N-ACETYLNEURAMINIC ACID ALDOLASE from Microorganism

PREPARATION and SPECIFICATION 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 PROPERTIES 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, Hg 2＋ , 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) APPLICATIONS 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） ASSAY Principle The elimination of NADH is measured at 340 nm by spectrophotometry. Unit definition One unit causes the oxidation of one micromole of NADH per minute under the conditions detailed below. Method Reagents 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・Na 2 ・3H 2 O (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 Procedure 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. Calculation 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 (cm 2 /micromole) 1.0 : Light path length (cm) df : Dilution factor C : Enzyme concentration in dissolution (c mg/mL) REFERENCES 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). Table 1. Effect of Various Chemicals on  N -Acetylneuraminic acid aldolase ［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 MgCl 2 107 CaCl 2 87 Ba(OAc) 2 95 FeCl 3 89 CoCl 2 93 MnCl 2 98 ZnSO 4 92 NiCl 2 99 CuSO 4 64 Pb(OAc) 2 87 AgNO 3 0 HgCl 2 0 Chemical Concn.(mM) Residual activity(%) PCMB 2.0 0 NEM 2.0 103 NaF 2.0 100 NaN 3 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 Ac, CH 3 CO; PCMB, p-Chloromercuribenzoate; NEM, N-Ethylmaleimide; EDTA, Ethylenediaminetetraacetate;SDS, Sodium dodecyl sulfate. 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