α-GLUCOSIDASE(MALTASE) from Microorganism

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 -4 M (p-Nitrophenyl-α-D-glucopyranoside) Inhibitors Ag ＋ , Hg 2＋ , 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 PNPG 2 205.0 Maltotriose 203.0 PNPG 3 284.0 Maltotetraose 168.0 PNPG 5 164.0 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 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. Na 2 CO 3  solution 0.2 M (21.2g Na 2 CO 3  /1,000 mL of H 2 O) 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 Na 2 CO 3  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 Na 2 CO 3  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 (cm 2 /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 MgSO 4 97 CaCl 2 71 Ba(OAc) 2 106 FeCl 2 50 CoCl 2 63 MnCl 2 69 ZnCl 2 104 CdCl 2 47 NiCl 2 110 CuSO 4 39 Pb(OAc) 2 75 AgNO 2 0.3 HgCl 2 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 NaN 3 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, CH 3 CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; NEM, N-Ethylmaleimide; IAA, Iodoacetamide; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride. Fig.1. Stability (Powder form) (kept under dry conditions) Fig.2. Stability (Powder form) (kept under dry conditions) Fig.3. Stability (Liquid form) in 50mM PIPES buffer solution, pH7.0 (contg. 0.5mM CaCl 2 , 0.1 % detergent) enzyme concn,:5U/mL 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 15 min-reaction in 100mM phosphate buffer, pH7.0 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 15min-treatment with 0.2M K-phosphate buffer, pH7.0 contg. 1mM EDTA and 0.05 % Tween20. enzyme concn.: 5U/mL