BME 34 Midterm 2

Salting Out

Protein precipitation caused by an increase in the salt concentration

Salting Out: Process

- Proteins remain in aqueous solution bc of interactions between hydrophilic amino acids and surrounding water molecules

- If ionic strength of solvent is increased by adding an agent, some of the water molecules will interact with the salt ions, decreasing the \# of water molecules available to interact w the protein

- So, when protein molecules can't interact with the solvent, they interact w each other and form a precipitate

- This precipitate (containing enzyme of interest & other proteins) can be filtered or centrifuged & separated from the supernatant

Ion-Exchange Chromatography

- Early stages of purification process

- Protein solution added to a column containing an insoluble polymer (like cellulose) that has been modified so that its ionic characteristics will determine the type of mobile ion (cation or anion) it attracts

- Proteins whose net charge is opposite to that of the ion-exchange material will bind to it, and other proteins will pass thru the column

Size Exclusion Chromatography

- Later stages of purification

- Columns containing bed of cross-linked gel particles are used, gel particles exclude large protein molecules while allowing entry of smaller molecules

- Separation occurs bc larger protein molecules follow a path down the column between the gel particles

- Larger molecules have shorter elution time and are recovered first from gel filtration column

Affinity Chromatography

- Column packed with a particulate stationary phase to which a ligand molecule like a substrate analogue, inhibitor, or cofactor of the enzyme of interest would be firmly bound

- As sample mix is passed thru the column, the enzyme interacts with and binds to the immobilized ligand, being retained within the column as all the other components pass thru

- Then, a solution of the ligand is introduced to the column to release (elute) and recover the bound enzyme from the column

L-Asparginase

Treatment for cancer (ALL - acute lymphoblastic leukemia)

Takes advantage of fact that leukemia cells can't make asparagine but normal cells can

Leukemic cells need asparagine, asparginase deprives leukemic cells of asparagine --\> cell death

Luciferase

Enzyme used in bioluminescence

- Luciferases (classified as oxidoreductases) do not require an external light source, but need addition of luciferin (consumable substrate)

- Used as reporter to assess transcriptional activity in cells or can be used to detect level of ATP in assays

Enzyme Immobilization

A technical process in which enzymes are fixed to or within solid supports, creating a heterogenous immobilized enzyme system; this stabilizes enzyme structure & maintains their activities

Compared to free enzymes, immobilized enzymes

Show lower activity and higher apparent Michaelis constants bc of a relative difficulty accessing the substrate

Advantages of Immobilized Enzymes

- Robust

- Resistant to environmental changes

- Fixed in place & localized

- Easy recovery of enzymes and products

- Multiple re-use of enzymes

- Continuous operation of enzymatic processes

- Rapid termination of reactions

- Greater variety of bioreactor designs

Disadvantages of Immobilized Enzymes

- Uses in industrial applications are limited

- Loss of catalytic properties in some enzymes

- Some enzymes become unstable

- High cost for the isolation, purification, and recovery of the active enzyme

What supports are used in enzyme immobilization?

Natural polymers, synthetic polymers, inorganic materials

Methods of Immobilization

1. Adsorption

2. Covalent Bonding

3. Entrapment

4. Copolymerization

5. Encapsulation

Adsorption

- Simplest

- Enzymes are adsorbed to external surface of the support

- Weak low energy bonds stabilize enzymes on support (hydrogen, van der waals, ionic)

Methods of Adsorption

1. Static process - put enzyme in close proximity

2. Dynamic batch process - stirring

3. Reactor loading process - reactor promotes adhesion

4. Electrode position process - electrode simulates bonding

Adsorption - Advantages

- Easy to carry out

- No reagents needed

- Minimum activation steps involved

- Comparatively cheap method

- Less disruptive to proteins than chemical methods

Adsorption - Disadvantages

- Desorption of enzymes from support

- Lower efficiency

Covalent Bonding

Formation of covalent bonds between enzyme and support

- Widely used

- Chemical groups in enzymes form covalent bonds w support

Covalent Bonding - Advantages

- Strong linkage of enzyme to support

- No leakage or desorption problems

- Comparatively simple

- Variety of support w different functional groups available

- Wide applicability

Covalent Bonding - Disadvantages

- Chemical modification of enzyme leads to functional conformation loss

- Enzyme inactivation by changes in conformation when the enzyme undergoes rxns at active sites

- Can be overcome thru immobilization in presence of the enzyme substrate or a competitive inhibitor

Entrapment

Enzymes are physically entrapped inside a matrix

- Bonds involved may be covalent or noncovalent

- Support used is a polymer

Entrapment - Advantages

- Fast

- Cheap

- Mild conditions required

- Less chance of conformational changes in enzyme

Entrapment - Disadvantages

- Enzyme leakage

- Pore diffusion limitation

- Chance of microbial contamination

Cross Linking

Covalent bonding btwn various groups of enzymes via polyfunctional reagents; no support material involved

Encapsulation

Enclosing enzymes in a semipermeable membrane capsule

Encapsulation - Advantages

- Cheap and simple

- Large quantity of enzymes can be immobilized

Encapsulation - Disadvantages

- Pore size limitation

- Only small substrate molecule is able to cross the membrane

Whole Cell Immobilization Advantages

- Multiple enzymes can be introduced in a single step

- Extraction and purification of enzymes not required

- Enzymes stable for a long time

- Native conformation of enzyme is best maintained

- Cell organelles like mitochondria and chloroplasts can be immobilized

Whole Cell Immobilization Disadvantages

- Concentration of enzymes will be lower

- Production of unwanted enzymes and unwanted products

- Modification of end products by other enzymes

Advantages of nanoscale particles include:

- Sustained drug delivery

- Hydrophilic and hydrophobic multi-drug co-incorporation and programmed delivery

- Prolonged circulation time in vivo

- The ability to be engineered with functionalized ligands

- Stimuli responsive controlled drug release

Which of the following is a disadvantage of immobilized enzymes over free enzymes?

Enzyme may become inactive

The immobilized enzyme produced by micro encapsulation technique provides

A large surface area