α淀粉酶检测操作规程

1、-淀粉酶活力检测操作规程1 酶活定义1g固体酶粉（或1mL液体酶），于60、pH=6.0条件下，1h液化1g可溶性淀粉，即为1个酶活力单位，以u/g(u/mL)表示。2 原理在一定温度和PH值条件下，-淀粉酶将淀粉分子链中的-1，4葡萄糖苷键随机切断成长短不一的短链糊精、少量麦芽糖和葡萄糖，而使淀粉对碘呈篮色的特异性反应逐渐消失，呈红棕色，其颜色消失的速度与酶活力有关，故可通过固定反应后的吸光度计算其酶活力。3 仪器和设备3.1 分析天平：感量0.0001g3.2 精密pH计：精确至0.013.3 分光光度计：应符合GB9721有关规定3.4 电热恒温水浴锅：30100，±0.23.

2、5 计时器：每小时误差不超过5秒4 试剂与溶液4.1 原碘液：称取碘（I2）11g、碘化钾（KI）22g，用少量水使碘完全溶解，然后定容至500mL，贮于棕色瓶中。4.2 稀碘液：吸取原碘液2.00ml，加碘化钾20g，用水溶解并定容至500mL，贮于棕色瓶中。4.3 20g/L可溶性淀粉溶液： 称取可溶性淀粉2.000g，精确至0.001g，用水调成浆状物，在搅动下缓缓倾入70mL沸水中，然后，以30ml水分几次冲洗装淀粉的烧杯，洗液并入其中，加热至完全透明，冷却，定容至100mL。此溶液需要当天配置。4.4 磷酸缓冲液（pH=6.0） 称取磷酸氢二钠（Na2PO4·12H2O）4

3、5.03g、柠檬酸（C6H8O7·H2O）8.07g，使水溶解并定容至1000mL。配好后用pH计校正。5 分析步骤5.1待测酶液的制备：称取酶粉1 g，精确至0.0002g（或吸取液体酶1.00mlL），先用少量的磷酸缓冲液溶解，并用玻璃棒捣研，将上请液小心倾入容量瓶，沉渣部分在加入少量缓冲液，如此捣研34次，最后全部移入100mL容量瓶中，用缓冲液定容到刻度（将估计酶活力除以4，即酶活理应在3.75.6u/mL范围内），摇匀，通过4层纱布过滤，滤液供测定用。5.2 测定：5.2.1 吸取可溶性淀粉溶液20.0mL于试管中，加入缓冲液5.00mL，摇匀后，于（60±0.2

4、）的恒温水浴中预热5min。5.2.2 加入稀释好的待测酶液1.00mL，立刻记时，摇匀，准确反应5min。5.2.3立即吸取反应液1.00mL于稀碘液5.00ml中，摇匀，并以稀碘液做空白，于660nm波长下，用10mm比色皿，迅速测定其吸光度。根据吸光度查附表1，求得测试酶液的浓度。6 计算样品酶活力（u/g或u/mL）= c×n式中： c 表示测试酶液的浓度（u/mL）n 表示样品的稀释倍数 7 结果的允许差平行实验相对误差不得超过2%。附表 1 吸光度与测试-淀粉酶酶浓度对照表吸光值酶浓度(u/ml)吸光值酶浓度(u/ml)吸光值酶浓度(u/ml)吸光值酶浓度(u/ml)0.

5、1004.6940.1404.4920.1804.3010.2204.1320.1014.6890.1414.4870.1814.2970.2214.1280.1024.6840.1424.4820.1824.2920.2224.1240.1034.6790.1434.4770.1834.2880.2234.1200.1044.6740.1444.4720.1844.2830.2244.1160.1054.6690.1454.4670.1854.2790.2254.1120.1064.6640.1464.4620.1864.2750.2264.1080.1074.6590.1474.4570.

