Tutorial 3 Enzyme Immobilization Flashcards
Describe the importance of immobilized enzymes in the food industry
- Long term storage of enzymes as they are more robust and more resistant to environmental changes.
- Improve efficiency of catalyzed reaction
- Enhanced reproducibility
- Cost effectiveness
- Resuability: Heterogenous immobilized enzymes allow easy recovery of both enzymes and products, multiple reuse of enzymes, continuous operation of enzymatic processes, rapid termination of reactions and greater variety of bioreactor designs.
- The solid support systems generally stabilize the structure of the enzymes and consequently maintain their activities
- Compared to their free forms, immobilized enzymes are generally more stable and easier to handle
- It is easily isolated from the solution and pure product is not contaminated.
What are the factors that affect the nature of immobilized enzymes and describe with an example?
- Type of enzyme, whether it is carrier bound or carrier free enzyme.
A carrier such as synthetic polymer immobilized on a enzyme can make is highly hydrophilic and stable both chemically and mechanically. - Carrier’s nature
Enzymes are immobilized onto a high surface area of nanostructured materials such as nano porous materials, the large surface area resulted in improved enzymes loading increasing apparent enzyme activity per unit.
A carrier like magnetic nanoparticle are used to immobilize enzymes in order to enhance bioelement stability, ease separation from reaction mixture and increase catalyst stability. - Immobilization technique
Physical entrapment technique avoid any negative influence on the structure of an enzyme, enzyme is encapsulated to prevent aggression and denaturation.
Compare the advantages and disadvantages of the immobilized enzymes using carrier-bound immobilized enzymes and carrier-free immobilized enzymes?
Carrier-bound immobilized enzymes are chemically/ physically bound to a carrier.
Advantages: Reusability, improved catalytic activity and stability.
Disadvantage: More costly, may cause contamination to pure product
Carrier-free immobilized enzymes are constructed on its own mass.
Advantages: No need for extra inactive mass, less contamination caused, cost effectiveness
Disadvantages: Not as stable or have an improved catalytic activity as carrier-bound enzymes.
What would be the easiest method to separate silica particles of different particle size in large scale?
Physical entrapment with size exclusion
Repeating mesoporous structure entrap large molecules within their pores separating silica particles of different particle size.
Does it affect the active site of enzyme during immobilization?
Use of harsh conditions and reagents during immobilization can cause confirmational changes and denaturation of active site of enzymes.
For example, protein immobilization by absorption on mesoporous silicates have highly variable pore size distribution, their preparation can involve the use of harsh conditions or reagents, which can cause denaturation of proteins.
Four methods of enzyme immobilization
- Non-covalent adsorption and deposition
- Classical covalent immobilization
- Physical entrapment
- Bioconjugation
Describe one selected matrix and describe how it is used in real life usages.
Carrier: Hydrogels
Enzymes can also be immobilized in natural or synthetic hydrogels or cryogels in non-aqueous media.
For example, Polyvinyl alcohol cryogels formed by free thawing method and have been used widely for immobilization of whole cells.
Carrier: Inorganic support: Silica
Silica have higher mechanical strength that allow their utilization for a much wider range. Their preferential use in the preparation of high-performance liquid chromatography media.
Compare the four methods of enzyme immobilization and discuss the usage of each method for industrial purposes?
Non covalent methods of protein involve passive adsorption onto hydrophobic surfaces, does not involve any formation of covalent bonds like Classical covalent immobilization method that is formed through reaction with functional groups. The other method are also relatively different. Physical entrapment involves encapsulating the enzyme avoiding any negative influence on the structure. Bioconjugation involves the bioconjugation of enzymes for example with use of enzymatically active fusion proteins. In this case, a protein of interest is fused to an enzyme that reacts selectively with an immobilized substrate analogue/inhibitor. This reaction forms a covalent bond between the enzyme and the substrate on the surface like the method mentioned previously. Bioconjugation is used is medical industry and less important than the other methods.
What are the matrices used while immobilizing the enzyme by covalent binding?
Synthetic polymers: Immobilization by covalent attachment to acrylic resins to variety of enzymes for industrial application.
Biopolymers: Mainly water-insoluble polysaccharides such as cellulose, agarose have been used as supports for immobilizing enzymes
Smart Polymers: Enzymes immobilized via covalent attachment to stimulus responsive or mart polymer.
What are immobilization techniques that could be used when substrates molecules are much smaller than enzyme?
Hollow fiber units have been used to entrap an enzyme solution between thin and semipermeable membranes. A semipermeable membrane is used to retain enzyme, hence hollow fiber is filled with enzyme and then merged with substrate. As it is semipermeable, there will be reaction causing the immobilization of enzyme.
What are the method for producing immobilized enzymes with multifunctional reagents?
Cross linking: It is one of the chemical methods of enzyme immobilization in which enzyme is attached to each other through covalent bond via bi- or multifunctional reagents.
Covalent modification immobilization method involves formation of covalent bonds that are formed through reaction with functional groups present on the protein surface. Hence multifunctional reagents are being used.
Name Commonly employed water insoluble supports for the covalent attachment of enzyme?
Acrylamide
Polypeptides
Dextran
