生物化学原理教学（杨荣武）Chapter13 Enzyme applications and research methods.pptVIP

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生物化学原理教学（杨荣武）Chapter13 Enzyme applications and research methods.ppt

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Chapter 13 Enzyme applications and research methods Outline Assay of enzyme activities Methods for expressing enzyme activities Methods for determining enzyme activities Separation and purification of enzymes (can be skipped) Enzyme engineering (self-study) Enzyme activity Enzyme activity = moles of substrate converted per unit time = rate × reaction volume. Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified. The SI (The International System ) unit is the katal, 1 katal = 1 mol s?1, but this is an excessively large unit. A more practical and commonly used value is 1 enzyme unit (U) = 1μmol min?1. 1 U = 16.67 nanokatals and 1 kat = 6×107U Specific activity The specific activity of an enzyme is the activity of an enzyme per milligram of total protein (expressed in μmol min?1mg?1). Specific activity gives a measurement of the activity of the enzyme. It is the amount of product formed by an enzyme in a given amount of time under given conditions per milligram of total protein. Specific activity is equal to the rate of reaction multiplied by the volume of reaction divided by the mass of total protein. The SI unit is katal kg?1, but a more practical unit is μmol mg?1 min?1. Enzyme activity assay All enzyme assays measure either the consumption of substrate or production of product over time Enzyme assay conditions A large excess of the substrate Initial velocity Optimal reaction conditions (optimal pH, optimal temperature, optimal ionic strength, etc). Assay methods Direct assay Indirect assay Coupled assay Enzyme purification is extraction of a single enzyme/protein from e.g. cells, tissue, etc. which may contain more than 1000 different proteins and lots of other biomolecules Keep structure and activity intact Each enzyme requires a specific strategy for purification How is purification measured ? Select the sources Establish the essay method Design the strategy Preparing extracts f