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![Page 1: Models for More Complex Enzyme Kinetics Allosteric enzymes - Some enzymes have more than one substrate binding site. - Allostery or cooperative binding:](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649f515503460f94c749a2/html5/thumbnails/1.jpg)
Models for More Complex Enzyme Kinetics
• Allosteric enzymes- Some enzymes have more than one substrate binding site.
- Allostery or cooperative binding:the binding of one substrate to the
enzyme facilitates binding of other substrate molecules.
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Models for More Complex Enzyme Kinetics
The rate expression in this case is
nSmK
nSmV
dt
Sdv
]["
][][
''ln]ln[ln mKSnvmV
v
vmV
v
ln
n is cooperativity coefficient; n>1 indicates positive cooperativity;n can be determined by rearranging the above equation as
And plotting versus ln[S].
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Models for More Complex Enzyme Kinetics
• Insoluble substratewood chips, cellulosic residues
-Access to the active site on these biopolymers by enzyme is limited by enzyme diffusion.
-The number of reaction sites exceeds the number of enzyme molecules.
-This is opposite that of the typical situation with soluble substrates, where access to the enzyme’s active site by substrate limits reaction.
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Models for More Complex Enzyme Kinetics
• Insoluble substrate
If considering- the initial product formation rate and - the reaction is first order in terms of
the enzyme-substrate complex concentration, yields,
][
][max,
EeqK
ESVv
][2max, 0SkSV adskdeskeqK /
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Factors Affecting Enzyme Kinetics • pH effects
- on enzymes
- enzymes have ionic groups on their active sites.
- Variation of pH changes the ionic form of the active sites.
- pH changes the three-Dimensional structure of enzyme.
- on substrate
- some substrates contain ionic groups
- pH affects the ionic form of substrate
affects the affinity of the substrate to the enzyme
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Factors Affecting Enzyme Kinetics • Temperature
- on the rate enzyme catalyzed reaction
k2=A*exp(-Ea/R*T)
T k2
- enzyme denaturation
T
][][
2ESk
dt
Pdv
v
][][
Edkdt
Ed
Denaturation rate:kd=Ad*exp(-Ea/R*T)
kd: enzyme denaturation rate constant;
Ea: deactivation energy
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Immobilized Enzyme Systems• Enzyme immobilization:
To restrict enzyme mobility in a fixed space.
Advantages:- Easy separation from reaction mixture, providing the
ability to control reaction times and minimize the enzymes lost in the product.
- Re-use of enzymes for many reaction cycles, lowering the total production cost of enzyme mediated reactions.
- Ability of enzymes to provide pure products.
- Possible provision of a better environment for enzyme activity
- Study of the action of membrane-bound intracellular enzyme
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Immobilized Enzyme Systems
• Methods of Enzyme Immobilization:
- Entrapment
- Surface immobilization
- Cross-linking
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Entrapment immobilization is based on the localization of an enzyme within the lattice of a polymer matrix or membrane.
- to retain enzyme - allow the penetration of substrate.
It can be classified into matrix and micro capsule types.
Immobilized Enzyme Systems
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Immobilized Enzyme Systems
Entrapment - matrix entrapment - membrane entrapment
(microencapsulation)
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Immobilized Enzyme Systems- matrix entrapment
Matrix materials:
organics: polysaccharides, proteins, carbon, vinyl and allyl polymers, and polyamides. e.g. Ca-alginate, agar,
K-carrageenin, collagenEnzyme + polymer solution → polymerization
→ extrusion/shape the particles
inorganics: activated carbon, porous ceramic and diatomaceous earth.
Shapes: - particle- membrane - fiber
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Immobilized Enzyme Systems- membrane entrapment
- Regular semipermeable membrane: nylon, cellulose, polysulfone and polyacrylate.
- Microencapsulation: Microscopic hollow sphere are formed.The sphere contain the enzyme solution
and is enclosed within a porous membrane.
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Immobilized Enzyme Systems
Entrapment
challenges:- enzyme leakage into solution
- diffusional limitation
- reduced enzyme activity and stability
- lack of control micro-environmental conditions.
It could be improved by modifying matrix or membrane.
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Immobilized Enzyme Systems
Surface immobilization
According to the binding mode of the enzyme, this method can be further sub-classified into:
- Physical Adsorption - Ionic Binding - Covalent Binding
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Immobilized Enzyme SystemsSurface immobilization
Physical Adsorption is based on the physical adsorption of enzyme protein on the surface of water-insoluble carriers.
- If a suitable carrier is found, this method can be both simple and cheap.
- The active sites and activity of enzymes are less affected.
- Desorption of enzyme takes place because of weak attraction.
- Non-specific of other protein or substances will affect the properties of enzyme.
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Immobilized Enzyme SystemsPhysical Adsorption:
Weak forces: Van der Waals or dispersion
Materials:
Inorganic: almumina, silica, porous glass, ceramics.
Organic: e.g. cellulose, starch, activated carbon.
Carbon nano-tube (Kim, Jeong Yun, Special Publication - Royal Society of Chemistry, 2004)
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Immobilized Enzyme Systems
Ionic binding:
Interaction forces: ionic bonds.
Features: similar to that of physical adsorption.
Polysaccharides and synthetic polymers having ion-exchange centers are usually used as carriers.
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Immobilized Enzyme Systems
Covalently binding is the formation of covalent bonds between the enzyme and the support matrix.
Interaction forces: covalent bonds.
Features: - may alter the conformational structure and active
center of the enzyme, resulting in major loss of activity and/or changes of the substrate.
- the binding force between enzyme and carrier is so strong that no leakage of the enzymes occurs.
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Immobilized Enzyme Systems
Covalent binding:
To select the type of reaction for enzyme covalent immobilization:
- do not cause loss of enzymatic activity. - the active site of the enzyme must be
unaffected by the reagents used.
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Immobilized Enzyme Systems
Covalent binding:
The functional groups on the supports that may take part in this binding are listed below:
e.g. amino group, carboxyl group, sulfhydryl group, hydroxyl group, phenolic
group etc.
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Immobilized Enzyme Systems
Cross-linking: to cross link enzyme molecules with each other using agents such as glutaraldehyde.
Features: similar to covalent binding.
Several methods are combined.
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Summary of Immobilization Methods
Methods of Enzyme immobilization:
- Entrapment - matrix- membrane (microencapsulation)
- Surface immobilization- physical adsorption- ionic binding- covalent binding
- Cross-linking
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Recycle packed column reactor: - allow the reactor to operate at high fluid velocities.- a substrate that cannot be completely processed on a single pass
Immobilized Enzyme Reactors
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Fluidized Bed Reactor:- a high viscosity substrate solution - a gaseous substrate or product in a continuous reaction system- care must be taken to avoid the destruction and
decomposition of immobilized enzymes
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- An immobilized enzyme tends to decompose upon physical stirring.
- The batch system is generally suitable for the production of rather small amounts of chemicals.