Bahir Dar University

Bahir Dar Institute of Technology

Department of Chemical Engineering
Introduction to Biochemical Engineering course for 5 th year chemical
engineering students
Bahir Dar, Ethiopia 2016
 Biotechnology and Biochemical Engineering
It is dealing with an intersection between biology (Study of life)
and Engineering
Introduction
Definition of biotechnology:
 It is a broad field that involves the application of biological systems, organisms, or
derivatives to develop or create products and technologies for various practical
purposes.
 It is an interdisciplinary area that governs the application of biology and chemistry in
engineering sciences.
 It is the knowledge of the exploitation of living microorganisms and their by-products,
such as enzymes, secondary metabolites, and any product from the pathway of living
organisms. These bio-based products are expanding as safe food additives, medicines,
and cosmetics.
 In the past decades, the application of biotechnology focused only on animal biotechnology or plant cell
technology and horticulture. But today, the development of biotechnology has enhanced and moved beyond the
borders. The knowledge has expanded in many fields of engineering as well as in advance biomaterial and
Nano-biotechnology products.
Definition of biochemical engineering
 Biochemical engineering is a specialized branch of chemical engineering that applies principles and techniques
of chemical engineering to the production of materials, chemicals, pharmaceuticals, and energy from biological
systems. It involves the use of living cells, enzymes, and biological systems in conjunction with engineering
principles to design, optimize, and scale up processes for the production of biologically derived products.
 Key components of biochemical engineering include the design of bioreactors, optimization of fermentation
conditions, downstream processing, and the integration of biological systems into industrial processes.
 The goal is to efficiently and economically produce bio-based products on a large scale while considering
factors such as yield, purity, and sustainability.
The intersection of biology and engineering, represented by fields like biotechnology and
biochemical engineering, is of paramount importance due to its numerous implications and
contributions across various sectors. Here are several reasons highlighting the significance of
this intersection:
• To create new innovative technologies
• Gene therapy
• Bio sensors (medical imaging techniques), glucose level
• Genetically modified organisms (pest resist, harsh environment resist)
• Crop variety
• Biofuel production, bio-remediation
• Developing artificial organ
• Economic development (bio based products supply)
 Over view of biotechnology
Ancient biotechnology practice:
Fermentation, biological control of pest (cats), lupine (medication for animal’s skin),
medicinal plant use (damakasei, aregresa, tenaadame), selective breeding, dye production,
traditional medicine (herbs, animal extracts).
Modern biotechnology practice:
Modern biotechnology practices involve the use of advanced techniques and tools to
manipulate biological systems at the molecular and cellular levels for various applications.
Here are some key examples of modern biotechnology practices: genetic engineering
 Ethical breeding involves careful selection, proper care, and consideration of the long-
term well-being of the animals
 In Ethiopia: GM crops, selective breeding, genetic testing
Key principles and techniques in biotechnology
 Biotechnology encompasses a wide range of principles and techniques that involve the
manipulation of living organisms, cells, and biological systems to develop products and
technologies for various applications. But the two commonly principles are: genetic
engineering and bioprocess engineering.
 Genetic engineering
Principle: The manipulation of genes and genetic material to achieve specific traits or
outcomes.
Techniques: The Recombinant DNA technology, gene cloning, gene editing and genetic
modification.
manipulation of genes and genetic material to achieve specific traits or outcomes.
 In the process of genetic engineering desired genes are isolated and these genes are used to create
recombinant DNA.
 Multiple copies of gene are called gene cloning.
 Transfer of desired gene to the host organism called gene transfer.
 The newly formed DNA called recombinant DNA transfer in the host organism and it does not multiply itself
until it is become part of the genome of the host cell. Hence for the multiplication of DNA in an organism it
should be the part of the chromosome which has a specific sequence known as origin of replication.
 Origin of replication: is a specific DNA sequence which is responsible for initiating replication.
 So the Alein DNA linked to the origin of replication, and then it gets in to the genome of the recipients and
multiply and be inherited along with the host DNA.
 Cloning making identical copies of any template of DNA.
