实验---纤维素酶活力的测定(共14页).doc
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1、精选优质文档-倾情为你奉上实验 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,2
2、00g酒石酸钾钠,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
3、取0.0,0.20,0.40,0.60,0.80,1.00m 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
4、25mL试管中各加入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 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
5、单位(g / mgmin)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 determinat
6、ion of reducing 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 am
7、ino to form orange 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
8、(6) electric 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 r
9、edistilled 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 use.(2) 0.1mol/L pH4.5 acetic acid-sodium acetate buffer solution.(3) 0.5%CMC-Na liquid: Weigh
10、0.50g sodium 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
11、 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 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, respect
12、ively. 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 optic
13、al density as 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,
14、 dilute with 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 s
15、ubsequent operation 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 th
16、e 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 glucose standard curve to get glucose g number
17、of samples5. Results CalculationUnder the above conditions, 1 mg of cellulase catalyzes hydrolysis of cellulose to form 1 microgram glucose within 1 minute is defined as a unit of activity.Activity of Cellulase Units (g/mgmin)= N Dilution multiple of enzyme liquid, here is 10030 mashing time used, m
18、in1 mL number of enzyme liquid 6. Notice(1) whether standard or sample solution, the other ingredients except glucose should be removed to elimilate the influence effectly. The standard curve obtained should go through the origin of coordinates.(2) When using pipettes to add reagents, dont mix pipet
19、tes with pipette tips. 实验 食品中黄酮含量的测定-可见分光光度法一、 实验目的掌握可见分光光度法测定食品中总黄酮含量的方法。二、实验原理黄酮类化合物中的酚羟基能与Al3+ 的碱性溶液中生成红色络合物,其颜色深浅与黄酮含量成正比,在510 nm波长处有最大吸收,故可比色测定。三、适用范围适用于食品中总黄酮含量的测定。四、仪器及试剂1. 仪器:可见分光光度计,分析天平,10 mL 比色管,容量瓶、移液管等。2. 试剂:10亚硝酸钠溶液,10硝酸铝溶液,1molL氢氧化钠溶液,乙醇,芦丁标准品等。五、实验方法1.标准溶液的制备精密称取在120减压干燥至恒重的芦丁标准品100
20、mg,置100mL容量瓶中,加甲醇70mL,置水浴上微热使溶解,放冷,加甲醇至刻度,摇匀。精密吸取10mL,置100mL容量瓶中,加水至度,摇匀,即得0.1mg/ mL芦丁。2.标准曲线的制备准确吸取芦丁标准溶液(0.1mgmL) 0.0mL、1.0mL、2.0mL、3.0mL、4.0mL、5.0mL,分别置于10mL比色管中,各加30乙醇使成5.0mL,各准确加入10亚硝酸钠溶液0.3mL,充分摇匀,放置6min。再准确加入10硝酸铝溶液0.3mL,充分摇匀,放置6min。各加1 molL氢氧化钠溶液4.0mL,用蒸馏水稀释至刻度,充分摇匀,放置15min,用分光光度计,在510nm波长处测
21、定吸光度。以吸光度为纵坐标,浓度为横坐标,绘制标准曲线。 3.试样测定称取一定量的试样置三角瓶中, 精确加入70% 乙醇50 mL, 称定重量, 超声提取30 min, 再称定重量, 加70% 乙醇补足减失重量, 摇匀, 过滤, 取滤液备用。准确吸取滤液(浓度约0.1mgmL) 2mL4mL,置10mL比色管中,按标准曲线制备项下自“各加30乙醇使成5.0mL。“起依法操作直至测定出样品的吸光度。 六、结果计算X=式中: X) 样品中总黄酮的含量( 以芦丁计) , mg/ 100g( mL) ;A) 吸取滤液中黄酮的量,g;m ) 样品的质量, g(mL);F) 样品的稀释倍数。七、说明及注意
22、事项1.可见分光光度法测定黄酮含量,应注意控制反应时间、显色时间以及试剂用量等条件。 实验 双波长法测定混合颜色液中的单色素 一、 实验目的掌握双波长法测定混合颜色液中的单色素及分光光度计的操作使用。二、实验原理双波长测定法是在同一时间内用两个不同波长的光束交替照射同一样品液。其中一个波长为测定波长,另一个为参比波长。并由检测器测出两波长下样品液的吸光度差值A,A与被测物质的浓度成正比。若被测物中含有某种干扰物,但干扰物的最大吸收峰波长与被测物的最大吸收波长之间相差30nm以上,则可用作图法求出适当的测参比波长对,以消除干扰组分的影响。三、仪器与试剂 1. 仪器:分光光度计(带自动扫描功能)
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