β-羟丁酸脱氢酶（β-HBDH）

用途 用于β-羟丁酸试剂的研发和大量配制。 酶学性质 来源： 微生物 酶学委员会编号： EC1.1.1.30 分子量： 27 kDa (SDS-PAGE) 等电点： 6.1 Km值： 1.46×10-2M (D-3-Hydroxybutyrate)，9.5×10-5M (NAD+) 抑制剂： Hg²⁺，Ag⁺，SDS 最适pH： 8.5 图1 最适温度： 60℃ 图2 pH稳定性： 5.0-10.0 (25℃,16h) 图3 热稳定性： 50℃以下稳定(pH8.5，30min) 图4 稳定性： -25～-15℃静置保存12个月保持 90%以上活性 图5 活性测定方法 1、原理 反应生成的NADH的量可用分光光度计在340nm检测。 2、酶活定义 单位酶活定义为在下述反应条件下，每分钟生成 1μmol NADH 所需的酶量。 3、试剂准备 试剂I：100mM pH8.5 Tris-HCl缓冲液。 试剂II：158mM 3-羟丁酸钠溶液（用试剂I溶解）。 试剂III：27.9mM NAD+（用试剂I溶解）。 试剂IV：100mM Tris-HCl，pH8.5，含0.1%BSA。 待测样品：用试剂IV将酶液稀释至0.1-0.5 U/mL。 4、操作步骤 4.1 在3mL比色皿中加入3mL试剂I，0.5mL试剂II，0.2mL试剂III，37°C预热5min。 4.2 加入100μL待测样品，混匀。 4.3 在340nm处37°C反应，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 Vt：反应液总体积 (3.1 mL)； Vs：酶液体积 (0.1 mL)； 1.0：光路长度(cm)； df：稀释倍数； C：酶浓度 (mg/mL)； 6.22：标准反应条件下，生色基团在340nm处毫摩尔吸光系数(cm2/μmol)。 Application It is used for the development and mass formulation of β-hydroxybutyrate reagents. Enzymatic properties Source: Microorganism Enzymology Committee Number: EC1.1.1.30 Molecular weight: 27 kDa (SDS-PAGE) Isoelectric point: 6.1 Km value: 1.46×10-2M (D-3-Hydroxybutyrate), 9.5×10-5M (NAD+) Inhibitor: Hg²⁺，Ag⁺，SDS Optimal pH: 8.5 Figure 1 Optimum temperature: 60℃ Figure 2 pH stability: 5.0-10.0 (25℃,16h) Figure 3 Thermal stability: Stable below 50℃ (pH8.5, 30min) Figure 4 Stability: -25 ~ -15℃ standing store for 12 months. More than 90% activity Figure 5 Assay method for activity 1. Principle The amount of NADH produced by the reaction can be measured by spectrophotometer at 340nm. 2. Definition of enzyme activity Unit enzyme activity is defined as the amount of enzyme required to produce 1μmol of NADH per minute under the following reaction conditions. 3. Reagent preparation Reagent I: 100mM pH8.5 Tris-HCl buffer. Reagent II: 158mM sodium 3-hydroxybutyrate solution (dissolved with reagent I). Reagent III: 27.9mM NAD+ (dissolved with reagent I). Reagent IV: 100mM Tris-HCl, pH8.5, containing 0.1%BSA. Sample to be tested: Dilute the enzyme solution with reagent IV to 0.1-0.5 U/mL. 4. Operation procedure 4.1 Add 3mL reagent I, 0.5mL reagent II and 0.2mL reagent III into a 3mL colorimetric dish and preheat it at 37°C for 5min. 4.2 Add 100μL sample to be tested and mix well. 4.3 Reaction at 340nm at 37°C, record absorbance change within 1min (∆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 The formula can be captured or edited in this position Vt: total volume of reaction liquid (3.1mL); Vs: enzyme liquid volume (0.1mL); 1.0: optical path length (cm); df: dilution ratio; C: Enzyme concentration (mg/mL); 6.22: Under standard reaction conditions, the millimolar absorption coefficient (cm2/μmol) of the color-producing group at 340nm.

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