 Generally there are 3 steps of genetically modifying an organism
1. identifying the DNA with desirable genes
2. introducing the identified DNA in to the host
 Bioprocess Engineering: During the process of biotechnology, it’s important to maintain
sterile condition.
To allow the growth of only desired microbes or you can carry out excel large quantities of
microbes for the manufacture of biotechnological products.
Principle: The application of engineering principles to biological systems for the production of
valuable products.
Techniques: Optimization of production conditions, downstream processing, and scale-up
strategies.
How the first recombinant DNA molecule was created?
 In the year 1972, two scientists of USA, Stanley Cohen and Herbert Beyer created the first recombinant
DNA.
 The isolated antibiotic resistance from a plasmid of a bacteria of Salmonella typhimurium.
 A plasmid – is an autonomously replicating circular extra chromosomal DNA.
 A gene is responsible for conferring antibiotic resistance.
 The gene was isolated by cutting the desired piece of DNA from the plasmid using restriction enzyme
(molecular scissors) were used.
 A piece of DNA cut was then linked with the plasmid DNA (act as a vector).
 A vector behaves as a vehicle to transfer the piece of DNA attached to it in to another cell, where it can be
replicated and or expressed.
 The linking of fragment DNA with plasmid DNA was done with another enzyme DNA ligase (act as a gum to
join the cut ends of the DNA molecules).
 These result the new formation of circular autonomously replicating DNA.
 It is created in vitro and is known as recombinant DNA and then it’s transferred to E Coli a bacterium closed
 In its new cell the DNA could make multiple copies by using the new host DNA polymerase
enzyme.
 It is resulted multiple copies of antibiotic resistance gene in E Coli, this process of
multiplication of gene or piece of DNA is known as Cloning.
Basic concepts in biotechnology
DNA and Genetic Material:
Definition: Deoxyribonucleic acid (DNA) is the genetic material that carries the instructions for the development
and functioning of living organisms.
Significance: Understanding DNA structure and function is crucial for manipulating genes in biotechnological
applications.
Recombinant DNA Technology:
Definition: Recombinant DNA technology involves the combining of DNA from different sources to create new
genetic combinations.
Significance: It is a key technique in genetic engineering, allowing the introduction of specific genes into host
organisms.
Genetic Modification (GM) and Genetically Modified Organisms (GMOs):
Definition: Genetic modification involves altering the genetic makeup of an organism, often by introducing
specific genes.
Gene Expression:
Definition: Gene expression is the process by which information from a gene is used to synthesize a functional
product, such as a protein.
Significance: Understanding and manipulating gene expression is crucial for producing desired proteins and
controlling cellular functions.
PCR (Polymerase Chain Reaction):
Definition: PCR is a laboratory technique used to amplify a specific DNA sequence, producing multiple copies for
analysis.
Significance: PCR is widely used in various biotechnological applications, including DNA sequencing,
diagnostics, and gene cloning.
Bioreactors:
Definition: Bioreactors are vessels or systems designed for the cultivation of cells, microorganisms, or enzymes
under controlled conditions.
Significance: Bioreactors are essential for large-scale production in industries such as pharmaceuticals,
biotechnology, and biofuel.
Fermentation:
Definition: Fermentation is a metabolic process that converts sugars into acids, gases, or alcohol in the presence
of microorganisms.
Significance: Industrial fermentation is widely used in biotechnological processes for the production of various
products, including antibiotics and biofuels.
Enzymes and Biocatalysts:
Definition: Enzymes are biological catalysts that speed up chemical reactions. Biocatalysts involves using
enzymes for chemical transformations.
Significance: Enzymes are crucial in biotechnological processes, including the production of pharmaceuticals,
food processing, and biofuel production.
Cloning:
Definition: Cloning involves creating identical copies of a gene, cell, or organism.
Significance: Cloning techniques are used in genetic engineering and the production of organisms with specific
traits.
Tools and techniques in biotechnology
 Recombinant DNA Technology:
Description: Involves the manipulation of DNA to create new genetic combinations by combining DNA from
different sources.
