植酸酶活性的测定--钼蓝法.doc

植酸酶活性的测定钼蓝法1 原理针对预混料复杂的物料体系，用不同的缓冲液对其进行抽提，最大限度的减少饲料中无机磷，微量元素，多维及其他成分对植酸酶测定的影响，用钼蓝法对抽提液中植酸酶活性进行定量。植酸酶在一定温度和pH条件下，水解底物植酸钠生成正磷酸和肌醇衍生物，在酸性环境中与钼酸铵显色剂反应生成蓝色的（Mo2O3MoO3）复合物，在波长700nm下比色测定。酶活力单位定义：在37、pH5.0条件下，每分钟从5.0mM植酸钠溶液中释放出1微摩尔的无机磷定义为1个酶活力单位（U）2 试剂 本规定中所用试剂，在没有注明其它要求时，均指分析纯试剂；所用溶剂和水无注明时，均指蒸馏水。 清洗实验用容器不要用含磷清洗剂。2.1 乙酸缓冲液A（0.25mol/L）：称取14.355g无水乙酸钠，加入0.5gTriton X-100和0.5g牛血清白蛋白，900ml水溶解，冰乙酸调pH值至5.000.01，水定容至1000ml。2.2乙酸缓冲液B（0.25mol/L）：称取14.355g无水乙酸钠，加入1.0g吐温-20和30gEDTA，900ml水溶解，冰乙酸调pH值至5.000.01，水定容至1000ml。2.3 植酸钠溶液（6.25mmol/L）：称取577.4mg肌醇六磷酸钠，加入574.2mg无水乙酸钠，90ml水溶解，冰乙酸调pH值至5.000.01，水定容至100ml，现用现配（实际反应液中的最终浓度为5.0mmol/L）。2.4 终止液：5%三氯乙酸（5%TCA）。2.5 1.5钼酸铵（试剂A）：7.5g钼酸铵溶于400ml水中，慢慢加入22ml浓硫酸，水定容到500ml，冰箱储存，有效期1个月。2.6 2.7硫酸亚铁（试剂B）：冰箱储存，有效期1个月。2.7 显色剂：移取4份试剂A（2.5），1份试剂B（2.6）混合后使用，现用现配。2.8 磷酸二氢钾：称量前于烘箱中烘至恒重，用乙酸缓冲液（2.2）配制50mmol/L标准液，再用50mmol/L配制成4.0mmol/L磷酸二氢钾，溶剂为乙酸缓冲液（2.2），冰箱储存。3 仪器设备恒温水浴锅，分光光度计（有10mm比色皿），磁力搅拌器，涡流式混合器，酸度计（精确至小数点后两位），离心机（最高转速4,000rpm以上），其它实验室常用设备。4 标准曲线绘制 将4.0mmol/L磷酸二氢钾标准溶液用乙酸缓冲液（2.2）稀释成0.0、0.8、1.6、2.4、3.2、4.0mmol/L的溶液，按表1的操作步骤一起反应。以无机磷含量为纵坐标（0.2ml以上稀释液无机磷含量分别为：0.00、0.16、0.32、0.48、0.64、0.80mol），以吸光值为横坐标，绘制标准曲线，列出直线回归方程（Y=KX+B）。5 样品测定5.1 试样溶液的制备 建议称取5-10g含酶饲料，精确至0.01g，置于100mL容量瓶中，加入乙酸缓冲液（2.1）定容（之前需充分搅拌20min），取其中2-10ml置于另一100ml容量瓶中用乙酸缓冲液（2.2）定容。稀释的最终结果应使样液浓度保持在0.02-0.06U/mL.左右，待反应。5.2 反应按下面的反应顺序进行操作，在反应过程中，从加入底物（2.3）开始，向每支管中加入试剂的时间间隔要绝对一致，37保温30min。反应步骤及试剂、溶液用量见表A1。表1反应顺序样品，标准样品空白标准空白样品稀释液（ml）0.20.2乙酸缓冲液 (2.2)（ml）0.237预热5min依次加入底物（2.3）（ml）0.80.8（第二步）0.8（第二步）混合37保温30min依次加入终止液（2.4）（ml）1.01.0（第一步）1.0（第一步）依次加入显色剂（2.7）（ml）1.01.01.0混合总体积（ml）3.03.03.0A5.3 样品测定 反应后的试样在室温下静置10min，如出现混浊需在离心机上以4,000rpm离心10min，上清液以标准空白调零，在分光光度计700nm波长处测定样品空白（A0）和样品溶液（A）的吸光值，AA0为实测吸光值。用直线回归方程计算样品植酸酶的活性。A6 结果计算和表示植酸酶活性U按下式计算： K（AA0）V Sm30 U = F其中：U样品植酸酶活性，U/g； K标准曲线斜率；F样品溶液反应前的总稀释倍数；S样品测试量，表1中S0.2ml；V反应总体积，表1中V=1ml；m试样质量，g；A0测定样品空白吸光值；A样品溶液吸光值；30反应时间，min。两个平行样品的测定结果用算术平均值表示，保留整数。A7 允许差 同一样品两个平行测定值的相对偏差不大于8。Determination of Phytase ActivityMolybdate-Blue MethodA1. PRINCIPLEDetermination of phytase activity is based on the colorimetrical reaction between ammonium molybdate and free phosphorus which released from by the hydrolysis of phytate. The product solution painted in blue is measured by spectrophotometer at a wave-length 700nm, the measured absorbency quantificates with the amount of free phosphorus.1 unit of phytase activity (U) is the amount of enzyme which releases 1 umol inorganic orthophosphate from substrate (sodium phytate ,50mM) at the temperature 37 and pH5.0 in 1 minute.A2. REAGENTSAll the reagents used must be analytical prue. Detergents containing phosphate should not be used in washing container. A2.1. WaterDistilled water, or equivalent.A2.2. Buffer solution (0.1 mol/L)Dissolve 5.742 g sodium acetic acid, 0.5 g Triton X-100 and 0.5 g bovine serum albumin in 900 mL water; adjust to pH 5.0 with acetic acid (100%), and dilute to 1 L with water.A2.3. Substrate solutionDissolve 577.4 mg sodium phytate (C6H6O24P6Na12) from rice (Cat. No. P-3168, Sigma Chemical Co., St. Louis, MO) and 574.2 mg sodium acetic acid in 90 mL water, adjust the pH to 5.0 with acetic acid (100%), and dilute to 100 mL with water. Prepare this solution fresh daily.A2.4. Reaction stop solutionTrichloroacetic acid (5%).A2.5. Ammonium heptamolybdate solution (Solution A)Dissolve 7.5 g ammonium heptamolybdate (N6H24Mo7O24.4H2O) in 400 mL distilled water, slowly add 22 mL sulfuric acid (98%), and dilute to 500 mL with water. This solution may be kept at 4 shielded from light for 1 month.A2.6. Ferrous sulfate solution (Solution B)Ferrous sulfate (2.7%). This solution may be kept at 4 and shielded from light for 1 month.A2.7. Color solutionMix 100 mL solution A and 25 mL solution B. Prepare this solution fresh daily.A2.8. Potassium dihydrogen phosphate stock solutionPrepare potassium dihydrogen phosphate to constant weight at 60 before dissolving it to a final concentration of 4.0 mmol/L using buffer solution (A2.2). Prepare this solution fresh daily.A3. APPARATUSA3.1. Waterbath: thermostatically controlled to 37.0 0.1 by circulating water.A3.2. Ultraviolet-visible spectrophotometer A3.3. Centrifuge: can be used at a relative centrifugal force of 3000gA3.4. pH meterA4. PREPARATION OF STANDARD SOLUTIONS AND CURVEPrepare working standards of 0.0、0.8、1.6、2.4、3.2、4.0mmol/L potassium dihydrogen phosphate solution by serial dilution of stock solution (A2.8). Carry out the procedure which described in Table A1, and then plot the absorbance difference of the standard solutions (X-axis) against the corresponding exactly calculated amount of potassium dihydrogen phosphate (Y-axis). Draw the best fitting curve through the origin and give the regression equation (Y=KX+B). A5. PREPARATION OF SAMPLEIt is suggested that weithted 5-10g enzyme sample ,place it in a volumetric of 100mL, adjust the volume to the mark by the acetate buffer solution and mix thoroughly.Dilute the weighted sample in duplicate (sample and blank) with buffer solution to the phytase activity within 0.03-0.08 U/mL.A standard sample with exactly calculated activity is recommended to be determined as the same procedure to test the accuracy.A6. ASSAYThe assay is carried out according to the procedure in Table A1. In this procedure, interval of adding reagents to every tube should be the same after the substrate adding to the reaction solution.Table A1ProcedureSample，StandardsSample blank Standards blank Sample solution（mL）0.20.2Buffer solution（A2.2）（mL）0.25min at 37Substrate solution（A2.3）（mL）0.80.8（the second step）0.8（the second step）Mixing30min at 37Stop solution（A2.4）（mL）1.01.0（the first step）1.0（the first step）Color reagent（A2.7）（mL）1.01.01.0MixingTotal volume（mL）3.03.03.0Centrifuge all the tubes for 10 min at 4,000 rpm before standing for 10 min at room temperature. Measure the absorbance of sample （A）and its blank （A0）at 700 nm with the spectrophotometer after zeroing the instrument with standards blank of. Determine the enzyme