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Purpose of enzyme immobilization
Economic puropose Enzymes can be reused Recovery of product is easier Choice of immobilization
7 pH 7. Adsorption Simple method and high enzyme loading Incubating supporter with enzymes in an appropriate pH and ionic strength Driving force is hydrophobic intxn and salt bridge DEAE-Sephadex anion exchanger 0 100 100 CM-Sephadex cation exchanger 100 75 34 % bound at pH 2. 3.5 pH 4. Absorption Covalent linkage Enzyme entrapment Encapsulation of enzyme 1. 4.Methods of immobilization 1. 2.0 Preparation of immobilised invertase by adsorption 2 .
~ ++ Exposure ++ ++ ± ++ ++ ++ + ± ++ ++ ++ Reactivity + ++ ± + + ++ ± ± ± + + ++ + Stability of couple + ± ± + + ++ + + ± _+ + ++ + .~ ++ Use + + + ++ ± ± + + + ± - 2. CNBr method CNBr. Covalent coupling Residue Aspartate Arginine Cysteine Cystine Glutamate Histidine Lysine Methionine Serine Threonine Tryptophan Tyrosine C terminus N terminus Carbohydrate Others Content + + + + ± ++ ++ ++ + . Covalent coupling Maximum 0.~ ++ .activated Sepharose 3 .2.2 g enzyme/g matrix Very little leakage The most common methods in laboratory scale 1.
carbodiimide 2. ethyl chloroformate : less toxic than CNBr 3. Covalent coupling 2. Covalent coupling 4. glutaraldehyde 4 .2.
3-aminopropyltriethoxysilane 3. Covalent coupling 5.2. easy to use and convenient for any kinds of enzymes Liposome can be used for this purpose 5 . Entrapment More than 1 g enzyme/ 1 g gel Only for small substrate and product Enzyme can be covalently linked to acrylamide gel 4. Membrane confinement Hollow fiber type of membrane can be used Although expensive.
Comparison between the methods Covalent binding Difficult High Strong No Selective Low Yes No No Membrane confinement Simple High Strong No Very wide High No Yes Yes Characteristics Preparation Cost Binding force Enzyme leakage Applicability Running Problems Matrix effects Large diffusional barriers Microbial protection Adsorption Simple Low Variable Yes Wide High Yes No No Entrapment Difficult Moderate Weak Yes Wide High Yes Yes Yes Immobilization enhances the stability of the enzyme Covalently linked Adsorption Acryloyl chloride Activities of the chymotrypsin at 60 °C 6 .
diffusion rare is limiting If Da<<1. but into 10 as immobilized form) Kinetics of immobilized enzyme in nonporous solid support Assuming steady state. J s = k L ([ S 0 ] − [ S ]) = Vmax [ S ] K m + [S ] Michaelis-Menten eqn is defined in moles per unit time per unit area Defining dimensionless Damköhler number Da = Vmax k L [ S0 ] Maximum reaction rate/maximum diffusion rate If Da>>1. reaction rate is limiting 7 .Kinetics of immobilized enzyme Km and Vmax are changed Specificity can be changed (trypsin hydrolyze pepsinogen into 15 fragments in solution.
Kinetics of immobilized enzyme in nonporous solid support Free enzyme Immobilized enzyme The slope = kL Kinetics of immobilized enzyme in nonporous solid support As kL decreases. 8 .
Kinetics of immobilized enzyme in porous matrix Assuming steady state. V [S ] d 2[S ] 2 d[S ] J s = De ( + ) = max 2 dr r dr K m + [S ] Assuming that no external diffusion limitation Michaelis-Menten eqn is defined in moles per unit time per unit volume Defining dimensionless numbers d2S dr Where 2 + R 2Vm S S 2 dS = =φ2 [ S 0 ]De β + S r dr β +S S= [S ] r K . 9 .β = m [S0 ] R [S0 ] Kinetics of immobilized enzyme in porous matrix As φ increases.r = .
Kinetics of immobilized enzyme in porous matrix η (effectiveness factor) is defined as the rate with diffusion limitation versus the rate w/o diffusion limitation Kinetics of immobilized enzyme in porous matrix 10 .