Applications: Gene cloning, production of genetically modified organisms (GMOs), and synthesis of recombinant
proteins.
 Polymerase Chain Reaction (PCR):
Description: A laboratory technique used to amplify specific DNA sequences, producing millions of copies for
analysis.
Applications: DNA sequencing, gene expression analysis, and diagnostics.
 DNA Sequencing:
Description: Determining the precise order of nucleotides in a DNA molecule.
Applications: Genomic studies, personalized medicine, and identification of genetic variations.
 Gene Expression Analysis:
Description: Studying how genes are turned on or off to produce functional products like proteins.
Applications: Understanding cellular processes, disease mechanisms, and drug development.
 Gel Electrophoresis:
Description: Separation of DNA, RNA, or proteins based on their size and charge using an electric field.
Applications: DNA fingerprinting, analysis of gene expression, and protein characterization.
 Bioreactors:
Description: Controlled environments for the cultivation of cells, microorganisms, or enzymes for large-scale
production.
Biotechnology has diverse applications across various fields, contributing to advancements in medicine,
agriculture, and industry.
Here are key areas where biotechnology plays a significant role:
 Medicine :( gene therapy, pharmaceutical, diagnostic tools)
 Agriculture: (genetically modified crops)
 Industrial application: enzyme production, bioremediation
 Environmental: (biological waste treatment, biosensors)
 Food and beverage industry
 Introduction to biochemical engineering
Biochemical engineering plays a crucial role in industrial processes by integrating principles from chemical
engineering with biological systems to design, optimize, and scale up processes for the production of various bio-
based products. Its importance in industrial settings is evident in several key areas:
Bioprocessing and Fermentation, biopharmaceutical production, bio based materials and etc.
Biochemical engineering principles
Industrial application of biochemical engineering
Biochemical engineering has a wide range of industrial applications, contributing to the production of various bio-
based products and processes. Here are some key industrial applications of biochemical engineering:
biopharmaceutical, bio based materials, biogas, biofuel, bio based detergents, bioremediation, biopolymers and etc.
…
Challenges and ethical considerations
 Challenges:
Ethical Concerns and Public Perception: The use of genetically modified organisms (GMOs), gene editing
technologies, and other advanced biotechnological tools raises ethical concerns and may face resistance from the
public.
Environmental Impact: The release of genetically modified organisms into the environment may have
unintended ecological consequences, affecting biodiversity and ecosystems.
 Ethical concern:
Informed Consent: Ensuring that individuals participating in biotechnological research or clinical trials provide
fully informed and voluntary consent.
Equitable Access to Benefits: Fair and equitable distribution of benefits arising from biotechnological
advancements, especially in cases involving indigenous knowledge or genetic resources.
Environmental Stewardship: Implementing practices that prioritize environmental sustainability and minimize
the impact of biotechnological processes on ecosystems.
Openness and Transparency: Consideration: Fostering openness and transparency in scientific research, data
sharing, and communication to build public trust.
Ethical Animal Use: Consideration: Ensuring ethical treatment and use of animals in biotechnological research,
following established guidelines and standards.
Cultural Sensitivity: Consideration: Respecting and incorporating cultural values and perspectives, particularly
when working with indigenous communities or diverse cultural groups.
Education and Public Engagement: Consideration: Promoting education and public engagement initiatives to
enhance awareness and understanding of biotechnological advancements and their ethical implications.
Conclusion
 In conclusion, biotechnology and biochemical engineering are dynamic and
interdisciplinary fields that play a pivotal role in shaping the present and future of science,
medicine, industry, and environmental sustainability. These fields leverage the principles of
biology and engineering to address a wide range of challenges and contribute to
advancements across diverse sectors.
 Modern and traditional biotechnology
 History of biotechnology
 Principles of biotechnology and biochemical engineering
 Industrial applications of both
 Challenges and ethical consideration during applying those fields
2. Enzyme kinetics
Understanding Enzyme kinetics
Definition of enzymes and applications
 A number of amino acids form proteins (20 a.a), 100 or more a.a structured to form many proteins, which has
different functions.