6、1874.2700.2274.1050.1084.6540.1484.4520.1884.2660.2284.1010.1094.6490.1494.4470.1894.2610.2294.0970.1104.6440.1504.4420.1904.2570.2304.0930.1114.6390.1514.4380.1914.2530.2314.0890.1124.6340.1524.4330.1924.2480.2324.0850.1134.6290.1534.4280.1934.2440.2334.0820.1144.6240.1544.4230.1944.2400.2344.0780.

7、1154.6190.1554.4180.1954.2350.2354.0740.1164.6140.1564.4130.1964.2310.2364.0700.1174.6090.1574.4080.1974.2270.2374.0670.1184.6040.1584.4040.1984.2220.2384.0630.1194.5990.1594.3990.1994.2180.2394.0590.1204.5940.1604.3940.2004.2140.2404.0560.1214.5890.1614.3890.2014.2100.2414.0520.1224.5840.1624.3850.

8、2024.2050.2424.0480.1234.5790.1634.380.2034.2010.2434.0450.1244.5740.1644.3750.2044.1970.2444.0410.1254.5690.1654.3700.2054.1930.2454.0370.1264.5640.1664.3660.2064.1890.2464.0340.1274.5590.1674.3610.2074.1850.2474.0300.1284.5540.1684.3560.2084.1810.2484.0260.1294.5490.1694.3520.2094.1760.2494.0230.1

9、304.5440.1704.3470.2104.1720.2504.0190.1314.5390.1714.3420.2114.1680.2514.0160.1324.5340.1724.3380.2124.1460.2524.0120.1334.5290.1734.3330.2134.1600.2534.0090.1344.5240.1744.3290.2144.1560.2544.0050.1354.5180.1754.3240.2154.1520.2554.0020.1364.5130.1764.3190.2164.1480.2563.9980.1374.5070.1774.3150.2

10、174.1440.2573.9950.1384.5020.1784.3100.2184.1400.2583.9910.1394.4970.1794.3060.2194.1360.2593.988吸光值酶浓度u/ml吸光值酶浓度u/ml吸光值酶浓度u/ml吸光值酶浓度u/ml0.2603.9840.3003.8690.343.7500.383.6390.2613.9810.3013.8660.3413.7470.3813.6340.2623.9780.3023.8630.3423.7440.3823.6320.2633.9740.3033.8600.3433.7410.3833.6290.264

11、3.9710.3043.8570.3443.7390.3843.6260.2653.9680.3053.8540.3453.7360.3853.6230.2663.9640.3063.8510.3463.7330.3863.6210.2673.9610.3073.8480.3473.7300.3873.6180.2683.9580.3083.8450.3483.7270.3883.6150.2693.9540.3093.8420.34937240.3893.6120.2703.9510.3103.8390.3503.7210.3903.610.2713.9480.3113.8360.3513.

12、7180.3913.6070.2723.9440.3123.8330.3523.7160.3923.6040.2733.9410.3133.8300.3533.7130.3933.6020.2743.9380.3143.8270.3543.7100.3943.5990.2753.9350.3153.8240.3553.7070.3953.5960.2763.9320.3163.8210.3563.7040.3963.5940.2773.9280.3173.8180.3573.7010.3973.5910.2783.9250.3183.8150.3583.6990.3983.5880.2793.

13、9220.3193.8120.3593.6960.3993.5850.2803.9190.3203.8090.3603.6930.403.5830.2813.9150.3213.8060.3613.6900.4013.580.2823.9130.3223.8030.3623.6870.4023.5770.2833.9220.3233.8000.3633.6840.4033.5750.2843.9190.3243.7970.3643.6820.4043.5720.2853.9150.3253.7940.3653.6790.4053.5690.2863.9120.3263.7910.3663.67

14、60.4063.5670.2873.9090.3273.7880.3673.6730.4073.5640.2883.9060.3283.7850.3683.6700.4083.5590.2893.9030.3293.7820.3693.6680.4093.5560.2903.9000.3303.7790.3703.6650.4103.5540.2913.8970.3313.7760.3713.6620.4113.5510.2923.8940.3323.7740.3723.6590.4123.5480.2933.8910.3333.7710.3733.6560.4133.5460.2943.8880.3343.76