INTRODUCTION TO BIOCHEMISTRY AND BIOCHEMICAL ENGINEERING

The Kinetics of Enzyme-Catalyzed Reactions

The Enzyme-Substrate Complex and Enzyme Action

Enzymes are remarkable biological catalysts that enable life by accelerating biochemical
reactions by many orders of magnitude. Their high specificity arises from their three-
dimensional structure, allowing selective binding and transformation of substrates into
products.

Mechanism: E + S ES → E + P

This three-step model underpins most enzymatic reactions:

• Binding of substrate to enzyme (formation of the enzyme-substrate complex, ES)

• Conversion to product

• Release of product and regeneration of enzyme

The active site is typically a cleft or pocket formed by the tertiary structure, involving
residues that engage the substrate through:

• Hydrogen bonds (e.g., between amino and carbonyl groups)

• Ionic interactions (charged amino acid side chains)

• Hydrophobic interactions (nonpolar side chains)

Lock-and-Key Model: Suggests the active site is rigid and only fits the correct substrate.

Induced Fit Model: Recognizes that enzyme structure is dynamic—substrate binding

induces conformational changes enhancing catalytic efficiency.

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Transition State Theory: Enzymes stabilize the high-energy transition state intermediate
(‡), reducing the activation energy (G‡) and enhancing the forward rate without shifting
equilibrium.

Simple Enzyme Kinetics with One and Two Substrates

Michaelis-Menten Kinetics

The most foundational model in enzyme kinetics, developed by Michaelis and Menten in
1913, assumes:

𝑑[𝐸𝑆}
• A steady-state concentration of ES ( = 0)
𝑑𝑡

• Irreversible conversion to product (no back reaction)

Where:

• v is the rate of product formation

• Vmax = kcat[E]0 , the maximum rate when all enzyme is saturated

• , reflecting the affinity of enzyme for substrate

• kcat
(turnover number): number of substrate molecules converted per enzyme per
second

Catalytic efficiency: kcat/ Km

• Highvalues indicate efficient enzymes (diffusion-limited enzymes like catalase,

superoxide dismutase)

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Evaluation of Parameters

Experimental analysis often involves transforming the Michaelis-Menten equation into

linear forms:

• Lineweaver-Burk:

• Eadie-Hofstee: Vmax

• Hanes-Woolf:

Each has pros and cons—Lineweaver-Burk is sensitive to small errors in low [S] regions,
while Hanes-Woolf spreads data more evenly.

Reversible Reactions, Two-Substrate Reactions, and Cofactor

Activation

• Reversible Reactions: When product accumulates and reversibly binds to enzyme:

E+P⇌

EP → ES → E + S The equation becomes: v =

• Two-substrate reactions:

• Sequential (Ordered or Random): A + B + E → EAB →E+P+Q

• Ping-pong: One product leaves before second substrate binds.

• Cofactors: Enzymes such as dehydrogenases require NAD+, FAD, or ATP for activity.

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Determination of Elementary-Step Rate Constants

Relaxation Kinetics

Relaxation kinetics techniques analyze how an enzyme system returns to equilibrium after
a small perturbation, such as a temperature or pressure jump. These are especially useful for
reversible reactions.

For a simple reversible reaction: A+B ⇌ C Relaxation time τ is related to the forward and
reverse rate constants: = kf +kr

This approach allows determination of rate constants when direct observation of

intermediates is challenging.

Some Results of Transient-Kinetics Investigation

Transient kinetics captures rapid events before steady-state is reached. Commonly studied
with stopped-flow or quenched-flow techniques, this method is used to measure pre-steady
state bursts and to observe intermediates.

Example: In burst kinetics of serine proteases, a rapid initial product formation indicates a
fast acylation step followed by slower deacylation.

Mathematical tool: Fit observed time courses to exponential terms for extracting rate
constants.

Other Patterns of Substrate Concentration Dependence

Substrate Activation and Inhibition

Some enzymes exhibit substrate activation where binding of one molecule enhances binding
or activity at another site. Others experience inhibition at high substrate concentrations
(substrate inhibition).

Rate expression with inhibition: v =

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Multiple Substrates Reacting on a Single Enzyme

Complex enzymes may catalyze different reactions depending on the substrate. This requires
kinetic modeling that accounts for mutually exclusive or cooperative substrate binding.

Modulation and Regulation of Enzymatic Activity

The Mechanisms of Reversible Enzyme Modulation

Regulation can occur by non-covalent allosteric effectors or reversible covalent

modifications.

• Allosteric sites: Effectors bind here to change enzyme conformation.

• Phosphorylation: Alters enzyme activity in response to signaling.

