Biochemical Engineering

Kinetics of enzymes:

⎯ _______________ reaction is the controlling step in enzyme kinetics.

⎯ Plot of V vs. S is linear which goes to max. at which velocity (reaction rate) is max. and
then it becomes non-linear.
⎯ The plot of 1/V vs. 1/S is a straight line which has positive (positive/negative) slope and
which cuts Y-axis on positive (positive/negative) and X-axis on negative
(positive/negative).
⎯ Plot of K vs. 1/T is a straight line with slope = - E/R and Y-intercept = r.
⎯ In complex enzyme kinetics oxidation-reduction and regeneration step are involved.
⎯ In fermentation silicon emulsion is used as uniform agent.
⎯ The most of the fermentation processes were delayed due to contamination problem.
⎯ In rate of production to the rate of substrate reduced with time.
⎯ Denaturation is constant for balancing reaction if temp. is raised in between 30 – 400C
then enzyme activity is raised 1 – 8 times.
⎯ In special type of hydrogenation fermentation production of nucleic acid is proving to be
an important source for flavor enhancing components.
⎯ In complex enzyme kinetic reaction, slop is same in competitive and non-competitive
inhibition but intercept is different.
⎯ In industrial media sterilization, electromagnetic radiation such as X-ray etc. is not
applicable due to large volume of fluid.
⎯ Optimum substrate concentration is used for better reaction rate and maximum product
formation.
⎯ In principle of biosensors, after molecules reorganization heat thermistors are converted
to signal.
⎯ As reaction rate increases, change in pH leads to denaturation of proteins, oxidation and
reduction.
⎯ In complex enzyme kinetics oxidation reduction and regeneration steps are involved.
⎯ A small pressure in a bioreactor around 1.2 absolute can help in preserving asepsis
condition.
⎯ The problem faced by the engineer in the development of fermenter was design and
operation.
⎯ As per the scientist Gaden, citric acid production is indirectly related to carbohydrate
utilization.
⎯ As per scientist Moyer, in non – growth reaction glucose is converted to gluconic acid w.
r. t. time only.
⎯ Enzyme is a biochemical material that reacts with substrate and form free enzyme
product.
 ⎯ Transducers convert sensor stimuli to electrical signals.
⎯ In pH measurement, calomel electrodes are used because they are heat stable at 1210C
and 15psi pressure.
⎯ In 1923, scientist Pfizer obtained citric acid with the help of aspergillus niger species.
⎯ Enzyme is a biochemical material that react with substrate and form new product.

True/false: (statements are TRUE unless otherwise stated)

⎯ First stage of enzyme kinetics is reversible.

