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s090604054铁杆木虫 (著名写手)
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The determined method of dextran activity 1. Method 2 mL 3% dextran 70 KDa solution which prepared by pH 5.0 acetate buffer solutions is insulated 10 minutes at 35 ℃. Then 0.5 mL diluted enzyme solution is added into the dextran solution and insulated 1 h. The reducing sugars are determined by 3, 5-dinitrosalicylic acid (DNS) method. One enzyme activity unit (u) is the amount of enzyme corresponding to produce 1 μmol reducing sugar (equivalent to glucose) per minute under the above conditions. 2. Determined steps 2.1 Treatment method of control sample Ⅰ. The treatment of zymotic fluid: zymotic fluid is centrifugalled for 10 min at 4000 rpm. 1 mL supernatant is drawn and diluted 20-400 times with deionized water, and denoted as 1# solution. Ⅱ. 2 mL 3% dextran 70 KDa solution is added into a 10 mL centrifuge tube, and then kept 35 ℃ for 10 min, denoted as 2# solution. Ⅲ. 375 μL DNS solution is drawn into 20 mL cuvette with a 1000 μL finnpipette, and denoted as 3# solution. Ⅳ. 0.5 mL 1# solution is added into 2# solution and stirred well，and denoted 4# solution. Ⅴ. 0.5 mL 4# solution is added into 3# solution, denoted 5# solution. Ⅵ. The rest 4#solution is kept at 35 ℃ for 1h, and denoted 6# solution. Ⅶ. 5# solution is mixed thoroughly and kept at 100 ℃, the enzyme has been inactivated for 5 minutes, then cooled immediately in cold water, and 5.375 mL deionized water is added and stirred well, and denoted 7#. The 7# solution is used as a blank-control solution. 2.2 Treatment method of fermentation sample Ⅰ. 375 μL DNS solution is drawn into 20 mL cuvette with pipette, and denoted 8# solution. Ⅱ. 0.5 mL 6# solution is introduced into 8# solution and stirred well, then keep at 100 ℃ for 5 minters, when the enzyme has been inactivated, cooled immediately in cold water, and 5.375 mL deionized water is added and stirred well, and denoted 9# solution. 2.3 Detection of sample The spectrophotometer is preheated for 20 min. The 7# solution is used as reference, 9 # solution is detected. Each sample is measured at 540 nm for three times. 2.4 Calculation of enzyme activity Formula: enzyme activity (u/mL) = (absorbance / 0.6023)* dilution Ratio/ 60/ 180* 1000*10 2.5 Explanation of the formula Dilution Ratio is the dilution ratio of zymotic fluid，which depends on the enzyme activity unit, and the absorbance is less than 1; 0.6023 is calculated constant. 60 is reaction time of enzyme. 180 is the molecular weight of glucose. 1000 is the conversion between mole and micromole; 10 is the conversion between 0.1mL of enzyme solution and 1mL of enzyme solution. |
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