions)
Fig.3. pH-Activity
37 ℃, 5 min-reaction in 50 mM buffer solution : pH 5.0-6.0, acetate; pH 6.0- 9.0, K-phosphate; The enzyme activity was assayed by the 2,4-dinitroplenylhydradine method.
Fig.4. Temperature activity
5 min-reaction in 50 mM K-phosphate buffer pH 7.5, The enzyme activity was assayed by the 2,4-dinitroplenylhydradine method.
Fig.5. pH-Stability
10 ℃, 25 hr-treatment with 50 mM buffer solution : pH 4.0-6.0, acetate; pH 6.0-9.0, K-phosphate ; pH 9.0-10.0, borate.
Fig.6. Thermal stability
30 min-treatment with 50 mM K-phosphate buffer, pH 7.5, enzyme concentration.: 20 U/mL
| Size | 1 MG, 10 MG, 5 MG |
|---|
| Name | α2,8-sialyltransferase; Cstll |
| Catalog Number | EN01003 |
| E.C. | 2.4.99.8 |
| Product Description | E. coli recombinant α2,3/8-sialyltransferase from Campylobacter jejuni |
| Unit Definition | One unit is defined as the amount of enzyme that catalyzes the formation of 1 µmol Siaα2,3Lac from CMP-Sia and lactose per minute at 37 °C. |
10 in stock
| Name | UDP-Glc dehydrogenase; UDPDH |
| Product Code | EN01020 |
| E.C. | 1.1.1.22 |
| Product Description | E. coli recombinant uridine-5-diphosphoglucose dehydrogenase from Streptococcus pyogenes |
| Unit Definition | One unit will oxidize 1.0 μmole of UDP-glucose to UDP-glucuronic acid per minute at pH 8.7 at 25 °C. |
10 in stock
| Name | Inorganic pyrophosphatase (EcPPA) |
| Catalog No. | EN01018 |
| E.C. | 3.6.1.1 |
10 in stock
| Name | α2,3-sialyltransferase; PmST1 |
| Catalog Number | EN01002 |
| E.C. | 2.4.99.4 |
| Product Description | E. coli recombinant α2,3-sialyltransferase from Pasteurella multocida (P-1059) |
| Unit Definition | One unit is defined as the amount of enzyme that catalyzes the formation of 1 µmol Siaα2,3Lac from CMP-Sia and lactose per minute at 37 °C. |
10 in stock
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