⎯ Use of catalyst increases the value of Ea.
⎯ Values of Ea and A for any fermentation system are constant (?).
⎯ Only two values of T & K are enough to find the values of Ea and A (?).
⎯ As the temperature increases the rate of reaction increases.
⎯ Range of E value is 10 – 21Kcal/mol.
⎯ Michele Menton equation is V = Vmax. S/Km + S
⎯ Arrhenius equation is K = A. e-Ea/RT.
⎯ Arrhenius equation deals with temperature dependency.
⎯ In modern techniques, a resistance or reduction probe is used to detect buildup foam on
the top surface of reactor (F). capacitance or conductance
⎯ As per scientist Moyer, in non-growth reaction glucose is converted to gluconic acid
w.r.t. temperature only (F). Time
⎯ Competitive type of inhibition is reversible and it can be over come by adding some
more substance (F). substrate
⎯ Reaction is inhibited by some inhibitors even when we use more substrate.
⎯ Thermistors of semiconductors work on the principle that resistance of any conductor
changes with temperature.
⎯ The heart of biochemical engineering lies in scale up and management of cellular
process.
⎯ In bioreactor /fermenter dissolved oxygen is measured by polerographic sterilizable
electrode.
⎯ In enzyme kinetics, reaction rate is maximum in presence of inhibitors (F).
⎯ Spectrophotometer is used for the measurement of gas flow rate in reactor (F). rotameter
⎯ In growth associated model, a substrate is stoichemetrically converted to a single product.
⎯ In enzyme kinetics, half of the substrate is utilized at maximum velocity.
⎯ In fermentation process 180gm of sugar gives 92gm of alcohol (F).
⎯ In enzyme kinetics, regeneration step is used in complex enzyme kinetics for product
formation.
⎯ Stage gauge is used for measurement of shaft power input in biochemical reactor (F).
⎯ The rate of product formation is equal to the rate of substrate reduced with respect to
time.
⎯ In chemical foam breaking, ultrasonic thermal action is used (F). physical
⎯ Simultaneous action are those in which less than one product is produced (F). more
⎯ Cellular activities consider only kinetics of growth and product formation (?).
⎯ F-value is the time in minutes required to destroy the organic in the specified medium at
2500C (F). 2500C/1210F
⎯ Fermentation kinetics is concerned with the rate of cell synthesis and/or fermentation
product formation and effect of environment on the rates.
⎯ Two types of kinetics: (1) batch type/dynamic (2) continuous/ steady state
⎯ Shaken flask experiment (batch type kinetics):
⎯ Control measures for enzymes kinetics:
1. concentration of enzymes:
2. conc. of substrates
3. temperature
4.time
5. pH
6. pressure
7. O2 conc.
⎯ Simple enzyme kinetics: a simple kinetic model for enzyme substrate interaction is
proposed by Michealis Manton as,
V  =  Vmax S  At Km = S,
        Km + S 
V= Vmax/2

Where,

V= rate of reaction
Vmax = maximum velocity of the reaction
S= substrate concentration
Km = MM constant (moles/lit. or mass/volume)

⎯ Graph of 1/V vs 1/S is a straight line with ordinates 1/Vmax while abscissa is equal to -
1/Km and slope Km/Vmax.
⎯ The procedure for determining the values of Km and Vmax is experimental and is called
Lineweaver burk Plot.
1/V = Km/Vmax × 1/S × 1/Vmax

If 1/V = 0, then S = Km ,

V = Vmax /2
 ⎯ Factors affecting rate of reaction:
1. Substrate: rate of reaction α substrate
2. pH: every enzyme has its own pH values at which it has its max activity change in pH
leads to denaturation of proteins and decreases enzyme activity.
3. Temperature: rate of reaction α temperature. For product development pH-
Temperature profile is necessary. It can be given by Arrhenous equation:
T = A. e(-E/RT)
If, T > 800C, enzyme denatures and E = 40-130 kCal/gmol.

Enzyme inhibitors:
(A). Competitive/ Substrate analogue inhibition:
⎯ Competition for active site between substrate and inhibitor.
⎯ Can be overcome by addition of more substrate and removal of substrate.
V= Vmax . S
Km + S + Km/Ki . I
Where, I = conc. of inhibitor
Example: effect of glucose on action of invertase on sucrose
(B). Reversible/ noncompetitive inhibition:
V = Vmax . S . Ki
(Km + S)(Ki + I)
⎯ Typical effect of organic acids such as acetate, propionate, and lactate on hydrolytic
enzymes.

