Biochemical Engineering

INTRODUCTION OF BIOCHEMICAL ENGINEERING

ENZYME KINETICS

Engr. A. Corpuz 09/2021

Chemical Engineering
Cagayan State University – Carig Campus, Tuguegarao City

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1. Introduction
2. Bioprocessing Applications
3. Biotechnology
4. Scaling up of Bioprocessing Technologies
5. Biological Process
6. Fermentation

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Introduction
Biochemical Engineering
• concerned with conducting biological
processes on an industrial scale,
providing the link between biological
sciences and chemical engineering
• Increasing relevance due to dramatic
developments of biotechnology in the
recent years
• An interdisciplinary field

9/15/2021 ARC Aug2021 Netherlands (2020): Algae unit for Algae production as sustainable alternative
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biomass to produce fuel, oil and protein (https://www.alamy.com/)
 Bioprocessing

• Essential part of many foods, chemicals,

and pharmaceutical industries
• The operations may make use of
microbial, animal, and plant cells and
their components (proteins, enzymes,
metabolites) to manufacture new useful
products and destroy harmful wastes.
• Also signifies the utilization of waste
(production of alcohol, biofuels,
therapeutics chemicals such as
antibacterial, vaccines, etc)

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 Bioprocessing Applications

From: Advances in bioreactors for lung bioengineering:

From scalable cell culture to tissue growth monitoring
(Mahfouzi et al, 2021).

➢ Pharmaceuticals and medicines (Insulin, Penicillin, other Antibacterials, Proteins)

➢ Organ growth in reactors (Pancreas for transplanting islet cells for Type I Diabetes; Lung transplantation)
➢ Foods (Fermented foods, Beverages; Functional foods)
➢ Computers based on biological molecules rather than silicon chips (DNA vs silicon: biochips for new-age computers)
➢ Other industrial processes (superorganisms for removal of pollutants; biofuels)
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Insulin story
Slow-
Type 1 release Biosynthetic
diabetes insulin 95% of
insulin global users
(poor (added a prefer
chance of protein found (rat insulin gene
human
normal in fish sperm, spliced into a
protamine) bacterium) E. coli insulin
life)

1921 1936 1970’s 1977 1982 2001

Human insulin
insulin insulin
first approved
from a from genetically
dog's pancreas engineered
pancreas of cattle, pharmaceutical
F. G. Banting, pigs product
Risk:
C. H. Best
animal Eli Lilly Corporation

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diseases ARC Aug2021 6
 Biotechnology
• Traditional biotechnology (Congress of the United States, 1984)
• Commercial techniques that use living organisms, or substances from those organisms, to make or
modify a product, including techniques used for the improvement of the characteristics of economically
important plants and animals, and for the development of microorganisms to act on the environment
Modern biotechnology refers to

1. recombinant DNA
- allows the direct manipulation of genetic material of individual cells, which
may be used to develop microorganisms that produce new products as well as
useful organisms
❖ Genetic Engineering - the genetic manipulation within living cells
Genetic engineering

2. cell fusion
- a process to form a single hybrid cell (hybridoma) with nuclei and
cytoplasm from two different types of cells in order to combine the
desirable characteristics of the two

❖specialized cells of the immune system can produce useful antibodies

- for disease diagnosis, treatment, and protein purification Hybridoma technology
(use of tumor cells: immortality and rapid proliferation)
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 Bioprocessing Technologies
Successful commercialization requires the development of a
large-scale process that is technologically viable and
economically efficient.
Components:
➢ Raw materials – biomass (nutrients, energy source)
➢ Medium – liquid mixture (growth factors)
➢ Cell culture – pure strain (microorganism, animal or plant cell,
or enzyme catalyst)
➢ Bioreactor or fermenter – with multiple components to control
fermentation (optimal design, operation and control)
➢ Product recovery and purification (max purity, minimal cost)

process development and design

✓ Basic sciences for the process
(microbiology, biochemistry, molecular
biology, genetics)
✓ Reaction kinetics
✓ Process control
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✓ Separation processes ARC Aug2021
 Biological Process
o Biological processes have advantages and disadvantages over traditional chemical processes.

Advantages Disadvantages
Mild reaction condition Complex product mixtures

(room temperature, atmospheric (multiple enzyme reactions, various cell components and
pressure, and fairly neutral pH) metabolic by-products)

Specificity Dilute aqueous environments

(enzyme catalyst to chemical reaction/s) (small amounts of and heat sensitive products)

Effectiveness Contamination

(faster enzyme-catalyzed reaction) (molds, bacteria)

Renewable resources Variability

(biomass) (changing environment, sensitive enzymes)

9/15/2021 Recombinant DNA technology ARC Aug2021 10

 Fermentation
• the process for production of alcohol or lactic acid from glucose (traditional)

• Fermentation originally referred to the metabolism of an organic compound under anaerobic conditions
• an enzymatically controlled transformation of an organic compound (A Merriam-Webster, 1977)
• modern industrial fermentation includes both aerobic and anaerobic large-scale culture of organisms