 Among these enzymes are types of proteins, they are biological catalyst they catalyze biochemical reaction in
the body which are called critical molecule.
 Enzymes are biological catalysis.The high efficiency of enzymes makes them commercially valuable and their
specificity of action is offering diverse advantages in clinical medicine.
 Enzymes can be defined as biological polymers that catalyze biochemical reactions.
 Enzymes are highly specialized proteins that act as biological catalysts, facilitating and accelerating chemical
reactions within living organisms. They play a crucial role in metabolic processes, allowing life to exist and
thrive.
Properties of enzymes:
 Enzymes are the complex protein molecules, often called biocatalysts, which are produced by living cells.
 They are highly specific both in the reactions that they catalyze and in their choice of reactants, which are
 Enzymes are applicable in diverse areas
 One of the most common advantages of enzymes is their ability to function continuously
even after their removal or separation from the cells. This means that even after the
separation of cells from in vivo environments, they continue to work efficiently under in
vitro conditions; we can conclude that these biocatalysts remain in an active state even after
their isolation.
 Principally, enzymes are non-toxic, biodegradable and can be produced in ample amounts
by microorganisms for industrial applications.
 An enzyme typically catalyzes a single chemical reaction or a set of closely related
reactions.
 Side reactions resulting in the wasteful formation of by-products are rare in enzyme
catalyzed reactions, in comparison to unanalyzed ones.
Structure of enzymes
 Enzymes are a linear chain of amino acids that generate the three-dimensional structure. The
sequence of amino acids enumerates the structure which in turn identifies the catalytic
activity of the enzyme.
 The structure of the enzyme denatures when heated, leading to loss of enzyme activity,
which is typically connected to the temperature.
 Enzymes are larger than their substrates and their size vary, which range from sixty-two
amino acid residues to an average of two thousand five hundred residues present within fatty
acid synthase.
 Only a small section of the structure is involved in catalysis and are situated next to binding
sites.
 The catalytic site and binding site together constitute the enzyme’s active site.
 Every enzyme has specific substrate, every enzyme recognizes with extremely high specificity,
where like lock and key, where a specific molecule is a key that activate the enzyme function.
 Active site is a specific area where a substrate binds with an enzyme.
 Because it has the right size and composition (has functional groups that make favorable
electrostatic interactions with certain residues) in the active site of the enzyme (vanderwals
interaction, hydrogen bond).
 Substrate bind in to the active site as is or it might cause an induce fit, when the enzyme changes
shape slightly once the substrate is inside.
 Ones binding a number of things happen the bond becomes weaker and easier to break, so many
tasks involved by enzymes is breaking down large components.
 Once bind it forms enzyme substrate complex.
 Once the enzyme catalyze the reaction. The reaction proceeds, the altered substrate leaves, and
 Substrate recognition is stereospecific.
 Some enzymes need non protein component to carryout biological reaction. This non protein
component is called cofactors. And an enzyme along with its cofactor is known as Holoenzyme.
 When cofactor is removed from enzyme is called Apoenzyme.
 Apoenzyme is usually inactive and requires cofactor for its function.
 Cofactor divided in to two (organic and inorganic)
 Inorganic cofactors are metal ions.
 Organic cofactors sub groups in to two the one is tightly bound to the reaction which is prosthetic group
and the other one is release after reaction (coenzyme), most of them can not synthesized by the body
and derived from vitamins (NADH, NADPH, AND FAD).
 Enzyme-Substrate Interaction: The binding of the substrate to the active site is highly specific and often
involves multiple non-covalent interactions, such as hydrogen bonds, electrostatic interactions, and van
der Waals forces.
 Denaturation: Enzymes are sensitive to changes in temperature and pH. Extreme conditions can disrupt
Mechanisms of enzyme reaction
 Enzymes work by lowering the activation energy (Ea or ΔG✳) for a reaction.
 This increases the rate of reaction.
 Enzymes decrease the Gibbs free energy of activation, but they have no effect on the free energy of reaction.
