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1、生化工程第三章 固定化酶反应动力学第三章 固定化酶反应动力学Immobilized Enzyme Systems DefinitionThe restriction of enzyme mobility in a fixed space is known as enzyme immobilization. In general, immobilized enzymes are enzymes that are attached to, or entrapped within, a macro- scopic support matrix so that the resulting cataly
2、st can be reused.Two opposite conceptImmobilized enzymeFree enzyme3.1 Advantages of Immobilized Enzymes3.1 Advantages of Immobilized EnzymesAdvantages: 1. Enzyme reutilization: catalyst reuse;2. Elimination of enzyme recovery and purification processes: product purity , while effluent handling probl
3、ems ;3. Provide a better environment for enzyme activity;Disadvantages: Increase the diffusion resistance, so decreases the reaction rate.3.2 Applications of Immobilized Enzymes3.2 Applications of Immobilized EnzymesImmobilized enzyme are employed in many fields.Analytical devices:For example: in im
4、mobilized enzyme electrodes for the detection of various chemical substances. As catalyst in industrial processes:Among 2000 types of enzymes found, only 50 60 have been applied in industry, most of which are extracellular enzymes.3.3 Methods of Immobilization3.3 Methods of ImmobilizationImmobilized
5、 Soluble EnzymesMembrane- EntrappedBetween Macroscopic MembranesMicro- EncapsulatedCovalently- BoundTo SupportTo EnzymeEntrappedBoundAdsorbed (Physical or Ionic)Matrix- EntrappedEntrapment: The physical enclosure of enzymes in a small space.Ploymeric materials: Ca-alginate, agar, -carrageenin, polya
6、crylamide, collagen;Solid matrices: activated carbon, porous ceramic, diatomaceous earth.uMatrix entrapmentMatrices used for enzyme immobilization are usually:When immobilized in a polymer matrix, enzyme solution is mixed with polymer solution before polymerization takes place.uMembrane entrapmentMe
7、mbrane: nylon, cellulose, polysulfone, polyacrylate.In all cases, a semi-permeable membrane is used to retain high-MW compounds (enzyme) while allowing small-MW compounds (substrates or products) access to the enzyme.In this technique, microscopic hollow spheres are formed. The spheres contain the e
8、nzyme solution, while the sphere is enclosed with a porous membrane.Microencapsulation: a special form of mem- brane entrapment.Problems:1. Leakage of enzymes into solution:2. Considerable diffusional resistance emerges:Reducing the particle size of the matrices and/or capsules;3. Reduction of enzym
9、e activity and stability:A little difficult to handle.4. Lack of control of the microenvironment:Alter the unfavorable microenvironmental conditions;Reducing the MW cutoff of membranes or the pore size of solid matrices;Bound:Inorganic materials: alumina, silica, porous glass, ceramics, diatomaceous
10、 earth;Organic materials: cellulose (CMC, DEAE-cellulose), starch, activated carbon, ion-exchange resin (Amberlite, Sephadex, Dowex).uAdsorptionA physical attachment of enzyme on the surfaces of support particles by weak physical forces: van der Waals.Support materials:Desorption is quit common beca
11、use the binding forces are so week, especially in the presence of strong hydrodynamic forces.Advantages:Normally, active site is not affected, nearly all activity is retained after immobilization.Disadvantages:uCovalent BindingIt is the retention of enzymes on support surfaces by covalent bond forma
12、tion, via certain functional groups, such as amino, carboxyl, hydroxyl, and SH group. The functional groups must not be the active sites.uCross-LinkingThe enzyme molecules can also cross-link with each other. But this may cause significant changes in the active site of enzymes, and also severe diffu
13、sion limitations may result.SUMMARYThe most suitable support material and immobilization method vary depending on the enzyme and particular application.Then how to select support materials ?a. Binding capacity, which is a function of charge density, functional groups, porosity, and hydrophobicity;b.
14、 Stability and retention of the enzyme activity, which is a function of functional groups on support material and microenvironmental conditions.How to select method of immobilization ?CharacteristicCarrier Binding MethodsCross- linking MethodEntrapping Method Physical adsorptionIonic Binding Covalen
15、t BindingPreparationEasyEasyDifficultDifficultDifficultEnzyme ActivityLowHighHighModerateHighSubstrate SpecificityUnchangeableUnchangeableChangeableChangeableUnchangeableBinding ForceWeakModerateStrongStrongStrongRegenerationPossiblePossibleImpossibleImpossibleImpossibleGeneral ApplicationLowModerat
16、eModerateLowHighCost of ImmobilizationLowLowHighModerateLowTable: Comparison of the Characteristics of Different Methods of Enzyme Immobilization3.4 Immobilized Enzyme KineticsBioreactor kineticsIntrinsic kinetics (本征反应动力学)Mass transfer of substrate and productIllustration of Mass Transport and Chemical Reaction Interactions in a Symmetric Slab of Immobilized Enzyme.PHENOMENAPARAMETERSBulk FluidSt