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1. Enzymes as catalysts
2. Enzyme specificity
3. Classification and Applications of Enzymes
4. Catalytic effect
5. Factors affecting enzyme activity
6. Enzyme Kinetics

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 Enzymes (E) as catalysts
• proteins produced by living cells (animal, plant, and microorganisms: pure culture)
• act as catalysts (in small amounts) in biological reactions by decreasing activation energy, AE
• catalytic ability is based on protein structure
• specific chemical reaction is catalyzed at the active site, small portion of the surface of the enzyme
• rate of an enzyme-catalyzed reaction is usually much faster than that of nonbiological catalysts

Grooves as active sites

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(flipper.diff.org)
 Enzyme Specificity
• An enzyme catalyst is highly specific, and catalyzes only one or a small number of chemical reactions
E S P
α-amylase starch glucose + maltose + oligosaccharides
lactase lactose glucose + galactose
lipase fat fatty acids + glycerol
maltase maltose glucose
urease urea + H2O 2NH3 + CO2
cellobiase cellobiose glucose
rennin milk Curd (cheesemaking)
pepsin proteins at acidic pH Hydrolyzed protein
trypsin proteins at mild alkaline pH Hydrolyzed protein
glucose isomerase glucose fructose
glucose oxidase D-glucose + O2 + H2O gluconic acid
alcohol dehydrogenase Ethanol + NAD + acetaldehyde + NADH2
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 Classification of Enzymes (Crueger and Crueger, 1984)
➢ Industrial enzymes: amylases, proteases, glucose isomerase, lipase, catalases, and penicillin acylases

-tons usage

➢ Analytical enzymes: glucose oxidase, galactose oxidase, alcohol dehydrogenase, hexokinase,

muramidase, and cholesterol oxidase e.g., in biosensors
-milligrams to grams usage in their pure forms; high production costs

➢ Medical enzymes: such as asparaginase, proteases, lipases, and streptokinase for drug manufacture,
therapeutics, cleaning wounds, diagnosis
-milligrams to grams usage in their pure forms; high production costs

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 • Alkaline protease: cleaning aid in laundry detergents
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• Protease: meat tenderizer, cheese making
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• Modulators – substances which can combine with enzymes to alter their catalytic activities
• Others: shear, product concentration
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 ENZYME KINETICS
• Enzyme kinetics deals with the RATE of enzyme reaction and how it is affected by the various chemical
and physical reactions.
• Kinetic studies of enzymatic reactions provide information about the basic mechanism of the enzyme
reaction and other parameters that characterize the properties of the enzyme.
• The rate equations developed from the kinetic studies can be applied in calculating reaction time,
yields and optimum economic condition, which are important in the design of an effective bioreactor.

1. Simple Enzyme Kinetics

A. Michaelis Menten-approach
1. Batch PFR
2. CSTR
2. Complex Enzyme Kinetics
A. Competitive
B. Noncompetitive Inhibition
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 Simple Enzyme Kinetics

Rate or reaction, rs

Change of product and substrate conc. wrt time

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 (Brown, 1902)
k3
max. reaction rate rmax or vmax
k2

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conc. within the range of the enzyme tested.
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 Simple Enzyme Kinetics

The effect of substrate conc. on the initial reaction rate

• Empirical expression
(Henri, 1902)

Kinetic parameters rmax and KM to be experimentally determined.

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 Simple enzyme kinetics
➢ substrate or product inhibition not accounted for

1
To derive rate equation: 2
Measurable entities: CS, CE0

Michaelis-Menten approach: assumed that the product-releasing step (2) is much slower than
the reversible reaction (1) – justified by weak interaction within E-S complex; employed in heterogeneous catalytic
reactions in chemical kinetics

Briggs-Haldane approach: d(CES)/dt = 0, pseudo steady-state (or quasi-steady-state) assumption

in chemical kinetics; applied in homogeneous catalytic reactions

Numerical solution: solution of DE

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 Michael-Menten Kinetics
Rate-limiting step 2 1

Equilibrium rxn 1 2

Enzyme conservation 3
Kinetic parameters:
(2&3)
KM, Michaelis constant
Michaelis-Menten rmax, max. rxn rate
equation

when KM is equal to CS,

r =1/2 rmax

(4&1)
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Exercise 2.1
• Rate expression for numerical solution: expressed in terms of rate constant(s) and reactant concentration(s)
𝑑𝐶𝑝
=
𝑑𝑡
𝑑𝐶𝐸𝑆
=
𝑑𝑡
𝑑𝐶𝑆
=
𝑑𝑡
Conservation equation:
simultaneous differential equations can be solved numerically by using a computer; requires
the knowledge of elementary rate constants, k1, k2, and k3 and initial molar concentration of
enzyme. The elementary rate constants can be measured by the experimental techniques such
as pre-steady-state kinetics and relaxation methods

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 Example 2.1: Derive the rate equation

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 Evaluation of Michaelis-Menten Parameters
• Conduct a series of batch runs of different S concentrations at constant CE0
• Get initial reactions rates, r, measured at CS,0
• By regression

