Biocatalysis

Question 1

What is biocatalysis?

Answer 1

Using enzymes or whole cells to produce a product.

Question 2

Give 4 examples are where biocatalysis has been used historically or currrently.

Answer 2

Bread making using yeast.
Beer making using yeast.
Cheese making using rennins to induce coagulation and ripening of milk.
Detergents to remove stains.

Question 3

What are the advantages of biocatalysis?

Answer 3

They have high selectivity: entantioselectivity, stereo- selectivity, site specificity.
They have few side reactions and off-target reactions.
They use gentle conditions: cellular temperatures, cellular pHs and water as a solvent.

Question 4

What are the disadvantages of biocatalysis?

Answer 4

Enzymes and cells have evolved to be and work within living organisms so they may not do the function you desire or the function may not work in the conditions you desire.

Question 5

What is the EC system?

Answer 5

This is an enzyme classification system that classifies enzymes based on the reaction they carry out. They are classified based on their first reaction that was discovered so further functions may also occur e.g. the reverse reaction. Structurally different enzymes can be in the same category if they carry out the same reaction type.

Question 6

What type of reaction is EC1?

Answer 6

Oxidoreductases- oxidation or reduction reactions

Question 7

What type of reaction is EC2?

Answer 7

Transferases- adding a group to a molecule

Question 8

What type of reaction is EC3?

Answer 8

Hydrolases- Using water to cleave bonds (These often work in reverse, putting groups together and eliminating water)

Question 9

What type of reaction is EC4?

Answer 9

Lyases- Eliminating a group or breaking a chemical bond without water being involved.

Question 10

What type of reaction is EC5?

Answer 10

Isomerases- Rearranging bonds with no addition or elimination.

Question 11

What type of reaction is EC6?

Answer 11

Ligases- Joining two fragments, requiring ATP as a cofactor.

Question 12

Which EC classes are the most common in industry settings?

Answer 12

1-Oxidoreductases

3-Hydrolases

Question 13

Give 3 examples of enzymes that industry produces.

Answer 13

1) Deoxyriboaldolase- EC 2. This uses selectivity of enzymes to choose the correct chemical to continue in the process with. It is used to produce cholesterol lowering drugs, which have a market of $13 billion.
2) Synthesis of High Fructose Corn Syrup involves alpha-amylase and glucoamylase (EC3) which break starch to sugar and sugar to glucose, then glucose isomerase (EC5) to convert glucose to fructose. These enzymes are very stable, so don’t need replacing often. 11 million tons are produced per year.
3) Cellulases (EC2) are used in the textile industry to change the colour of jeans and also in biofuels. The textile market fluctuates but is worth $198 million

Question 14

What is kinetic resolution?

Answer 14

If a racemic mixture containing two enantiomers exists, kinetic resolution uses 1 enzyme to selectively choose the correct enantiomer. It can be done 2 ways:

• hydrolyse the incorrect enantiomer
• add another group to the correct one to produce a product or next step in product production.

Question 15

What is dynamic kinetic resolution?

Answer 15

If a racemic mixture containing two enantiomers exists, dynamic kinetic resolution uses 2 enzymes. The first enzyme converts incorrect enantiomers to the correct version. The second enzyme adds groups onto the correct enzyme or continues with the process.

Question 16

Describe enzyme kinetics.

Answer 16

In general if you add substrate the rate of reaction increases until saturation is achieved. However it’s a little more complicated. Michaelis-Menten kinetics and Quasi-steady state assumption can be used to define the product production in terms or substrate concentration, enzyme concentration and reaction rates.

Question 17

What is the equation describing protein production kinetics?

Answer 17

dP/dt = (K2.Eo.S)/(Km+S)

Question 18

Describe 3 enzyme performance parameters.

Answer 18

1) Activity: This describes the production of the product. The benchmark is 1 U which is equivalent to 1microMol of product/min/mg of enzyme/active site.
2) Proficiency: This describes the difficulty of the reaction. Calculated using Kcat/Kuncat where Kcat is the turnover number and Kuncat is the reaction rate that occurs if no enzyme is present. (This is often extrapolted at high temperatures)
3) Specificity: This compares the activity of 2 substrates. calculated using (Kcat/Km)1/(Kcat/Km)2 which compares two substartes not in 50:50 mix. ALternativeky use an E-value (benchmark 100) which compares enantiomers.

