酶的分離工程ppt課件-文庫吧資料
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【正文】 。 ? heat。 solubility increase! Salting out (Can be used for fractionation) Beyond a certain ionic strength（ ） , the charged molecules are quickly precipitated because the excess ions (not bound to the protein) pete with proteins for the solvent. Saltingout effect: ions take ?all‘ water, expose the nonpolar surface。Chapter 4 Purification of Enzymes Proteins are very diverse. They differ by size, shape, charge, hydrophobicity, and their affinity for other molecules. All these properties can be exploited to separate them from one another so that they can be studied individually. Objectives ?Purity ?Stable ?Cost ?Time Purification typically involves three steps 1) Preparation of a crude extract from harvested cells 2) Fractionation: Separation of a mixture of proteins into various fractions according to some property (. size, charge, solubility) 3) Separation of protein from solvents and concentration Unit 1 Preparation of Crude Enzymes Endoenzyme: intracellular Most enzymes of the metabolic pathways. Exoenzyme: extracellular Break down (hydrolyze) large food molecules or harmful chemicals. Example: cellulase, amylase, penicillinase. Solid/Liquid Separation When harvesting broth cultures, how are cells separated from the broth? Decanter Centrifuge Clarified liquid Rotating Bowl Rotating scroll Frame Filter How to improve filter velocity? 1） Flocculation and Agglomeration 2） Decrease viscosity 3） Filter aid Cell Disruption The main ponent of cell wall ? Bacteria : Peptidoglycan ? Yeast： Dextran， Mannose， Protein ? Mycelial fungus: Chitin, Dextran ? Gram Positive Bacterial Cell Wall Gram Negative Cell Wall Fungus Cell Wall Mechanical methods Grinding ( in liquid nitrogen, ball mill ）－ Dry way Homogenization （ mortar , homogenizer) Wet way Physical methods temperature difference （ freezing and thawing ） pressure difference( osmotic shock) ultrasonication Chemical treatment anic solvents detergents： Triton X100， Tween （ used if enzyme is in lipid membrane ） Enzyme lysis autolysis extra enzyme Ways to break cell Bead Mill Cascading beads Cells being disrupted Rolling beads Sonicator Sonicator Disrupts tissue by creating vibrations which cause mechanical shearing of the cell wall. Cell suspension Ultrasound tip Ultrasound generator After breaking the cell…… 1) Keep temperature low 2) Purify as soon as possible 3) Avoid oxidation 4) Avoid contamination Cooling and protease inhibition are important to recover the enzyme! Enzyme Extraction ? From plant and animal tissue. ? To achieve maximum solubility and activity of the enzyme. Extract methods Solvent or Solution extract target salting liquid ～ NaCl solution acid solution pH2～ 6 aqueous solution alkali solution pH8～ 12 aqueous solution anic solvent watermiscible anic solvent Methods for Extraction of Enzymes Unit 2 Methods of Purification Centrifugation ? Preparative centrifugation ? Analytical centrifugation Preparative centrifugation ? Collect material ? cells ? precipitated macromolecules ? Subcellular fractionation Analytical Centrifugation Sedimentation Coefficient (s) ? is the velocity per Fc, or s = v/ω2r ? unit is Svedberg ,where 1 S = 1013 sec Relative Centrifugal Force and Rotation Per Minute ? e
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