实验 纤维素酶活力的测定（3,5-二硝基水酸法）
一、实验目的
掌握还原糖的测定原理，学习用3,5-二硝基水酸法测定纤维素酶活力的法。
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. 3,5-二硝基水酸显色液：称取10.0 g 3,5-二硝基水酸，溶入200mL蒸馏水中，加入20g分
析纯氢氧化钠，200g酒酸钾钠，加水至500mL，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，放置一后使用。
2. 0.1mol/L pH4.5乙酸-乙酸钠缓冲溶液。
3.jupiter 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0.0，0.20，0.40，0.60，0.80，1.00m L 葡萄糖标准液于6支25mLsuffer比色管中，均用蒸馏水稀释至1mL，加3.5-二硝基水酸显色剂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μg数为横坐标，绘出标准曲线。
序号 | 1 | 2 | 3 | 4 | 5 | 6 |
葡萄糖标液 | 0.0 | 0.20 | 0.40 | 0.60 | 决定的英文0.80 | 1.00 |
蒸馏水 | 1.0 | 0.80 | 0.60 | 0.40 | 0.20 | 0.0 |
3,5-二硝基水酸 | 3.0 | 3.0 | 3.0 | 3.0 | 3.0 | 3.0 |
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A550nm | 0.0 | | | | | |
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2.空白管的测定： 在2支25mL试管中各加入school是什么意思英语1.0mL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 bi是什么意思mL 3,5-二硝基水酸显色液，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μg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（μg / mg·min）＝
式中：N—酶液的稀释倍数，此处为100
30—糖化所用时间，min
1—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.人力资源实操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 Cellula Activity
1. Purpo of Experiment
Master the principle of determination of reducing sugar,Learn the method of determinating cellula activity using 3,5-dinitro salicylic acid method.
2. Experimental Principle
Cellula can hydrolyze cellulo to produce reducing sugar such as cellobio and gluco. Tho sugars can reduce nitro of 3,5- dinitro salicylic acid into amino to form orange yellow amino compounds, so colorimetric method can be ud to determine the reduction product expresd as cellula activity .
3. The Main Instruments and Reagent
3.1 Experimental Instrument
(1) 25mL colorimetric tube type (2).722 spectrophotometer
(3) dropper (4) bath (5) pipette (6) electric furnace
3.2 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 tho reagents. Adding redistilled phenol 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 u.
(2) 0.1mol/L pH4.5 acetic acid-sodium acetate buffer solution.
(3) 0.5%CMC-Na liquid: Weigh 0.50g sodium carboxymethylcellulo, dissolved in water to make a colloidal solution, standing for a night. Shake well before using.
(4) 1.0mg/mL gluco standard solution: weigh 100mg anhydrous gluco dried to constant weight, dissolve in water to100mL.
(5) Cellula liquid: 0.05g enzyme dissolvevolte是什么意思 in 50 mL pH4.5 acetic acid-sodium acetate buffer solution, then suck 1.0mL to a 100mL volumetric flask，dilute with the buffer solution to scale.
4. Experimental Steps
4.1 Standard Curve Drawing:
Adding 0.0, 0.20, 0.40, 0.60, 0.80, 1.00m L gluco standard solution in 6 colorimetric tube of 25mL, respectively. In every 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 ordinate, gluco μg number as abscissa, to draw the standard curve.
Serial Number | 1 | 2 | 3 | 4 | 5 | 6 |
gluco standard solution (mL) | 0.0 | 0.20 | 0.40 | 0.60 | 0.80 | 1.00 |
brow是什么意思Distilled water (mL) | 1.0 | 0.80 | 0.60 | 0.40 | 0.20 | 0.0 |
3,5-dinitro salicylic acid (mL) | 3.0 | 3.0 | 3.0 | 3.0 | 3.0 | 3.0 |
Experiment Operation | Heatingeverythingido 10min in boiling water bath, dilute with water to scale after cooling, shake, colorimetric determination |
Absorbance A550nm | 0.0 | | | | | |
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4.2 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 subquent operation is same as sample.
4.3. Determination of Sample:
In each of 3 25mL tubes adding 0.5% CMC-Na liquid 3.0mL, cellula 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, mixing. Set empty tube zero, determine OD value at 550nm. Check on gluco standard curve to get gluco μg number of samples
