Development of Enzyme Immobilization Technique

Transcription

1 Development of Enzyme Immobilization Technique Professor SEUNG-WOOK KIM Laboratory of Bioprocess Department of Chemical and Biological In this presentation

2 Enzymes Enzymes are are biological biological catalysts. catalysts. They increase increase the the rate rate of of chemical chemical reactions reactions taking taking place place within within living living cells cells without without themselves themselves suffering suffering any any overall overall change. change. The reactants reactants of of enzyme-catalysed enzyme-catalysed reactions reactions are are termed termed substrates substrates and and each each enzyme enzyme on on a particular particular substrate substrate to to produce produce a particular particular product. product. Enzymes are are protein protein molecules molecules Enzyme Action Model

3 Application of Enzyme Advantages Problems Enzyme of high or low specificity can be selected to desired function. Little (or No) by-product formation is observed. Optimal activity occurs under very mild reaction condition. The cost of enzyme preparation is often high Enzyme are intrinsically unstable Enzyme are easily inhibited Substrate or products which has low solubility in aqueous solution can pose difficulties What is an Immobilized Enzyme? An An immobilized enzyme is is one one whose movement in in space has has been restricted either completely or or to to a small limited region. Attachment to to solid structure, incorporation in in gels gels etc etc for for use. use.

4 Benefits of Immobilizing an Enzyme There are a number of advantages to attaching enzymes to a solid support and several major reasons are listed below: Multiple or or repetitive use use of of a single single batch batch of of enzymes The The ability ability to to stop stop the the reaction rapidly rapidly by by removing the the enzyme from from the the reaction solution (or (or vice vice versa) versa) Enzymes are are usually stabilized by by binding Product is is not not contaminated with with the the enzyme (especially useful useful in in the the food food and and pharmaceutical industries) Easy Easy seperationof of enzymefrom the the product Allows Allows development of of a multienzyme reaction system Reduces effluent disposal problems The properties of immobilized enzymes Enzyme Carrier Biochemical properties Reaction type and kinetics Chemical characteristics Mechanical properties Immobilization method Mass-transfer effects Operational stability Performance

5 Selected characteristic parameters of immobilized enzymes Enzyme Biochemical properties Molecular weight, Functional groups on protein surface, Purity (inactivating/protective function of impurities) Enzyme kinetic parameters Specific activity, ph-, temperature profiles, Kinetic parameters for activity and inhibition, Enzyme stability against ph, temp., solvents, contaminants, impurities. Carrier Chemical characteristics Chemical basis and composition, Functional groups, swelling behavior, Accessible volume of matrix and pore size, Chemical stability of carrier Mechanical properties Particle diameter, Single-particle compression behavior, Flow resistance (for fixed bed application), Sedimentation velocity (for fluidized bed), Abrasion(for stirred tanks)

6 Immobilized enzyme Immobilization method Bound protein, Yield of active enzyme, Intrinsic kinetic parameters (properties free of mass transfer effects) Mass transfer effect Buffer effect (different concentrations of solutes inside and out- -side the catalyst particles), External and internal (porous) diffusion Stability Operation stability Storage stability Performance productivity Enzyme consumption Methods for Enzyme Immobilization Methods for Enzyme Immobilization Carrier Binding Cross Linking Entrapment Physical Ionic Covalent Lattice Microcapsule Adsorption Bonding Bonding Type Type

7 Carrier-Binding The carrier-binding method is is the the oldest oldest immobilization technique for for enzymes. In In this this method, the the amount of of enzyme bound bound to to the the carrier carrier and and the the activity after after immobilization depend on on the the nature nature of of the the carrier. The carrier-binding method can can be be further further sub-classified into: into: Physical Adsorption Ionic Ionic Binding Covalent Binding Physical Adsorption Mode This This method is is based based on on the the physical adsorption of of enzyme protein on on the the surface of of water-insoluble carriers. Advantages Disadvantages Little Little or or no no conformational change of of the the enzyme or or destruction of of its its active active center center Usually no no reagents and and only only a minimum of of activation steps steps are are required Simple and and cheap cheap Desorptionof of the the protein resulting from from changes in in temperature, ph, ph, ionic ionic strength Non-specific