There are two main models of enzyme action
 Lock-and-Key Model:
Lock-and-Key Model: Initially proposed by Emil Fischer, it suggests that the active site has a rigid, preformed
shape that perfectly fits the substrate.
Describes the initial understanding of enzyme-substrate interaction, where the substrate fits perfectly into the
active site like a key into a lock.
 Induced Fit Model:
Proposed by Daniel Koshland, it suggests that both the enzyme and the substrate undergo conformational changes
upon binding, leading to a more complementary fit.
Induced Fit Model: A more refined view, suggesting that the active site can undergo conformational changes to
accommodate the substrate, enhancing the specificity and efficiency of the catalytic process.
Enzyme kinetics
 Enzyme kinetics is concerned with the influence of enzymes on chemical reaction rates.
 Enzyme kinetics is the study of the chemical reactions that are catalyzed by enzymes.
 In enzyme kinetics, the reaction rate is measured and how get changes in response to changes in experimental
parameters such as substrate concentration, enzyme concentration etc.
 This is the oldest approach to understanding enzyme mechanisms and remains the most important.
 The initial rate (or initial velocity), designated Vo, when [S] is much greater than the concentration of enzyme
[E] can be measured by Michaelis–Menten kinetics. It is one of the simplest and best-known models of
enzyme kinetics.
 Michaelis-Menten equation, the rate equation for a one-substrate enzyme-catalyzed reaction.
 Transition state theory suggests that as molecules collide and a reaction takes place, they are momentarily in a
strained or less stable state than either the reactants or the products. During this transition state, the potential
energy of the activated complex increases, effectively creating an energy barrier between the reactants and
products.
 Products can only be formed when colliding reactants have sufficient energy to overcome this energy barrier.
The energy barrier is known as the activation energy (ΔG*) of a reaction. The greater the activation energy for
a given reaction is, the lower the number of effective collisions.
 The molecular model currently used to explain how an enzyme catalyzes a reaction is the induced-fit
hypothesis. In this model, the enzyme binds it’s substrate to form an enzyme–substrate complex where the
structure of the substrate is distorted and pulled into the transition state conformation. This reduces the energy
required for the conversion of a given reactant into a product and increases the rate of a reaction by lowering
the energy requirement and therefore increasing the number of effective collisions that can result in the
formation of the product.
 The Michaelis–Menten equation, as presented by Michaelis and Menten and further developed by Briggs and
Haldane, is fundamentally important to enzyme kinetics. The equation is characterized by two constants: the
Michaelis–Menten constant (Km) and the indirectly obtained catalytic constant, kcat. Although derived from a
simple, single-substrate, irreversible reaction, the Michaelis–Menten equation also remains valid for more
complex reactions.
 A very important term Kcat.
 Kcat (turnover number) is number of substrate molecules converted in to product per active site of an enzyme
in one second. And the unit of Kcat is S-1
Plotting graphs makes easy to understand the kinetics.
E+S-----ES------E+P
When we plot the graph of substrate or product against time we understand the levels of concentration.
 The graph of substrate concentration over time shows a decreasing profile, where the substrate concentration
declines over time during the reaction.
 The graph of product concentration over time shows an increasing profile, where the product concentration
increases over time during the reaction.
 If we take the slope of the graph what we get is the rate of reaction. The rate of reaction is the change of the
concentration of substrate or product per unit time.
 Rate=dp/dt=-ds/dt
 The unit of the rate of reaction is concentration per time.
 It can also called the velocity of reaction.
 It turns out the velocity of reaction changes when substrate changes.
 When the substrate concentration increases the velocity of the reaction also increases until it reaches its
plateau, this is called Vmax (maximum velocity) for a particular enzyme.
 And at the half of Vmax achieved the substrate concentration is equal to Km.
 The ratio of Kcat to Km is used to represent the efficiency of the enzyme and the ratio is called specificity
constant.
 When the ratio is very high, the enzyme is very efficient.
Michael Menten Equation
 The plot shows the velocity of the reaction at different substrate concentration.
