β-GLUCOSIDASE from Sweet almond

PREPARATION and SPECIFICATION Appearance Light yellow amorphous powder, lyophilized Activity GradeⅠ 15 U/mg-solid or more (containing approx. 30 % of BSA) Contaminant α-Amylase ≤ 5.0×10 -4  % Stabilizers BSA, glutathione (reduced) PROPERTIES Stability Stable at −20 ℃ for at least one year (Fig.1) Molecular weight approx. 110,000 Isoelectric point 7.3  1) Michaelis constants 2.8×10 -3  M (p-Nitrophenyl-β-D-glucopyranoside), 3.3×10 -3  M (2,4-Dichlorophenyl-β-D-glucopyranoside) Structure 2 subunits per enzyme molecule Optimum pH 5.5 (Fig.4) Optimum temperature 50−55 ℃ (Fig.5) pH Stability pH 6.0−9.0 (25 ℃, 64 hr) (Fig.6) Thermal stability below 50 ℃ (pH 7.3, 1 hr) (Fig.7) Effect of various chemicals (Table 1) APPLICATIONS This enzyme is useful for structural investigation of carbohydrates and for enzymatic determination of α-amylase in combination with α-glucosidase ( AGH-211 ) 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. Acetate buffer, pH 5.0 (at 25 ℃) 0.1 M B. PNPG solution 20 mM (603 mg p-nitrophenyl-β-D-glucopyranoside/100 mL of H 2 O)(Stable for two weeks if stored at 0−5 ℃) C. Na 2 CO 3  solution 0.2 M (21.2 g Na 2 CO 3  /1,000 mL of H 2 O) D. Enzyme diluent 10 mM phosphate buffer, pH 7.0 containing 0.2 % of BSA. 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 Acetate buffer, pH 5.0 (A) 0.5 mL Substrate solution (B) Concentration in assay mixture Acetate buffer 50 mM PNPG 5.0 mM BSA 0.05 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 400 nm 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 addition of the enzyme solution (OD blank). * Dissolve the enzyme preparation in ice-cold 50 mM Tris-HCl buffer pH 7.8 (approx. 1 mg/mL) and dilute to 0.006−0.022 U/mL with the enzyme diluent (D), 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 18.1×1.0×t×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)A.K.Grover, D.D.Macmurchie and R.J.Cushley; Biochim.Biophys.Acta, 482, 98 (1977). (Characteristics ofβ-Glucosidase from almond) 2)R.Heyworth and P.G.Walker; Biochem.J., 83, 331 (1962). 3)J.H.Hash and K.W.King; J.Biol.Chem., 232, 395 (1958) Table 1. Effect of Various Chemicals on β-Glucosidase ［Residual activity after 1 hr-treatment at 30 ℃.] Chemical Concn.(mM) Residual activity(%) None - 100 Metal salt 0.5 CaCl 2 92.7 FeSO 4 94.1 CoCl 2 95.5 ZnCl 2 95.0 CuSO 4 94.5 HgCl 2 99.8 CrCl 2 93.9 MgSO 4 96.8 SnCl 2 93.6 CdCl 2 93.0 AgNO 3 92.7 NiCl 2 95.5 Chemical Concn.(mM) Residual activity(%) MnCl 2 94.3 BaCl 2 93.9 FeCl 3 99.8 o-Phenanthroline 0.5 94.3 α,α′-Dipyridyl 0.5 94.3 Borate 25 94.1 PCMB 0.05 94.5 MIA 0.5 89.3 NaF 0.5 96.6 NaN 3 10 98.9 EDTA 5.0 96.1 Triton X-100 0.5 % 102.3 Na-cholate 0.5 % 99.5 PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate. Fig.1. Stability (Powder form) (kept under dry conditions) Fig.2. Stability (Powder form) (kept under dry conditions) Fig.3. Stability (Liquid form at 25 ℃) (enzyme concentration: 1.0 mg/mL buffer composition: 50 mM Tris-HCI buffer, pH 7.8) Fig.4. pH-Activity (37 ℃.15 min-reaction in 50 mM acetate buffer.) Fig.5.Temperature activity (15 min-reaction in 50 mM acetate buffer, pH 5.0) Fig.6. pH-Stability (25 ℃, 64 hr-treatment with 50 mM buffer solution:pH 3.5-6.0, acetate; pH 6.5-9.0, Tris-HCI) Fig.7. Thermal stability (1 hr-treatment with 50 mM Tris-HCI buffer,pH 7.3.)