8 Ionic Binding Mode This This method relies relies on on the the ionic ionic binding of of the the enzyme protein to to water-insoluble carriers containing ion-exchange residues. Advantages The The conditions are are much much milder milder than than those those needed for for the the covalent binding method. Little Little changes in in the the conformation and and the the active active site site of of the the enzyme. Disadvantages Leakage of of enzymes from from the the carrier carrier may may occur occur in in substrate solution of of high high ionic ionic strength or or upon upon variation of of ph ph High activities in in most most cases. cases. Covalent Binding Mode The The covalent binding method is is based based on on the the binding of of enzymes and and water-insoluble carriers by by covalent bonds. bonds. The functional groups Amino group Carboxyl group Sulfhydryl group, Hydroxyl group Imidazole group Phenolic group Thiol group Threonine group Indole group When trying to select the type of reaction by which a given protein should be immobilized, the choice is limited by two characteristics: (1) the binding reaction must be performed under conditions that do not cause loss of enzyme activity, and (2) the active site of the enzyme must be unaffected by the reagents used.

9 Covalent binding can be brought about by the following ; Diazotization : Amide bond formation : Alkylation and Arylation: Schiff's base formation : Amidation reaction : Thiol-Disulfide interchange : UGI reaction Mercury-Enzyme interchange Gamma-Irradiation induced coupling Carrier binding with bifunctional reagents : SUPPORT--N=N--ENZYME. SUPPORT--CO-NH--ENZYME SUPPORT--CH 2 -NH-ENZYME SUPPORT--CH 2 -S--ENZYME SUPPORT--CH=N--ENZYME SUPPORT--CNH-NH--ENZYME SUPPORT--S-S--ENZYME SUPPORT-O(CH 2 ) 2 N=CH(CH 2 ) 3 CH=N-ENZYME Advantages and Disadvantages of Covalent Binding Method Advantages Covalent binding is is usually thought to to be be stable method by by the the enzyme carrier carrier bond, bond, which which prevents elution of of proteininto into the the production stream. The wide range of of choicesis is possible by by selecting carrier carrier materials and and binding method. This This allows allows a great great deal deal of of flexibilityin in designing an an immobilized enzyme with with specific physical and and chemical properties, such such as as charge charge distribution, hydrophobe/hydrophileration, ration, spacer spacer arm arm separation, partitioning capabilities, etc. etc.

10 Disadvantages Covalent methods are are the the relatively expensive and and complicated in in procedureswhich are are involved. Also, Also, activity yields yields may may be be low low due due to to exposure of of the the enzyme to to harsh harsh environments or or toxic toxic reagent. Active site site may may be be modified through the the chemical reactions used used to to create create covalent bonding. Cross-Linking This This method is is based based on on the the formation of of covalent bonds between enzyme molecules, by by means means of of bi- bi-or or multi- multifunctional reagent, leading to to three- threedimensional crosslinked aggregates.

11 Advantages and Disadvantages Advantages Very little little desorption (Enzyme strongly bound) Cross-linking is is best best used used in in conjunction with with one one of of the the other other methods. It It is is used used mostly mostly as as a means means of of stabilizing adsorbed enzymesand also also for for preventing leakage. Disadvantages Cross-linking may may cause cause significant changes in in the the active active site site of of enzymes, and and also also severe severe diffusion limitationmay may lead lead to to significant loss loss of of activity. Loss Loss of of enzyme activityduring preparation. The most common reagent used for cross-linking is glutaraldehyde

12 The entrapment method of of immobilization is is based based on on the the localization of of an an enzyme within within the the latticeof of a polymer matrix matrix or or membrane. It It can can be be classified into into lattice and and micro capsule types. It is possible to confine the enzyme in a semi-permeable membrane which allows free passage of low molecular weight substrates and products but retains the high molecular weight enzyme. The formation of calcium alginate beads. Membrane Incorporation

13 Types of Reactors Batch reactors may include: Stirred Tank Tank for for Soluble enzymes Stirred Tank Tank for for Immobilized enzymes Stirred Tank Tank with with Immobilized enzyme Basket Basket Paddles Stirred Tank Tank with with Immobilized enzyme Basket Basket Baffles Baffles Total Recycle Packed Bed Bed Reactor Total Recycle Fluidized Bed Bed Reactor Continuous reactors may include: Stirred Tank Reactor with with Filtration Recovery Stirred Tank Reactor with with Settling Tank Recovery Stirred Tank Reactor with with Immobilized Enzyme Basket Paddles Stirred Tank Reactor with with Ultra filtration Recovery Packed Bed Bed Reactor Packed Bed Bed with with recycle Membrane Reactor using Hollow Fibers Flat Bed Bed Reactor Filter Bed Bed Reactor Fluidized Bed Bed Reactor, same but but better design(expanded top top section)

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