Enzyme Kinetics, Bioreactors & Fermentation Flashcards
List the factors affecting enzyme catalysis
- Concentration of substrate molecules (more substrate = quicker enzyme collisions & binding)
- Temperature (higher temps = faster kinetic energy until denature temp is reached)
- Inhibitors (competitive = prevent substrate binding, noncompetitive = reduce enzyme catalytic power)
- pH (this affects conformation of the protein)
Describe the characteristics of enzyme catalysts
- High efficiency
- High specificity (geometric, electronic & structure complementary)
- Regulatable (using inhibitors)
- Mild condition (most work best close to body temperature at at a particular pH)
Write the 2 elementary reactions occurring in the Michaelis Menten model and the rate equation for each of the components
A + E A.E
A.E R + E
Overall:
A + E A.E –> R + E
dCA/dt = - k1 CA CE + k-1 CA.E
dCE/dt = - k1 CA CE + (k-1 + k2) CA.E
dCR/dt = k2 CA.e
Write the concentration balance for the total concentration of enzymes present initially
C E0 = C E + C A.E
Write the assumptions related to quasi steady state
- The catalyst, E is recycled and the concentration is low
- The concentration of E and A.E change very little in absolute terms
- dCE/dt = dCA.E/dt = 0
Write the general equation for the Michaelis Menten model
dCR/dt = Vmax [CA / (CA + KM)]
where:
V max = k2 CE0
K M = (k-1 + k2)/k1
Derive the Michaelis Menten model equation from kinetics
Using the quasi-steady state assumption:
dCE/dt = - k1 CA CE + (k-1 + k2) CA.E = 0
CAE = k1 CA CE/(k-1 + k2)
CE = CE0 - CA.E
CAE = CE0 [CA/(CA + (k-1 + k2)/k1)]
dCR/dt = k2 CAE
Therefore:
dCR/dt = k2 CE0 [CA/(CA + (k-1 + k2)/k1)]
dCR/dt = Vmax [CA/(CA + KM)]
What happens when the initial rate is equal to half the maximum rate?
V max/2 = V max [CA/(CA + KM)]
KM = CA
What does it mean when KM is large?
The binding is weaker
Write the equation for the Lineweaver-Burke plot
1/rR = KM/V max (1/CA) + (1/V max)
Write the equation for the Eadie-Hofstee plot
Multiply the Michaelis-Menten by rR Vmax:
V max = (rR KM)/CA + rR
rR = V max - KM (rR/CA)
Define KM
The substrate concentration at which half the enzyme active sites are filled by substrate molecules
OR
The substrate concentration at which half the enzyme active sites are filled by substrate molecules
AND
It also shows when inhibitors are present if the value of KM changes for the same enzyme and substrate
What does it represent when:
- CA < KM
- CA = KM
- CA > KM
- Most enzymes are free
- Half of the active sites are free
- Most of the enzymes have formed complexes
Define V max
The maximum rate attainable and/or the rate at which the total enzyme concentration is present as the enzyme-substrate complex
What is k2 and what are the units in the Michaelis-Menten model?
k 2 = 1/s = first order rate constant
This is also known as the turnover number = the amount of substrate that are converted to product per unit time when the enzyme is fully saturated with the substrate.
Write the equation for enzyme kinetics in a batch reactor
CA0 - CA + KM ln (CA0/CA) = Vmax t
(CA0 - CA)/ln (CA0/CA) = k2 [CE0 t/ln (CA0/CA)] - KM
This can be plotted to determine the rate equation
Write the equation for enzyme kinetics in a CSTR
CA = (t Vmax CA)/(CA0 - CA) - KM
CA = k2 [CE0 t CA/(CA0 - CA)] - KM
This can be plotted to determine the rate equation
True or false: Michaelis-Menten kinetics can be applied to irreversible inhibition
False
The inhibitor forms a covalent linkage with the enzymes that cannot be removed.
List the 3 types of reversible inhibition
- Competitive (substrate and inhibitor are competing for the same active site)
- Non-competitive (inhibitor binds to a site other than the active site, reducing catalytic power)
- Uncompetitive (inhibitor binds only to the enzyme substrate complex)
List and briefly describe the 3 types of biotechnology
- Green - agriculture
- Red - medical
- White - industrial
List the 9 types of common bioreactor
- Stirred tank bioreactor (eg. pharmaceuticals)
- Incubator
- Photo-bioreactor
- Solid state bioreactor (eg. cheese)
- Bubble column bioreactor (eg. wastewater treatment)
- Hollow fibre cartridge
- Rocking bag bioreactor
- Tissue culture bioreactor
- 3D printing
List the 9 important parameters to control in bioreactor operation & design
- Temperature
- pH
- Sufficient substrate eg. carbon, sugars, proteins, fats
- Water availability
- Salts for nutrition
- Vitamins
- Oxygen
- Gas evolution
- Product & byproduct removal
What is the difference between enzyme fermentation and microbial fermentation?
In enzyme fermentation, the enzyme (catalytic agent) does not reproduce itself but in microbial fermentation, the microbe/cell (catalytic agent) does reproduce itself.
What is product poisoning?
Fermentation needs cells and food so in microbial fermentation, the cells multiply and also produce a waste product. Sometimes, the waste product produced can inhibit the action of the cells even if enough food present, causing product poisoning eg. wine making
Give an example of when each of the components in microbial fermentation is used:
A –> C + R
A - wastewater treatment breakdown
C - growing yeast and/or proteins
R - producing penicillin and other antibiotics
Write the general rate for the growth of microbial cells according to Monod kinetics
A –> C + R
rC = rR = (k CA CC)/(CA + CM)
C total = C pregnant + C resting
where k = k (1 - (CR/CR*))^n
where CR* is the concentration of R where all reactions stop
What does the value of the rate constant depend on in Monod kinetics?
Temperature, vitamins, presence of toxic substances, light intensity & presence of trace elements
Write the rate for enzyme fermentation according to Monod kinetics
A –> C + R
rR = (k2 CE0 CA)/(CA + KM) = V max [CA/(CA + KM)]
In general, what are the 2 factors that will cause reaction and cell multiplication in microbial fermentation to slow down?
- Depletion of A (famine)
2. Build up of R (environmental pollution)
Describe the stages involved in microbial batch fermentation
- Induction period (time lag where cells adjust)
- Growth period
- Stationary period
- Dying of cells
Draw the t vs. C curves for the following cases in batch fermentation:
- Substrate limiting
- Poison limiting
See W11 notes
