脂肪酶酶活检测MOA英文.docVIP

MOA for Lipase 1. 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 Definition 4.1.1 Lipase Lipase is an enzyme that hydrolyzes triglyceride or fatty acid ester 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 Activity One unit of lipase activity refers to1μmol of fatty acid released in 1 minute by 1g of solid lipase (or 1ml of liquid lipase) under 36℃ with a pH value of 9.4, shown as 1 u/ml, or 1 u/g. 4.2 Mechanism Under 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 titration) and the lipase activity can be denoted by the NaOH amount consumed. The equation is as follows: RCOOH+ NaOH → RCOONa +H2O 4.3 Apparatus 4.3.1 Digital thermostatic oscillator water bath, with precision of ±0.2℃ 4.3.2 Automatic pipettor 4.3.3 High speed disperser 4.3.4 pH meter: precision of 0.01pH unit 4.3.5 Magnetic stirring apparatus 4.3.6 Electronic balance 4.3.7 Micro-burette, scale of 20ml and minor tick of 0.1ml. 4.4 Reagents and Solutions Only analytical pure and distilled water, or deionized water or other kinds of water with equal purity can be used in the analysis, unless specified otherwise. 4.4.1 Polyvinyl alcohol (PVA), with a polymerization degree of 1750?0; 4.4.2 Olive oil: chemical pure; 4.4.3 95% ethanol; 4.4.4 Gly-NaOH buffer solution (pH=9.4) 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 solution (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