植酸酶活性的測定——鉬藍(lán)法

1、植酸酶活性的測定鉬藍(lán)法1 原理針對預(yù)混料復(fù)雜的物料體系，用不同的緩沖液對其進(jìn)行抽提，最大限度的減少飼料中無機(jī)磷，微量元素，多維及其他成分對植酸酶測定的影響，用鉬藍(lán)法對抽提液中植酸酶活性進(jìn)行定量。植酸酶在一定溫度和pH條件下，水解底物植酸鈉生成正磷酸和肌醇衍生物，在酸性環(huán)境中與鉬酸銨顯色劑反應(yīng)生成藍(lán)色的（Mo2O3MoO3）復(fù)合物，在波長700nm下比色測定。酶活力單位定義：在37、pH5.0條件下，每分鐘從5.0mM植酸鈉溶液中釋放出1微摩爾的無機(jī)磷定義為1個(gè)酶活力單位（U）2 試劑 本規(guī)定中所用試劑，在沒有注明其它要求時(shí)，均指分析純試劑；所用溶劑和水無注明時(shí)，均指蒸餾水。 清洗實(shí)驗(yàn)用容器不要

2、用含磷清洗劑。2.1 乙酸緩沖液A（0.25mol/L）：稱取14.355g無水乙酸鈉，加入0.5gTriton X-100和0.5g牛血清白蛋白，900ml水溶解，冰乙酸調(diào)pH值至5.00±0.01，水定容至1000ml。2.2乙酸緩沖液B（0.25mol/L）：稱取14.355g無水乙酸鈉，加入1.0g吐溫-20和30gEDTA，900ml水溶解，冰乙酸調(diào)pH值至5.00±0.01，水定容至1000ml。2.3 植酸鈉溶液（6.25mmol/L）：稱取577.4mg肌醇六磷酸鈉，加入574.2mg無水乙酸鈉，90ml水溶解，冰乙酸調(diào)pH值至5.00±0.01，

3、水定容至100ml，現(xiàn)用現(xiàn)配（實(shí)際反應(yīng)液中的最終濃度為5.0mmol/L）。2.4 終止液：5%三氯乙酸（5%TCA）。2.5 1.5鉬酸銨（試劑A）：7.5g鉬酸銨溶于400ml水中，慢慢加入22ml濃硫酸，水定容到500ml，冰箱儲(chǔ)存，有效期1個(gè)月。2.6 2.7硫酸亞鐵（試劑B）：冰箱儲(chǔ)存，有效期1個(gè)月。2.7 顯色劑：移取4份試劑A（2.5），1份試劑B（2.6）混合后使用，現(xiàn)用現(xiàn)配。2.8 磷酸二氫鉀：稱量前于烘箱中烘至恒重，用乙酸緩沖液（2.2）配制50mmol/L標(biāo)準(zhǔn)液，再用50mmol/L配制成4.0mmol/L磷酸二氫鉀，溶劑為乙酸緩沖液（2.2），冰箱儲(chǔ)存。3 儀器設(shè)備恒溫

4、水浴鍋，分光光度計(jì)（有10mm比色皿），磁力攪拌器，渦流式混合器，酸度計(jì)（精確至小數(shù)點(diǎn)后兩位），離心機(jī)（最高轉(zhuǎn)速4,000rpm以上），其它實(shí)驗(yàn)室常用設(shè)備。4 標(biāo)準(zhǔn)曲線繪制 將4.0mmol/L磷酸二氫鉀標(biāo)準(zhǔn)溶液用乙酸緩沖液（2.2）稀釋成0.0、0.8、1.6、2.4、3.2、4.0mmol/L的溶液，按表1的操作步驟一起反應(yīng)。以無機(jī)磷含量為縱坐標(biāo)（0.2ml以上稀釋液無機(jī)磷含量分別為：0.00、0.16、0.32、0.48、0.64、0.80mol），以吸光值為橫坐標(biāo)，繪制標(biāo)準(zhǔn)曲線，列出直線回歸方程（Y=KX+B）。5 樣品測定5.1 試樣溶液的制備 建議稱取5-10g含酶飼料，精確至0.

5、01g，置于100mL容量瓶中，加入乙酸緩沖液（2.1）定容（之前需充分?jǐn)嚢?0min），取其中2-10ml置于另一100ml容量瓶中用乙酸緩沖液（2.2）定容。稀釋的最終結(jié)果應(yīng)使樣液濃度保持在0.02-0.06U/mL.左右，待反應(yīng)。5.2 反應(yīng)按下面的反應(yīng)順序進(jìn)行操作，在反應(yīng)過程中，從加入底物（2.3）開始，向每支管中加入試劑的時(shí)間間隔要絕對一致，37保溫30min。反應(yīng)步驟及試劑、溶液用量見表A1。表1反應(yīng)順序樣品，標(biāo)準(zhǔn)樣品空白標(biāo)準(zhǔn)空白樣品稀釋液（ml）0.20.2乙酸緩沖液 (2.2)（ml）0.237預(yù)熱5min依次加入底物（2.3）（ml）0.80.8（第二步）0.8（第二步）混合

6、37保溫30min依次加入終止液（2.4）（ml）1.01.0（第一步）1.0（第一步）依次加入顯色劑（2.7）（ml）1.01.01.0混合總體積（ml）3.03.03.0A5.3 樣品測定 反應(yīng)后的試樣在室溫下靜置10min，如出現(xiàn)混濁需在離心機(jī)上以4,000rpm離心10min，上清液以標(biāo)準(zhǔn)空白調(diào)零，在分光光度計(jì)700nm波長處測定樣品空白（A0）和樣品溶液（A）的吸光值，AA0為實(shí)測吸光值。用直線回歸方程計(jì)算樣品植酸酶的活性。A6 結(jié)果計(jì)算和表示植酸酶活性U按下式計(jì)算： K×（AA0）×V S×m×30 U = ×F其中：U樣品植酸酶活

7、性，U/g； K標(biāo)準(zhǔn)曲線斜率；F樣品溶液反應(yīng)前的總稀釋倍數(shù)；S樣品測試量，表1中S0.2ml；V反應(yīng)總體積，表1中V=1ml；m試樣質(zhì)量，g；A0測定樣品空白吸光值；A樣品溶液吸光值；30反應(yīng)時(shí)間，min。兩個(gè)平行樣品的測定結(jié)果用算術(shù)平均值表示，保留整數(shù)。A7 允許差 同一樣品兩個(gè)平行測定值的相對偏差不大于8。Determination of Phytase ActivityMolybdate-Blue MethodA1. PRINCIPLEDetermination of phytase activity is based on the colorimetrical reaction bet

8、ween 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 a

9、mount 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 wa

10、ter, 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)

11、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 heptamo

12、lybdate 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 s

13、ulfate (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 befor

14、e 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 rela

15、tive 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 pl

16、ot 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 weit

17、hted 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 ca

18、lculated 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.T

19、able 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

20、.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 activity by reading