 At first the rate of the reaction increases linearly with the substrate concentration. This linear increase is called
first order kinetics.
 In the second plateau region the substrate concentration increases but no longer increases the velocity of the
reaction.
 At this point the velocity reaches Vmax.
 This plateau region is known as 0th order reaction kinetics.
 Which is velocity is independent of substrate concentration.
 For first Y=mx+b
 How ever this equation can not be used for the plateau region, as the velocity is independent of susbstrate.
 This is the reason why we need to derive M.M equation.
 The M.M equation explains this curve mathematically.
 The aim of this equation is to establish the mathematical relation between Vo, Vmax, and, Km such that both
 E+S……..ES reversible reaction under equilibrium also known as equilibrium assumption.
 According to law of mass action
KF [E][S]=KR[ES]
KR/KF= [E][S]/[ES]=Kd (dissociation constant)
 Second assumption pseudo steady state hypothesis (Briggs Haldane)
According to this assumption the concentration of ES complex remains constant during enzymatic reaction.
E+S……ES…..E+P
[ES] formation=[ES] breakdown
KF[E][S]=KR[ES]+Kcat[ES]
KF[E][S]=(KR+Kcat)[ES]
(KR+Kcat)/KF=[E][S]/[ES]
Km =[E][S]/[ES]
 The aim is to establish mathematical relation between Vmax, Vo, and Km.
Vo=dp/dt, product formation depends on dissociation of ES complex, or break down of ES complex.
Vo=Kcat[ES]
In the system there is free enzyme which are not bound in the substrate and other enzymes bound with substrates.
Total enzyme=Eo=E+ES
At Vmax when all the active sites of enzymes are occupied by substrates (E=0)
So, Eo=ES
Vmax= Kcat [Eo]
Km =[E][S]/[ES]
Km=[Eo-ES][S]/[ES]
Km=[Eo][S]-[ES][S]/[ES]
Km=[Eo][S]/[ES]-[S]
Km+[S]=[S]Vmax/Kcat[ES]
Km+[S]=[S]Vmax/Vo/[ES]*[ES]
Evaluation of kinetic parameters
Bioreactor design
Bioreactor design is a critical aspect of implementing enzymatic reactions on an industrial scale. The choice of
bioreactor design can significantly impact reaction efficiency, enzyme stability, and overall process economics.
Here are some common bioreactor designs used in enzyme kinetics:
Stirred-Tank Bioreactor:
Description: Stirred-tank bioreactors, also known as continuous stirred-tank reactors (CSTR), are widely
used in industrial processes. They consist of a tank with an agitator to ensure uniform mixing.
Applications: Suitable for both batch and continuous processes, allowing for precise control of reaction
conditions. However, shear forces from mechanical agitation may affect enzyme stability.
Packed-Bed Bioreactor:
Description: Packed-bed bioreactors involve immobilizing enzymes on a solid support material, creating a
column through which the substrate flows.
Applications: Ideal for continuous processes as they provide a high surface area for enzyme-substrate
interactions. However, mass transfer limitations may occur.
Fluidized-Bed Bioreactor:
Description: Similar to packed-bed reactors, fluidized-bed bioreactors use a solid support for enzyme
immobilization. The substrate flows upward, causing the support particles to become fluidized.
Applications: Effective for continuous processes, offering improved mass transfer compared to packed-bed
reactors.
Inhibitors:
 A modulator (or effector) is a substance which can combine with enzymes to alter their catalytic activities. An
inhibitor is a modulator which decreases enzyme activity.
 An inhibitor binds with the enzyme and decreases the catalytic activity of the enzyme.
 An inhibitor may be: Organic or Inorganic in nature.
 Enzyme inhibitors such as heavy metals (Pb, Cd, Hg) form stable complex with the enzyme and reduce the
activity.
 Such enzyme inhibition may be reversed only by using the chelating agent such as EDTA (ethylene diamine
tetraacetic acid) and citrate.
 The inhibitor can decrease the rate of reaction either competitively, noncompetitively, or uncompetitive.
 Basically, there are two types of inhibition observed, namely reversible and irreversible.