Example: Glycogen phosphorylase is activated by phosphorylation in response to adrenaline.

Analysis of Reversible Modulator Effects on Enzyme Kinetics

Allosteric enzymes often display sigmoidal (S-shaped) velocity vs [S] plots.

Hill equation:

Where n is the Hill coefficient (degree of cooperativity).

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Other Influences on Enzyme Activity

The Effect of pH on Enzyme Kinetics in Solution

pH affects ionization states of amino acids at active and binding sites. The enzyme may be
active only when specific residues are charged.

Bell-shaped curve: Reflects that both protonated and deprotonated forms are necessary for
activity.

Enzyme Reaction Rates and Temperature

Temperature increases molecular collisions but also causes denaturation above a threshold.

Arrhenius Equation: k = Ae−Ea/RT

Q10 rule: Rate approximately doubles for every 10°C rise within physiological range.

Enzyme Deactivation

Mechanisms and Manifestations of Protein Denaturation

Denaturation is loss of structure and function due to extreme conditions. Can be reversible
(e.g. urea removal) or irreversible (e.g. heating).

Molecular effects:

• Disruption of hydrogen bonds, hydrophobic core, disulfide bridges

Deactivation Models and Kinetics

Commonly modeled by first-order kinetics: E(t) = E0e−kdt

Where kd is the deactivation rate constant.

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Mechanical Forces Acting on Enzymes

Industrial reactors cause stress via stirring, shear, or interfacial adsorption. These can unfold
proteins or cause aggregation.

Strategies for Enzyme Stabilization

• Immobilization on beads or membranes

• Additives like glycerol or trehalose

• Genetic engineering to introduce disulfide bridges or increase hydrophobic core

Enzyme Reactions in Heterogeneous Systems

Enzymes can function in heterogeneous media such as immobilized systems, emulsions, or

membranes.

These systems improve reuse, stability, and separation but pose diffusion challenges.

Factors affecting kinetics:

• Surface area
• Enzyme loading
• Mass transfer limitations

Immobilized Enzyme Kinetics:

Why Immobilization?
Yes, why do we need an immobilized enzyme and why learn about it’s kinetics and
what’s an immobilized enzyme anyways?

Immobilization of an enzyme means that it has been confined or localized so that it

can be reused continuously. There are several reasons why this is desirable: for processing
with isolated enzymes, we need to retain an enzyme in immobilized form in the reactor. An
immobilized enzyme can be fixed in position near other enzymes participating in a catalytic
sequence, thereby increasing efficiency of multi-step conversions.
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Now let’s have a look at the actual kinetics.

First is the simplest case, enzyme is immobilized only on the external surfaces of slab
support. Thus, only the mass transport from the bulk solution to the support surface and the
reaction at that position needs to be considered.

One model for this, is the Nernst Diffusion layer which gives the following equation.

Where:

Ns - moles per unit time per unit area(flux)

S – substrate concentration at interface

S0 – substrate concentration in bulk fluid

Ks – mass transfer coefficient

Note : ks increases with increasing liquid flow rate through a packed column enzyme reactor.
It depends on other hydrodynamic conditions as well, which shall not be discussed here and
can be read through the provided reference.

In steady state, substrate cannot accumulate at catalyst interface. Thus, rate of substrate
supply by mass transfer must equal the rate of substrate consumption by the reaction at the
interface.

Assuming that Michaelis-Menten kinetics applies at the surface, and letting denote surface
reaction rate.

The number of parameters required to specify the system can be reduced from four (ks, s0 ,
vmax, Km) to two (Da and) by introducing dimensionless variables:

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Thus, the equation reduces two:

Da, also known as Damkohler number should be emphasized:

There are two special cases of this reaction, namely

Reaction-limited regime – max mass-transfer rate >> max reaction rate

Diffusion-limited regime – max reaction rate >> max transfer rate

With a bit more algebraic manipulation we get,

By tradition in chemical engineering, the influence of mass transfer on the overall reaction
process is represented using the effectiveness factor , which is defined physically by:

For our problem we have, after substituting values,

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For Da approaching zero, i.e., very slow reaction relative to maximum transfer rate show that
x must approach unity and so for this condition(reaction-limited case):

Many experimental reactors designed to operate in the reaction-limited regime have been
developed for ordinary heterogeneous catalysts. They all share the common concept of high
fluid flow rates near the catalyst to minimize mass-transfer resistance (larger ks, smaller Da).
A few drawbacks of said system is that fluid forces might cause partial or complete
denaturation of the attached enzymes and relative motion of catalyst particles may give
enzyme loss by particle-particle abrasion.