𝑟𝑚𝑎𝑥 𝐶𝑠
Linearization: 𝑟=
𝑘𝑀 + 𝐶𝑠
Langmuir plot 𝐶𝑠 𝑘𝑀 𝐶𝑠
= +
𝑟 𝑟𝑚𝑎𝑥 𝑟𝑚𝑎𝑥
1 𝑘𝑀 1
Lineweaver-Burk plot = +
𝑟 𝑟𝑚𝑎𝑥 𝐶𝑠 𝑟𝑚𝑎𝑥
Eadie-Hofstee plot
𝑟𝑘𝑀
𝑟 = 𝑟𝑚𝑎𝑥 −
𝐶𝑆
Nonlinear regression: Solver excel, see example (2.3)
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 Evaluation of Michaelis-Menten Parameters
• Conduct a series of batch runs of different S concentrations at constant CE0
• Get initial reactions rates, r, measured at CS,0
• By regression

𝑟𝑚𝑎𝑥 𝐶𝑠
Linearization: 𝑟=
𝑘𝑀 + 𝐶𝑠
Langmuir plot 𝐶𝑠 𝑘𝑀 𝐶𝑠
=
𝑟 𝑟𝑚𝑎𝑥 𝑟𝑚𝑎𝑥

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 Evaluation of Michaelis-Menten Parameters
• Conduct a series of batch runs of different S concentrations at constant CE0
• Get initial reactions rates, r, measured at CS,0
• By regression

𝑟𝑚𝑎𝑥 𝐶𝑠
Linearization: 𝑟=
𝑘𝑀 + 𝐶𝑠
1 𝑘𝑀 1
Lineweaver-Burk plot = +
𝑟 𝑟𝑚𝑎𝑥 𝐶𝑠 𝑟𝑚𝑎𝑥

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 Evaluation of Michaelis-Menten Parameters
• Conduct a series of batch runs of different S concentrations at constant CE0
• Get initial reactions rates, r, measured at CS,0
• By regression

𝑟𝑚𝑎𝑥 𝐶𝑠
Linearization: 𝑟=
𝑘𝑀 + 𝐶𝑠

𝑟𝑘𝑀
Eadie-Hofstee plot 𝑟 = 𝑟𝑚𝑎𝑥 −
𝐶𝑆

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Exercise 2.3
Linear and Non-linear regression to estimate kinetic parameters

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 Enzyme Reactor with Simple Kinetics
pH control

Residence
time
Assumptions
1. Batch reactor contents are well mixed.
2. At steady state PFR, the properties will be constant wrt time. Change of CS with time in a batch reactor can be predicted.

1. Batch Reactor
2. Steady-State Plug-Flow Reactor (PFR) or Tubular flow plot of (CS0-CS)/ ln
(CS0/CS) vs t / ln(CS0/CS)
• ideal reactor can approximate the long tube, packed-bed, and
hollow fiber, or multi-staged reactor
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 3. Continuous Stirred-Tank Reactor

the substrate concentration of the reactor should be constant

Dilution Residence
rate time
Assumption: reactor contents are well mixed.

plot of CS vs CS τ /(CS0 - CS)

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Models for More Complex Enzyme Kinetics
• Inhibited
▪ Inhibitor – decreases enzyme activity either competitively, noncompetitively, or partially
competitively
• Competitive - the reaction rate decreases due to the presence of inhibitor, but
a larger amount of substrate is required to reach the maximum rate

• Non-competitive- maximum reaction rate will be decreased by the presence

of a noncompetitive inhibitor while Michaelis constant KM will not be affected

• Other factors
concentration of various components (substrate, product, enzyme, cofactor, and so on), pH, T, shear

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Noncompetitive Inhibition
• Inhibitor can bind to the enzymes reversibly or irreversibly at the active site or at some other region.
• Assume same dissociation constant for the first equilibrium reaction with that of the third equilibrium reaction

Since S and I do not compete for a same site for the formation of E-S or E-I
complex, we can assume that dissociation constant for the 1st equilibrium rxn
is the same as that of the 3rd equilibrium rxn; similarly to 2nd and 4th rxns

slow reaction Partially Competitive Inhibition

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Competitive vs non-competitive

competitive noncompetitive
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ARC Aug2021
HW1:
kinetic parameters
evaluation

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HW1:
Deriving rate
equation

• Hints:
Meaning, k3>>k4
• Set up rate equation based on rate
limiting step
• Additional 2 relations from equilibrium
rxns
• Setup enzyme conservation
• One by one, aim to express rate
equation in terms of CS1, CS2, Vm, and
rate constants noting that

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HW1:
• mumol = µmol
• 1 unit activity = 1µmol product/min

• Hints:
• Initial rate is the slope
• To compute activity, how much enzyme
was used? Relate to initial rxn rate

µmol/mL.min

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HW1:

• Batch reactor
• CSTR
• Hints:
• Generation is zero in your OMB for CSTR.

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References
• Lee, J. M. (2009). Biochemical engineering. Englewood Cliffs,
NJ: Prentice Hall
• Doran, P. M. (2013). Bioprocess engineering principles.
Elsevier.

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