 In reversible inhibition, it is possible to eliminate the inhibitory action by decreasing its concentration (e.g.
dilution).
 In irreversible inhibition, there is no reversal of the inhibitory action due to the formation of covalent bonds.
 Two types of inhibition are possible:
Active site binding: In this case the inhibitor is a compound with a close structural and chemical similarity to the
substrate of the enzyme. As a result, the inhibitor binds to the active site in place of the substrate. The inhibitor
simply blocks the active site, and it is impossible for both of them to bind to the active site at the same time.
Conformational change: In this type, the inhibitor binds to another site which causes a conformational change
to the active site of the enzyme such that the substrate can no longer bind to the enzyme.
The mechanism of the reaction is: E + I………. EI
Competitive Inhibition:
Mechanism: In competitive inhibition, the inhibitor competes with the substrate for binding to the active site
of the enzyme.
Effect: This type of inhibition increases the apparent Km (Michaelis constant) of the enzyme-substrate
complex, but Vmax (maximum velocity) remains unchanged.
Example: Statins, used to lower cholesterol levels, competitively inhibit the enzyme HMG-CoA reductase.
Non-Competitive Inhibition:
Mechanism: Non-competitive inhibitors bind to a site on the enzyme distinct from the active site, causing a
conformational change that reduces the enzyme's activity.
Effect: Non-competitive inhibition decreases Vmax but does not affect Km.
Example: Heavy metals like mercury can act as non-competitive inhibitors.
Uncompetitive Inhibition:
Mechanism: Uncompetitive inhibitors bind only to the enzyme-substrate complex and prevent the release of
the product.
Effect: Both Km and Vmax decrease, and the slope of the Lineweaver-Burk plot changes.
Example: Some drugs used in cancer treatment exhibit uncompetitive inhibition.

Mixed Inhibition:
Mechanism: Mixed inhibitors can bind to either the free enzyme or the enzyme-substrate complex, leading to
different effects on Km and Vmax.
Effect: Km may increase or decrease, while Vmax decreases.
Example: Adenosine deaminase inhibitors can act as mixed inhibitors.
Irreversible Inhibition:
Mechanism: Irreversible inhibitors form strong covalent bonds with the enzyme, permanently inactivating it.
Effect: Irreversible inhibitors cause a permanent decrease in enzyme activity.
Enzyme regulation
Factors affecting enzyme activity
1. Enzyme Concentration: The rate of enzyme-catalysed reactions depends directly on the enzyme concentration.
In the presence of sufficient substrate, increase in enzyme concentration increases the rate of reaction.
2. Substrate concentration: With fixed enzyme concentration, an increase of substrate will result at first in a very
rapid rise in velocity or reaction rate. As the substrate concentration continues to increase, the rate of reaction
begins to slow down until, with a large substrate concentration, no further change in the velocity is observed.
3. Temperature: Enzyme are very sensitive to elevated temperatures, because of their protein nature. The rate of
enzyme-catalysed reaction increases with temperature up to a certain limit. Above a certain temperature, the
activity decreases with temperature because of enzyme denaturation. – For example, the optimum temperature for
most enzymes is between 40 − 45 oC. Hence, above 45 oC rapid denaturation will destroy the catalytic function of
the enzyme.
4. pH: Certain enzyme have ionic groups on their active sites and these ionic groups must be in a suitable form
(acid or base form) for the enzymes to function. Variation in pH of the medium results in changes in the ionic
form of the active site and changes the activity of the enzyme and hence the reaction rate. Changes in pH values
Understanding enzyme kinetics for biochemical engineers
 Process optimization
 Scale up process
 Bioreactor design
 Product yield and purity
 Understanding inhibition and activation
 Cost effective production.
3. Industrial application of Enzymes
Detergent Industry:
Enzyme Type: Proteases, amylases, and lipases.
Application: Enzymes in laundry detergents break down protein stains (proteases), starch-based stains (amylases), and fat/oil
stains (lipases), improving the efficiency of cleaning.