Now again by doing a bit of mathematical manipulation (which I suggest you try out), we can
arrive at equation for diffusion-limited case. (Da->, finite)

Thus, when Da is very large is first order in bulk substrate concentration and totally
independent of the intrinsic rate parameters vmax and Km. Thus, studies aimed at determining
activity retention or denaturation rates of immobilized enzymes should be conducted as
close as possible to reaction-limited regime.

Analysis of Intraparticle Diffusion and Reaction:

Now, enzymes are typically immobilized on the internal surfaces of porous systems or
entrapped in matrices through which substrates can diffuse. Unfortunately, this part cannot
be covered here but I do recommend checking out section 4.4.2 of Bailey, the reference used
for creating this module, it’s an interesting derivation.

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Effects of Inhibitors, Temperature and pH on Immobilized Enzyme

catalytic Activity and Deactivation:
The effect of pH on enzyme kinetics were previously discussed in this same module,
but when an enzyme is immobilized, it might affect some of its intrinsic properties. This
requires careful study using methods like Nernst Diffusion Layer (The one discussed
above). After all, immobilization might mean different rate constants and even alterations to
the form of the equation needed to describe the effects of reaction parameters on
immobilized enzyme intrinsic activity.

To understand the effect of mass transfer processes, let’s take a first order reaction.

Firstly, the kinetics of the reaction in reaction-limited regime will be same as actual kinetics.
On the other hand, in diffusion-limited regime, the apparent overall kinetics will still be first
order, but the apparent rate constant will be the square root of the intrinsic rate constant.

Thus, apparent activation energy will be ½ of the true activation energy. Similarly, a change
in pH which reduces kinetic rate constant by 4 will only reduce it by 2 under diffusion-limited
condition.

Thus, one must be very careful when studying effects of reaction parameters on the kinetics
of immobilized enzyme-catalyzed reactions. Adequate care is required so as to distinguish
the kinetic parameters altered due to mass-transfer effects and hence are different from the
actual kinetic parameters of the enzyme itself.

Bioprocess Engineering and Cell Culture Briefing

1. Cell Growth Kinetics and Modeling

Cell growth in bioprocesses follows distinct phases: lag, log (exponential), deceleration,
stationary, and death. Understanding and modeling these phases are critical for optimizing
product yield and process efficiency.

Growth Phases:

Lag Phase: A period of adaptation where cells synthesize new enzymes and increase
in mass and volume, but not in number. This phase can be prolonged by low inoculum
volume, poor inoculum condition, or nutrient-poor medium. Multiple lag phases,
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known as diauxic growth, can occur if a medium contains multiple carbon sources
that are utilized sequentially (e.g., glucose then xylose). To avoid diauxic growth,
consistent culture medium composition, highly active inoculum in the exponential
phase, and a large inoculum volume (5-10% of new medium volume) are
recommended.

Log (Exponential) Phase: Characterized by rapid cell doubling, where the growth
rate is independent of nutrient and substrate concentration. Carbon sources are
actively utilized, and products are formed.

Stationary Phase: Occurs when nutrients are exhausted (substrate concentration

approaches zero) and waste or secondary metabolic products accumulate. The
growth rate equals the death rate, resulting in no net increase in cell population. Cells
may remain metabolically active, producing secondary metabolites.

Death Phase: Follows the stationary phase, marked by a decline in viable cell
numbers due to nutrient exhaustion and accumulation of toxic by-products.

Modeling Cell Growth and Metabolism:

Monod Equation: A widely used model to describe specific growth rate (µ) as a
function of limiting substrate concentration (S): µ = µm S / (Ks + S).
[µm: Maximum specific growth rate, S: Concentration of limiting substrate, Ks: Half-
saturation constant (substrate concentration at which µ is half of µm)]

In high substrate concentration (S >>> Ks), growth occurs at maximum rate (dX/dt =
µmX).
In low substrate concentration (S <<< Ks), growth is linearly dependent on substrate
concentration (dX/dt = µm S X / Ks).

Modified Monod Kinetics for CHO Cells: For CHO cells, a Monod-type equation can
be used, but it's important to consider a threshold substrate concentration [S]t below
which growth is not observed: µ = µmax [S]* / (Ks + [S]*), where [S]* = [S]o - [S]t.

Studies on CHO cells show that kinetic parameters like µmax and Ks can vary with
temperature and cell type (naive vs. recombinant). For example, naive CHO cells at
37°C had µmax = 0.050 h⁻¹ and Ks = 1.023 g/L, while at 33°C, µmax = 0.038 h⁻¹ and
Ks = 0.286 g/L. Recombinant CHO cells showed less temperature dependence, with
µmax ≈ 0.040 h⁻¹ and Ks ≈ 0.664 g/L at both 33°C and 37°C.

