《实验---纤维素酶活力的测定》由会员分享，可在线阅读，更多相关《实验---纤维素酶活力的测定（14页珍藏版）》请在金锄头文库上搜索。
1、实验 纤维素酶活力的测定（3,5-二硝基水杨酸法）一、实验目的掌握还原糖的测定原理，学习用3,5-二硝基水杨酸法测定纤维素酶活力的方法。 二、实验原理纤维素酶水解纤维素，产生纤维二糖、葡萄糖等还原糖，能将3,5-二硝基水杨酸中的硝基还原成橙黄色的氨基化合物，故可利用比色法测定其还原物生成量来表示纤维素酶的活力。三、主要仪器与试剂(一)实验仪器1. 25mL比色管 2. 722型分光光度计 3. 滴管 4.水浴锅 5.移液枪 6.电炉(二)、试剂1. 3,5-二硝基水杨酸显色液：称取10.0 g 3,5-二硝基水杨酸，溶入200mL蒸馏水中，加入20g分析纯氢氧化钠，200g酒石酸钾钠，加水至5
2、00mL，升温溶解后，加入重蒸苯酚2.0g，无水亚硫酸钠0.50g。加热搅拌，待全溶后冷却，定容至1000mL。存于棕色瓶中，放置一周后使用。2. 0.1mol/L pH4.5乙酸-乙酸钠缓冲溶液。3. 0.5%羧甲基纤维素钠水溶液，溶解后成胶状液，静置过夜。使用前摇匀。4. 葡萄糖标准溶液：称取干燥至恒重的无水葡萄糖100mg，溶解后定容至100mL， 此溶液含葡萄糖1.00mg/mL。5. 纤维素酶液：将0.05g酶溶解定容至50 mL，从中取出1.0mL再定容至100mL，待检测用。（用pH4.5乙酸-乙酸钠缓冲溶液配制） 四、实验步骤1.标准曲线的绘制：分别吸取0.0，0.20，0.4
3、0，0.60，0.80，1.00m L 葡萄糖标准液于6支25mL比色管中，均用蒸馏水稀释至1mL，加3.5-二硝基水杨酸显色剂3mL，在沸水浴中煮沸显色10min，冷却，加蒸馏水21mL，摇匀。以空白管调零，在550nm处比色。以光密度为纵坐标，以葡萄糖g数为横坐标，绘出标准曲线。序号123456葡萄糖标液0.00.200.400.600.801.00蒸馏水1.00.800.600.400.200.03,5-二硝基水杨酸3.03.03.03.03.03.0实验操作沸水浴加热10min，冷却后，加水定容，摇匀，比色测定吸光度A550nm0.02.空白管的测定： 在2支25mL试管中各加入1.0
4、mL酶液，沸水浴5min，冷却后加3.0mL 0.5%CMC-Na，与样品管同时放入50水浴30min。其它操作同样品管。3.样品的测定：在3支25mL试管中各加入0.5%羧甲基纤维素钠溶液3.0mL，酶液1.0mL，于50水浴中糖化30min，取出，立即于沸水浴中煮沸10min使酶失活，得糖化液，冷却加入3.0 mL 3,5-二硝基水杨酸显色液，再沸水浴10min，冷却后加水定容至25mL，混匀，以空白管调零，在550nm处测OD值，查葡萄糖标准曲线得样品的葡萄糖g数。 五、结果计算在上述条件下，1酶每分钟催化纤维素水解生成1微克葡萄糖定为一个活力单位。纤维素酶活力单位（g / mgmin）
5、式中：N酶液的稀释倍数，此处为10030糖化所用时间，min1反应酶液的mL数六、注意事项1.无论是标准液还是样品液，都要去除葡萄糖外的其他各种成分的对OD值的影响。使得到的标准曲线经过坐标原点。2.用移液管或移液枪加各试剂时，不能将移液管或移液枪头混用。各比色管中，均用蒸馏水稀释至1mL，加3.5-二硝基水杨酸显色剂3mL，在沸水浴中煮沸显色10min，冷却，加蒸馏水21mL，摇匀。Determination of Cellulase Activity1. Purpose of ExperimentMaster the principle of determination of reduci
6、ng sugar,Learn the method of determinating cellulase activity using 3,5-dinitro salicylic acid method.2. Experimental PrincipleCellulase can hydrolyze cellulose to produce reducing sugar such as cellobiose and glucose. Those sugars can reduce nitro of 3,5- dinitro salicylic acid into amino to form o
7、range yellow amino compounds, so colorimetric method can be used to determine the reduction product expressed as cellulase activity .3. The Main Instruments and Reagent3.1 Experimental Instrument (1) 25mL colorimetric tube type (2).722 spectrophotometer (3) dropper (4) bath (5) pipette (6) electric
8、furnace3.2 Reagent(1) 3,5- dinitro salicylic acid solution: Weigh 10 g 3,5- two nitro salicylic acid, dissolved in 200mL of distilled water, adding 20g sodium hydroxide of analytically pure, 200g sodium potassium tartrate, add water to 500mL, heating to dissolve those reagents. Adding redistilled ph
9、enol 2.0g, sodium sulfite anhydrous 0.50g. Heating and stirring. When the reagents are fully dissolved, cooling. Dilute with water to 1000mL. Stored in a brown bottle, lay aside a week before use.(2) 0.1mol/L pH4.5 acetic acid-sodium acetate buffer solution.(3) 0.5%CMC-Na liquid: Weigh 0.50g sodium
10、carboxymethylcellulose, dissolved in water to make a colloidal solution, standing for a night. Shake well before using.(4) 1.0mg/mL glucose standard solution: weigh 100mg anhydrous glucose dried to constant weight, dissolve in water to100mL.(5) Cellulase liquid: 0.05g enzyme dissolve in 50 mL pH4.5
11、acetic acid-sodium acetate buffer solution, then suck 1.0mL to a 100mL volumetric flask，dilute with the buffer solution to scale.4. Experimental Steps4.1 Standard Curve Drawing: Adding 0.0, 0.20, 0.40, 0.60, 0.80, 1.00m L glucose standard solution in 6 colorimetric tube of 25mL, respectively. In eve
12、ry colorimetric tube, adding distilled water until 1.0mL,then adding 3.5- dinitro salicylic acid solution 3.0mL, boiled in boiling water bath 10min for color developing, then cooling, add 21mL of distilled water, shaking. Set empty tube zero, determine OD value at 550nm. Using the optical density as
13、 ordinate, glucose g number as abscissa, to draw the standard curve.Serial Number123456glucose standard solution (mL)0.00.200.400.600.801.00Distilled water (mL)1.00.800.600.400.200.03,5-dinitro salicylic acid (mL)3.03.03.03.03.03.0Experiment OperationHeating 10min in boiling water bath, dilute with
14、water to scale after cooling, shake, colorimetric determinationAbsorbance A550nm0.04.2 Determination of Blank: Adding 1.0mL enzyme liquid in each of the 2 tubes of 25mL. put in boiling water bath 5min, after cooling add 3.0mL 0.5%CMC-Na liquid, then put in 50 water bath for 30min. The subsequent ope
15、ration is same as sample.4.3. Determination of Sample: In each of 3 25mL tubes adding 0.5% CMC-Na liquid 3.0mL, cellulase liquid 1.0mL, put at 50 water bath exactly 30min for glycosylation. Then remove immediately to put in boiling water bath for 10min to inactivate the enzyme. After the saccharification liquid cooling, add 3.0 mL 3,5- dinitro salicylic acid solution, then put again in boiling water bath 10min to develop color, then cooling, dilute with water to 25mL, mix