⎯ Pressure: Mass transfer = driving force

Resistance of force by inhibitor
⎯ Oxygen requirement: aerobic or anaerobic condition dependant.
⎯ Time: depends on temp., pressure and oxygen requirement.
⎯ Slope is same in both competitive and non competitive inhibition bu intersect is different.
Competitive Enzyme Kinetics:
V= Vmax . S
Km + Km . I/ Ki + S
Patterns of various fermentation:
1. Gaden: product formation α carbohydrate utilization (ethanol)
2. Product formation α 1/ C. U. (Citric acid)
3. Product formation not associated with Carb. Utilization (Penicillin)
Simple reaction kinetics:
⎯ Nutrients are converted to products in a fixed stiochiometry without accumulation of
intermediate .
 Ex. Conversion of glucose from gluconic acid by A. niger mycelia., growth of aerobactor
colacea.
Simultaneous reaction:
⎯ These are those in which more product is produced and relative rate of product formation
vary with nutrient concentration. Ex. Growth of Rhodotorula Glutirus, nutrients are
converted to product with accumulation of intermediates.
Consecutive reactions:
⎯ Nutrients are converted to product with accumulatin of an intermediate to sme degree.
Ex. Glucose fermentation to gluconic acid by gluconolactonose, many antibiotic
fermentation follow consecutive reactions.
Stepwise reaction:
⎯ Nutrients completely converted to intermediates brfore conversion to product or nutrients
selectively converted to product on preferential order.
Ex.1. Hexoses + pentoses E.Coli first hexoses completely utilized then pentoses according to
scientist Monod.
2. Glucose Acetobactor suboxydus 5 ketogluconic acid
Complex cases:
Ex. Penicillin process
Expression for kinetic parameters for cellular activites:
⎯ Specific growth rate μ = 1/x dx/dt
dx/dt = f (X, S, I)
X = cell concentration
Td = mass doubling time = 0.693/μ = log 2/μ
Monod equation:
μ = μmax . S
Ks + S
Where, μs = max. specific growth rate
Ks = saturation constant
S = concentration of growth limiting substrate
Yield value:
Yxis = X/ - S
Product formation models:
1. Growth associated model: when a substrate is stoichiometrically converted to a single
product. Then,
dp/dt = α dx/dt
x = cell concentration
α = stoichiometry
2. Non-growth associated model: when rate of product formation is only dependent on
cell conc. than
dp/dt = βX.
 Where β = proportionality constant.
3. Combined model:
If surface area increases then product also increases.
For lactic acid fermentation,
dp/dt = αdx/dt + βX
specific rate of product formation, V = αμ + β
Media sterilization:
⎯ Commonly moist heat is used for sterilization of media.
⎯ Foaming and viscosity should be controlled
⎯ Spores are more resistant to heat.
⎯ Thermal death point (TDP): it determines thermal death temp. (pt.) of spore suspension,
which is exposed to heat for definite period of time.
⎯ Thermal death temperature (TDT): it measures the amount of time required for
inactivating a sproe suspension at a given temperature.
⎯ The survival rate is used for used to designing pasteurizer but not used in sterilization,
where no spore survives, so one must use probabilistic approach rather than deterministic
approach.
Batch sterilization:
⎯ Steam sparging: hyperbolic heating T=T0 (1+αt/1+rt)
⎯ Electric heating: linear heating. T=T0 (1+αT) α= 9/m4T0
⎯ Steam heating: exponential isothermal heating. T= TH(1+βe-αt).
⎯ Cooling with coolent: non isothermal coolant T=Tco(1+βe-αt) Tco= initial temp. of cooling
water.
Continuous sterilization of media:
⎯ Frequently employed in fermentation industry for sterilization of media.
⎯ Steam heated plates raise the temp. of medium and maintain it for certain period of time,
and then cooled in another section.
Mass transfer:
⎯ Extraction: ex. Solvent extraction of oil
⎯ Leaching: ex. leaching of sugar from sugar beet and sugarcane.
⎯ Evaporation: conc. of material.
⎯ Mass transfer depends upon: permeability dm/dt = D.A dc/dx, where D= dilution rate,
dc= conc. of both sides dx= membrane used to pass through.
⎯ Dialysis: separation of low mol. Wt. compound from high molecular wt. compounds by
semipermeable membrane.
⎯ Mass transfer rate = driving force/ resistance
⎯ Bubble aeration: single bubble: size of bubble emerging from an orifice into water was
confirmed by van krevelen et al. to be proportional to cube root of orifice diameter, d,
and independent on gas flow rate, c, over a range from 0.02-0.5 cm3/sec.
⎯ If liquid is agitated, the liwuid motion is expressed by ratio of inertial force to viscous
force per unit volume of liquid and expressed as modified Reynolds no.