Question 19

What plots can be used to determine kinetic parameters and do they have pros/cons?

Answer 19

Lineweaver-Burk –> high 1/S can cause problems
Eadie-Hofstee –> easy to find the constants
Hanes-Woolf–> removes /S dependence

Question 20

How does inhibiton work?

Answer 20

Organisms regulate everything to prevent overaccumulation (which causes problems, when you’re forcing overaccumulation).
Reversible inhibition: Binds and blocks, then unbinds
Irreversible inhibiton: Binds and chemically changes the enzyme
Competitive: Competes for the active site, Km changes with inhibitor conc.
Noncompetitive: Reduces activity of enzyme,Vmax changes
Uncompetitive: Binds to ES and stops further activity,Vmax and Km changes.

Question 21

Why is inhibition a problem in an industry setting?

Answer 21

This process occurs in a bioreactor where unnatural inhibitors may also occur. Substrate inhibition means adding more substrate slows the rate of production. Product inhibition means product accumulation slows the rate of production.
Semi-batch reactors often solve this problem but it can make optimising difficult.

Question 22

Describe enzyme deactivation, thermodynamic stability and kinetic stability.

Answer 22

Enzyme deactivation is where enzyme denature and stop being productive. Enzyme activity increases with temperature until the enzyme denatures. This means there is an optimum temperature.
Thermodynamic stability is the inherent stability from key residues, the fold structure and interactions of the protein. If E U meaning there is an equilibrium between the enzyme and the unfolded state then biophysical measurements (spectroscopy, CD, DSC) to measure the deltaG at different temperatures. Plot to find Ts, Tm’ and Tm.
Kinetic stability accounts for the fact that denaturing can’t be undone by reducing the temperature. EU–>D. D occurs from aggregation, cleavage and side chain loss. You can determine the rate by heating and decreasing the temperature and comparing activity levels. This gives Kdobs = k/(1+K) which can be found on a graph of ln(E/Eo) vs Time. The half life is then calculated using Thalf = (ln2)/Kdobs.

Question 23

Describe immobilisation and why it is useful?

Answer 23

Immobolisation attaches an enzyme to a surface (physically, chemically, reversibly or irreversibly). It uses membranes, gels, ionic carriers, covalent attachment, cross-linking, reverse micelles or hollow fibres.
It is useful because it increases stability, prevents unfolding, allows recycling and means the protein sin’t in the final mixture so doesn’t need to be purified out.

Question 24

What are mass transfer effects?

Answer 24

Although immobilisation means there’s a large surface area for activity. Mass transfer effects mean that there are limits on the concentration the enzyme actually sees which lowers the activity. It can often be solved by stirring,.

Question 25

Describe two ways to calculate productivity.

Answer 25

Weight of the product/volume/time, with a benchmark of 1g/L/day.
Total turnover number: moles of product/moles of catalyst deactivated, with a benchmark of 100,000.

Question 26

What is productivity?

Answer 26

Productivity is a measure of the profitability as a function of time and volume.

Question 27

Why are organic solvents important?

Why are they problematic?

Answer 27

Solvents are important as they affect the stability of the protein. It is suggested that this happens because of the loss of hydrophobic contacts and because flexibility required for catalysis is lost.
They are problematic because their effects depend on the protein and although there has been attempts the effects can’t be predicted.

Question 28

What’s medium engineering?

Answer 28

Ths is an area that looks at how medium affects cells and proteins. It attempts to predict the changes, find optimum and generic versions and understand the effect of media.

Question 29

What is regeneration and why is it useful?

Answer 29

Regeneration of co-factors and enzymes can occur which reduces costs and can be done by whole cells that already exist or by the addition of fairly cheap enzymes/ chemicals.

Question 30

What 3 solvent options are there and what pros/cons do they have?

Answer 30

1) Miscible –> mixed state which can affect stability but there’s no mass transfer effects.
2) Immiscible –> This has two separate states so requires mixing to increase activity. Stability is not really affected but there are mass transfer effects.
3) Lyophilised (freeze-dried enzymes) –> This forms lumps in the solvent so experiences mass transfer effects and stability is affected depending on the solvent and the conditions under which it was frozen.