Food and Beverage Industry:
Enzyme Type: Amylases, proteases, cellulases, pectinases.Application: Enzymes are used in various food processing
applications, including starch hydrolysis, brewing, baking, fruit juice extraction, and clarification of fruit juices.
Textile Industry:
Enzyme Type: Cellulases.
Application: Cellulases are used in the denim industry for stone washing, providing a faded and worn appearance to jeans by
selectively breaking down cellulose fibers.
Biofuel Production:
Enzyme Type: Cellulases, amylases, lipases.
Application: Enzymes are crucial in the production of biofuels, such as ethanol and biodiesel, by breaking down complex
polysaccharides and triglycerides into fermentable sugars and fatty acids.
Cellulose conversion
 Biochemical conversion uses enzymes and microorganisms to convert biomass in to sugars and those
sugars into biofuels or bioproducts that can replace products currently made from crude oil. Here is one
example of a biochemical conversion process. After biomass is collected and transported to the processing
facility, it undergoes a pretreatment process so the components of the biomass are easier to breakdown
with enzymes in subsequent steps. Steam or water sometimes in the presence of chemicals, is used to
breakdown the biomass in to cellulose, hemicellulose, and lignin. In one possible approach, acid is used
for pretreatment. However enzymes can’t operate in highly acidic conditions, so a base is used to balance
the pH and the mixture is cooled before the enzymes are added. Enzymes perform a chemical reaction
called hydrolysis.
 During this process the enzymes break the cellulose chains in to glucose and the hemicellulose chains in to
xylose. Glucose and xylose are the sugars that can most readily be fermented in to ethanol or other
biofuels. During the fermentation process, the mixture is inoculated wit microbes such as yeast or bacteria
that digest the sugars and secrete compounds that can be used as biofuels or biofuel components.
 This total conversion process takes approximately three to five days. In the case of ethanol, the liquid is
separated by distillation, which is a method of separating mixtures based on differences in their boiling
points. The resulting ethanol is collected and purified for use in blending with fuel.
 The sugars can also be fermented or chemically converted in to longer chain molecules. With the addition of
hydrogen, these molecules are processed in to renewable gasoline and diesel. The solids and liquids
remaining after distillation, known as stillage, then undergo a series of steps to remove water from the
mixture.
 Solid and liquid separation whether by centrifugation or other means is used to recover the insoluble lignin
rich residue. This residue can then be sent to an onsite combustion system where it is burned to generate
steam and electricity that can be used for power.
Starch conversion
4 main enzymes used in brewing process
Beta-glucanase, alpha amylase, b amylase, protease
Alpha amylase in malting and fermentation process
Alpha amylase released from the starch granules with optimum temperature 73.89 0c and pH: 5.2
Catalyzes the hydrolysis of amylose and amylopectin in to dextrins. Increase the yield of carbohydrate that can be
fermented during fermentation.
B amylase in malting and mashing process. Released from the starch granuels. Optimum ph 5.5 and opt temp
62.78 0c.
Catalyzes the hydrolysis of amylose and amylopectin in to maltose.
B glucanase in malting and mashing process (found endogeneously in barely itself, opt pH: 6 and opt temp: 45-
50).
Helps in breaking the turbidity system by hydrolyzing the beer haze.
Lowers the viscosity of the warts.
Protease: in malting and mashing process
Present naturally in the barely kernel (opt temp: 520c, and opt pH: 7.5-8)
Digestion of protein for clarification and facilitation of malting.
Increase the degree of solubility of the proteins, lowers the viscosity of the beer.
Softens the kernel layer by hydrolyzing the cell wall protein during mashing.
Additional enzymes used in brewing: papain, FICIN, alpha-acetolactate decarboxylase (ALDC)
Papain, FICIN (extracted from figs and pawpaw latex)
Used as chill proof enzymes, hydrolyze the proteins that cause the chilling haze.
(ALDC)- reduce the fermentation time faster beer production.
Catalyze the conversion of alpha-acetolactic acid to acetoin
chill haze doesn't affect the flavor, it does affect the shelf-life of the beer.
Thank you