乳酸氧化酶（LOX）

用途 用于乳酸含量的检测。 酶学性质 来源： 微生物 酶学委员会编号： EC 1.1.3.2 分子量： 42 kDa (SDS-PAGE) 等电点： pH 4.6 Km值： 7.5 ×10 -4 M (L-Lactate) 抑制剂： Hg²⁺ , Pb²⁺ 最适pH： 6.0-7.0 图1 最适温度： 50℃ 图2 pH稳定性： 6.0-8.5 (25℃,16h) 图3 热稳定性： 50℃以下稳定(pH7.0，30min) 图4 稳定性： -25～-15℃静置保存12个月保持 90%以上活性 图5 活性测定方法 1、原理 反应生成的醌亚胺（Quinoneimine dye）的 量可用分光光度计在 555 nm 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 1 μmol H2O2 所需的酶量。 3、试剂准备 试剂 I：0.2 MpH 6.5 磷酸钾缓冲液。 试剂 II：1kU/mL 过氧化物酶（POD）溶液。 试剂 III：50 mM4-AA溶液。 试剂 IV：0.5 MDL-乳酸溶液，pH6.5。 试剂 V：50 mMTOOS 溶液。 酶稀释液：10 mMpH7.0 磷酸钾缓冲液含10 μM FAD。 样品：用酶稀释液将酶稀释至0.05-0.2U/mL。 按如下配制反应混合物： 试剂 I 10 ml 试剂 II 0.25 mL 试剂 III 1.5 mL 试剂 IV 5 mL 试剂 V 1.5 mL 双蒸水补充到 50 ml 4、操作步骤 4.1 向 1 mL 比色皿中加入反应1mL反应混合液。 4.2 将反应混合物在37 °C预热5min。 4.3 加入 20 μL 待测酶液，混匀。 4.4 在 555 nm处测定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 1.020：反应液总体积（mL）； 0.020：酶液体积（mL）； 1：光路长度 (cm)； 1/2：1 摩尔过氧化氢生成 1/2 摩尔醌亚胺染料； df：稀释倍数； C：酶浓度( mg/mL)； 39.2：标准反应条件下，生色基团在 555 nm 处毫摩尔吸光系数(cm2/μmol)。 Application For the detection of lactic acid content. Enzymatic properties Source: Microorganism Enzymology Committee Number: EC 1.1.3.2 Molecular weight: 42 kDa (SDS-PAGE) Isoelectric point: pH 4.6 Km value: 7.5 × 10-4 M (L-Lactate) Inhibitors: Hg²⁺ , Pb²⁺ Optimum pH: 6.0-7.0 Figure 1 Optimum temperature: 50℃ Figure 2 pH stability: 6.0-8.5 (25℃,16h) Figure 3 Thermal stability: Stable below 50℃ (pH7.0, 30min) Figure 4 Stability: -25 ~ -15℃ standing store for 12 months More than 90% activity Figure 5 Enzyme activity definition Unit enzyme activity is defined as the amount of enzyme required to catalyze the production of 1μmol H2O2 per minute under the following conditions. Assay method for activity 1. Principle The amount of Quinoneimine dye produced by the reaction can be measured by spectrophotometer at 555 nm. 2. Definition of enzyme activity Unit enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol H2O2 per minute under the following conditions. 3. Reagent preparation Reagent I: 0.2 MpH 6.5 potassium phosphate buffer. Reagent II: 1kU/mL peroxidase (POD) solution. Reagent III: 50 mM4-AA solution. Reagent IV: 0.5 MDL- lactic acid solution, pH6.5. Reagent V: 50 mMTOOS solution. Enzyme diluent: 10 mMpH7.0 potassium phosphate buffer containing 10 μM FAD. Sample: Dilute the enzyme with enzyme diluent to 0.05-0.2U/mL. Prepare the reaction mixture as follows: Reagent I is 10 ml Reagent II 0.25 mL Reagent III 1.5 mL Reagent IV is 5 mL Reagent V 1.5 mL Double steam water to 50 ml 4. Operation procedure 4.1 Add 1mL reaction mixture into 1mL colorimetric dish. 4.2 Preheat the reaction mixture at 37 °C for 5min. 4.3 Add 20μL of enzyme liquid to be measured and mix well. 4.4 The reaction is measured at 37 °C at 555 nm and the absorbance change (∆As) within 1min is recorded. * 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 1.020: total volume of reaction liquid (mL); 0.020: enzyme liquid volume (mL); 1: optical path length (cm); 1/2:1 mole of hydrogen peroxide to generate 1/2 mole of quinone imide dye; df: dilution ratio; C: Enzyme concentration (mg/mL); 39.2: Under standard reaction conditions, the millimolar absorption coefficient of the color group at 555 nm (cm2/μmol).

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