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Yield Coefficients: Quantify the efficiency of converting substrate to biomass or

product.
Yx/s: Grams of cells produced per gram of substrate consumed.
Yp/s: Grams of product produced per gram of substrate consumed.
Yp/x: Grams of product produced per gram of cells produced.

For B. sacchari on xylose, the xylose to PHA yield was approximately 0.37
gP(3HB)/gxyl under both nitrogen and phosphorus limitation.
For CHO cells, Yx/s was observed to be a function of substrate concentration, ranging
from 0.27–1.08 × 10⁷ cells/mL for naive cultures and 0.72–2.79 × 10⁶ cells/mL for
recombinant cultures. (Using simple models to describe the kinetics of growth,
glucose consumption, and monoclonal antibody formation in naive and infliximab
producer CHO cells)

[Specific Product Formation Rate (qp): Can be growth-associated (qp = Yp/x * µg),
non-growth-associated (qp = β = constant, often during stationary phase), or mixed-
growth-associated (qp = α µg + β - Luedeking-Piret equation).]

Monoclonal antibody (mAb) production in CHO cells can be described by a

Luedeking–Piret model: d[mAb]/dt = αd[X]/dt + β[X], with specific values for α and
β. (Using simple models to describe the kinetics of growth, glucose consumption, and
monoclonal antibody formation in naive and infliximab producer CHO cells)

Hybrid Cybernetic Approach (HCM): Combines physiological models (logistic

growth, external metabolite mass balances) with metabolic models (Elementary
Mode Analysis, EMA) to dynamically describe cell behavior. This approach can
segregate viable cells into growth and stationary metabolic states, each with different
specific rates of consumption/production.

2. Bioreactors and Bioprocess Design

Bioreactors are crucial for providing optimal environmental conditions for microbial growth
and product formation. Their design and operation are influenced by the organism and
desired product.
Bioreactor Types: Generally categorized into lab-scale or industrial-scale, with
variations including photobioreactors for light-dependent organisms.

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Key Parameters and Control:

Agitation: Essential for mixing nutrients, oxygen, and cells, and for temperature
distribution. Improper agitation can lead to shear stress on cells.

Temperature: Directly impacts metabolic rates and overall mass transfer.

pH: Controlled by acid/base addition.

Dissolved Oxygen (DO): Maintained above a certain saturation level (e.g., 40%) by
varying agitation speed or gas flow.

Oxygen Mass Transfer: Overall Mass Transfer Coefficient (KLa): A critical parameter
characterizing oxygen transport from the gas to the liquid phase. NA = KLa (C* - CL),
where NA is oxygen transfer rate, C* is oxygen saturation concentration, and CL is
dissolved oxygen concentration. The liquid side often presents the greatest resistance
to mass transfer, so KL ≈ kL.

Factors Affecting KLa: Gas hold-up (volume fraction of gas in total volume),
superficial gas velocity, power input from agitator (which influences interfacial area),
and temperature. Smaller bubbles and higher gas hold-up generally lead to higher
mass transfer rates.

3. Metabolic Engineering and Advanced Analysis Techniques

Metabolic engineering aims to modify cellular metabolism for improved product yield or
desired phenotypes. Advanced techniques like 13C Metabolic Flux Analysis (MFA) and
Elementary Mode Analysis (EMA) are vital tools.

13C Metabolic Flux Analysis (MFA): Uses labeled tracers (e.g., 13C) to track
metabolic pathways and determine intracellular flux distribution.

Applications: Identifying metabolic engineering targets. For instance, inhibiting PDH

kinase in CHO cells reduced lactate production and glycolytic fluxes while
maintaining TCA cycle flux and specific productivity (qP), leading to higher
volumetric titer and viable cell density (VCD) due to reduced lactate and delayed
glucose depletion.
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Future Directions: Mini Bioreactors: High-throughput systems like AMBR mini

bioreactors provide industrially relevant scale-down models (over 10,000L
commercial scale) and allow for multiple replicates and parallel tracer studies,
overcoming cost and representativeness issues of larger or smaller systems.

Expansion of Metabolic Models: Increasingly accounting for compartmentalization

within eukaryotic cells (cytosol and mitochondria) to accurately determine
intracellular flux landscapes. Methods for isolating subcellular compartments and
combining their labeling data with compartmentalized models enhance
understanding, such as identifying cytosolic isocitrate dehydrogenase as a major
source of cytosolic citrate.

