测酶活方法（Enzyme measuring method）.doc

测酶活方法（Enzyme measuring method） Determination of fresh litter and soil enzyme activity Application of Bob and Sinsabaugh - Lab, 1994 Steven Allison, 2001 1) remove the litter bag / samples of soil from the freezer compartment from the refrigerator, put the sample into 2 parts. 2) 1 copies for weighing, recording quality, and then put into the kraft paper bag or the aluminum box into the soil, 70-150 drying to constant weight (2 days), said dry weight. 3) the other 1 weigh about 2g (wet weight), quality records accurately, into the jar of small (blender Mini-jar) [] can use the flask, adding 60ml acetate buffer 1min, stirring with a magnetic stirrer (stem words stir 2min). The mixture is mixed into the tagged bottle. 4) substrate solution preparation: 1.0N NaOH: 40gNaOH 1L solution dissolved in deionized water (pure water, DI water) 50mM PH=5 acetate buffer: 4.374g sodium acetate trihydrate +1.1ml acetic acid dissolved in deionized water (pure water, 1L DI water (pH) acetate regulation by ice) Note: the purpose is to determine the index in the substrate solution saturated (conduct assay). Overall, 5mM substrate solution is sufficient, but because the samples are not familiar with or in suspension or not in the training process is not fully mixed, so it is necessary to use a high concentration of substrate solution to ensure that zero order kinetics. Determination of indicators Enzyme activity index substrate Phosphatase (phosphatase) 5mM of two sodium phosphate (pNP-phosphate) nitrobenzene 186.5mg /100 ml Cellulase (cellobiohydrolase; CBH) 2 mM of nitrobenzene in two sugar glucoside (pNP-cellobioside) fiber 92.7 mg/100ml B- glucosidase (B-glucosidase; BG) 5mM -B- of nitrobenzene glucoside (pNP-B-glucopyranoside) 150.7 mg/100ml B-N- Acetylglucosaminidase (B-N-acetylglucosaminidase; NAG) 2 mM B - N- of nitrobenzene acetylglucosamine glucoside (pNP-B-N-acetylglucosaminide) 68.5mg/100ml Polyphenol oxidase (polyphenol oxidase; PPOD) 5mM levodopa (L-DOPA) 98.6mg/100ml Perox