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1、MOA for Lipase1. Aim: Set up a standard operating procedure for lipase activity assay.2. Range： Specified the assay method of lipase activity.3. Responsible By: All staff of the QC department.4. Content:4.1 Term & Definition4.1.1 LipaseLipase is an enzyme that hydrolyzes triglyceride or fatty acid e
2、ster into fatty acid, diglyceride or monoglyceride, or hydrolyzes natural oil into fatty acid and glycerin, as well as catalyzing the synthesis and interchange of ester.4.1.2 Lipase ActivityOne unit of lipase activity refers to1 ymol of fatty acid released in 1 minute by 1g of solid lipase (or 1ml o
3、f liquid lipase) under 36C with a pH value of 9.4, shown as 1 u/ml, or 1 u/g.4.2 MechanismUnder definite condition, lipase can hydrolyze glyceride into fatty acid, diglyceride, monoglyceride and glycerin. The released fatty acid amount can be measured via standard alkaline solution titration (NaOH t
4、itration) and the lipase activity can be denoted by the NaOH amount consumed. The equation is as follows:RCOOH+ NaOH f RCOONa +H2O4.3 Apparatus4.3.1 Digital thermostatic oscillator water bath, with precision of 0.2C4.3.2 Automatic pipettor4.3.3 High speed disperser4.3.4 pH meter: precision of 0.01pH
5、 unit4.3.5 Magnetic stirring apparatus4.3.6 Electronic balance4.3.7 Micro-burette, scale of 20ml and minor tick of 0.1ml.4.4 Reagents and SolutionsOnly analytical pure and distilled water, or deionized water or other kinds of water with equal purity can be used in the analysis, unless specified othe
6、rwise.4.4.1 Polyvinyl alcohol (PVA), with a polymerization degree of 1750 50;4.4.2 Olive oil: chemical pure;4.4.3 95% ethanol;4.4.4 Gly-NaOH buffer solution (pH=94)Solution A: 0.2 mol/L NaOH solution (Weigh 8.0g NaOH and make the volume to 1000ml with distilled water);Solution B: 0.2mol/L glycine so
7、lution (Weigh 15.014g glycine and make the volume to 1000ml with distilled water);Gly-NaOH buffer: Mix 16.8ml of solution A and 50ml of solution B to have a constant volume of 200ml with distilled water. Adjust the pH value to 9.4.4.4.5 Substrate solutionStep 1: Weigh 40g of polyvinyl alcohol (PVA)
8、with a precision of 0.1g and add 1000ml of Gly-NaOH buffer solution (pH=9.4). Put them into the thermostatic oscillator and stir until dissolved completely. Then have a constant volume of 1000ml after cooling it down. Filter the solution with double gauze and the filtrate is for later use.Step 2: Me
9、asure 60ml of the above filtrate and add 30ml of olive oil. Disperse the solution with a high speed disperser for 12 minutes (4 treatments with 3 minutes each, every interval of 6 minutes) to get the emulsion solution, which is only for current use.4.4.6 Standard NaOH solution (0.01 mol/L)4.5 Analys
10、is Procedure4.5.1 Enzyme solution preparationStep 1: Dissolve 1.000g of lipase powder with 80ml of Gly-NaOH buffer (4.4.4) and stir with a magnetic stirring apparatus for 10 minutes. Transfer it to a 100ml volumetric flask to make a constant volume of 100ml with Gly-NaOH buffer (AVOID BUBLE), and th
11、en rest for 10 minutes.Step 2: The enzyme samples shall be taken by its supernatant after being shaken well in the later measurement.4.5.2 DeterminationStep 1: Calibrate the apparatus with the pH meter.Step 2: Take three triangular flasks (100ml each), one as Blank and another two as Test #1 andTest
12、 #2 (for parallel experiment). Add 4.00ml of Gly-NaOH buffer (4.4.4) and 5.0 ml of substrate solution (4.4.5) emulsified into each of the three flasks. Preheat them in the thermostatic oscillator under 36C 0.2C for 5 minutes.Step 3: For flask Test #1 and Test #2, measure 1.0ml of enzyme solution (4.
13、5.1) in two test tubes and transfer them into each flask, shake well immediately and then start timing. Shake lightly in the thermostatic oscillator (80 times per min) for 10 minutes for the enzyme to react.Step 4: Add 20ml of 95% ethanol (4.4.3) into flask Test #1 and Test #2 to terminate reaction.
14、 Step5: Meanwhile, for flask Blank, measure 1.0ml of enzyme solution (4.5.1) in a test tube and add 20ml of 95% ethanol (4.4.3) to terminate reaction. Then, transfer the inactivated solution into flask Blank and shake it well.Step 6: Rest the three flasks for 5 minutes, add 10ml of saturated NaCl so
15、lution into each flask and shake well.Step 7: Titrate Test 1# and Test 2# with Standard NaOH solution (0.01 mol/L) (4.4.6) till the pH value is the same as the Blank. The titration should be finished within 10 minutes. Record the volume of NaOH solution consumed.The NaOH solution consumed shall be i
16、n the range of 4.5ml5.5ml for each time of titration. Once the NaOH consumption is beyond the range, we shall go back to Enzyme solution preparation (4.5.1) to adjust the dilution times.4.5.3 Calcula tionThe calculation formula is as follows:v x c x n x w x 1000X =-10 x mWhere,X, refers to the activity of testing enzyme sample, shown in u/g or u/ml;v , refers to the titration v
