3-α-hydroxysteroid dehydrogenase (3α-HSD)

用途 用于总胆汁酸（TBA）试剂的研发和大量制备。 酶学性质 来源： 微生物 酶学委员会编号： EC1.1.1.50 分子量： 30 kDa (SDS-PAGE) 等电点： 5.2 Km值： 3.6×10-5M (雄酮)，4.7×10-5M (NAD+) 抑制剂： Cu²⁺，Ag⁺，Hg²⁺，Zn²⁺，Fe³⁺ 最适pH： 8.5-9.5 图1 最适温度： 55℃ 图2 pH稳定性： 6.0-9.0 (25℃,16h) 图3 热稳定性： 37℃以下稳定(pH9.0，30min) 图4 稳定性： -25～-15℃静置保存12个月保持 90%以上活性 图5 保护剂： BSA 活性测定方法 1、原理 反应生成的NADH可以用分光光度计在340nm检测。 2、酶活定义 单位酶活定义为在下述条件下，每分钟催化生成1μmolNADH所需的酶量。 3、试剂准备 试剂I：0.1M焦磷酸钠（用HCl调pH至8.9）。 试剂II：将319mg NAD+溶解至25mL双蒸水中，用固体NaHCO3调节pH至7.0-7.5，用双蒸水定容至30mL。 试剂III：将30mg溶解至100mL甲醇中。 酶稀释液：10mMTris-HCl，pH 9.0。 4、操作步骤 1.向3mL比色皿中依次加入2.6mL试剂I，0.2mL试剂II，0.1mL试剂III，混匀。 2.将反应混合物在25°C预热5min。 3.向反应混合物中加入0.1mL酶液，混匀，25°C反应，并用分光光度计在340nm下，记录1min内的吸光度变化（∆As） *用酶稀释液替代酶液，其它步骤相同，所得溶液吸光度为空白吸光度（∆Ab） ∆A=∆As-∆Ab 5、 活力计算 3.00：反应液总体积（mL）； 0.10：酶液体积（mL）； 1.0：光路长度(cm)； df：稀释倍数； C：酶浓度(mg/mL)； 6.22：NADH在340nm处毫摩尔吸光系数(cm2/μmol)。 Application It is used for the development and mass preparation of total bile acid (TBA) reagents. Enzymatic properties (note that 2+, +, 3+ are superscripts) Source: Microorganism Enzymology Committee Number: EC1.1.1.50 Molecular weight: 30 kDa (SDS-PAGE) Isoelectric point: 5.2 Km value: 3.6×10-5M (androsterone), 4.7×10-5M (NAD+) Inhibitors: Cu²⁺，Ag⁺，Hg²⁺，Zn²⁺，Fe³⁺ Optimum pH: 8.5-9.5 Figure 1 Optimum temperature: 55℃ Figure 2 pH stability: 6.0-9.0 (25℃,16h) Figure 3 Thermal stability: Stable below 37℃ (pH9.0, 30min) Figure 4 Stability: -25 ~ -15℃ standing store for 12 months More than 90% activity Figure 5 Protective agent: BSA Assay method for activity 1. Principle The NADH produced by the reaction can be detected with a spectrophotometer at 340nm. 2. Definition of enzyme activity Unit enzyme activity is defined as the amount of enzyme required to catalyze the production of 1μmolNADH per minute under the following conditions. 3. Reagent preparation Reagent I: 0.1M sodium pyrophosphate (adjust pH to 8.9 with HCl). Reagent II: Dissolve 319mg NAD+ into 25mL double steaming water, adjust the pH to 7.0-7.5 with solid NaHCO3, and adjust the volume to 30mL with double steaming water. Reagent III: Dissolve 30mg into 100mL methanol. Enzyme diluent: 10mMTris-HCl, pH 9.0. 4. Operation procedure 1. Add 2.6mL reagent I, 0.2mL reagent II and 0.1mL reagent III into a 3mL colorimetric dish and mix well. 2. Preheat the reaction mixture at 25°C for 5min. 3. Add 0.1mL enzyme liquid to the reaction mixture, mix it well, react at 25°C, and record the absorbance change within 1min at 340nm with a spectrophotometer (∆As). * Replace enzyme liquid with enzyme diluent, other steps are the same, the absorbance of the resulting solution is blank absorbance (∆Ab) ∆A=∆As-∆Ab 5. Vitality computing 3.00: total volume of reaction liquid (mL); 0.10: enzyme liquid volume (mL); 1.0: optical path length (cm); df: dilution ratio; C: Enzyme concentration (mg/mL); 6.22: Nanomolar absorption coefficient of NADH at 340nm (cm2/μmol).

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