Elementary Mode Analysis (EMA): A method to decompose a metabolic network

into elementary flux modes (EMs), which are minimal sets of reactions that can
operate in a steady state.
Polar Space Analysis Predictors (PSYA & EMPA): A novel approach that
transforms the traditional Cartesian yield solution space of EMs into a polar
coordinate system (using modules and angles). This "polar space" offers expanded
visualization, better discrimination of EMs with specific traits, and allows for the
identification of "cluster-paths" of EMs with similar characteristics.

Genetic Modification and Metabolic Engineering Prediction: EMPA (Elementary

Mode Perturbation Analysis) uses the polar solution space to predict the impact of
genetic modifications. It operates on the assumption that cells minimize metabolic
change while adapting, searching for the closest EM that maximizes or minimizes a
particular reaction. This theoretically provides a path with the lowest overall
metabolic modification rate while achieving a desired change in a specific reaction.

Application Example: EMPA analysis successfully identified attractive modification

targets to reduce lactate production in CHO-S cells by increasing mitochondrial
pyruvate transport (v39). This perturbation was found to reduce lactate production
(after a two-fold increase in v39 flux yield), increase TCA flux (up to 15-fold), and
affect glutamine transport and related reactions, consistent with previous reports.

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4. Nutrient Limitation Strategies

Nutrient limitation is a common strategy to enhance product accumulation, particularly for

secondary metabolites or storage compounds like polyhydroxyalkanoates (PHAs).
Burkholderia sacchari and PHA Production: B. sacchari LMG19450 is a promising
microbial platform capable of producing poly(3-hydroxybutyrate) [P(3HB)] from
xylose and other carbohydrates.

Nitrogen (N) vs. Phosphorus (P) Limitation: N-limited fed-batch experiments

resulted in higher specific cell growth (around 0.19 1/h) and substrate consumption
rates (around 0.24 1/h) compared to phosphorus-limited conditions.
Phosphorus limitation, while leading to higher P(3HB) concentration (g/L) and
overall cell dry weight (CDW), required approximately 20 hours longer to achieve an
equivalent P(3HB) content compared to nitrogen limitation. This suggests that pre-
adapting seed cultures to diminished phosphorus could avoid this delay.

Yields: The residual biomass yield from nitrogen (YXr/N) for B. sacchari was around
7.5 g/g, similar to E. coli and P. putida. The YXr/P for B. sacchari was considerably
higher (around 80 g/g) than for E. coli (33 g/g), suggesting lower phosphorus
amounts in B. sacchari biomass or possible polyphosphate accumulation.

Phosphate Transport Genes: Genome analysis of B. sacchari revealed the presence

of a complete pst locus (high-affinity phosphate uptake system) and polyphosphate
kinase genes, indicating a sophisticated system for phosphate acquisition and
storage.

5. Sterilization in Bioprocesses

Sterilization is a critical step to eliminate transmissible agents (fungi, bacteria, viruses,

spores) from media and equipment, ensuring purity and preventing contamination in
biological cultures.

Methods of Sterilization:

Physical Sterilization:

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Heat Sterilization: Moist Heat: Boiling (for metallic devices), Pasteurization (for milk,
alternative heating and cooling), Autoclaving (steam under pressure, 115°C for 60
min or 121°C for 20 min at 15psi, highly efficient and can kill bacterial spores).

Dry Heat and Radiation Sterilization

Chemical Sterilization: Uses agents like ethylene oxide, ozone, chlorine bleach,
glutaraldehyde, formaldehyde, hydrogen peroxide, peracetic acid.

Mechanical Methods:

Filtration: Using filters like Seitz filters (asbestos or other materials, pad-like, thicker
than membrane filters, may absorb solution) or sintered glass filters (glass, brittle, do
not absorb liquids). Candle filters (made of diatomous mud with minute pores) trap
microbes. In summary, the provided sources emphasize the importance of kinetic
modeling, advanced analytical techniques like 13C MFA and EMA, and strategic
nutrient management (e.g., limitation) in optimizing cell culture bioprocesses for
improved product yield and efficiency, particularly in industrially relevant cell lines
like CHO cells and promising microbial factories like Burkholderia sacchari. The
increasing use of mini bioreactors and compartmentalized metabolic models further
highlights the trend towards more rigorous and high-throughput bioprocess
development.

That’s it for Module – 3, hope you liked it. Do check out the videos in reference and stay tuned
for Module – 4.
Happy Learning!!!

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REFERENCES

#1: Biochemical Engineering Fundamentals by Bailey and Ollis

#2: Michaelis-Menten Model
#3: Immobilized Enzyme Kinetics

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