Introduction to Biochemical

Engineering
ChEg5136
Chapter One
Introduction to biotechnology and
Biochemical
Outline

1. Introduction to Biotechnology and

Biochemical Engineering
 Biotechnology
 Biochemical Engineering
 Biological Process
Why? Biochemical Engineering
Objectives

 Understand the fundamental basics of microbial

kinetics, metabolic stoichiometry and energetics.
 Understand the basics of bioreactor engineering
with knowledge on design and operation of
fermentation processes.
 Develop bioengineering skills for the production
and purification of biochemical product using
integrated biochemical processes
Introduction
Biochemical Engineering is concerned with the
industrialize biological processes.
This field connects biological Science and
chemical engineering
Since the rapid advancements in biotechnology,
the function of biochemical engineers has grown
in importance in recent years
Biotechnology
 Biotechnology is broadly defined as
Commercial techniques that use living organisms
or substances derived from those organisms to
create or modify a product, including techniques
for improving the characteristics of economically
important plants and animals and developing
microorganisms to act on the environment.
(Congress of united state 1984GC)
People have known how to use microorganisms to
ferment
 Beverages
 Food
 Crossbreed plants and animals for better yield
Since ancient times, but they had no idea what was
causing those biological changes.
Biotechnology has recently been used to refer to
innovative techniques such as recombinant DNA and
cell fusion.
Recombinant DNA
Recombinant DNA allows for the direct modification of
individual cells' genetic material,
Can be utilized to create microbes that produce new
products as well as useful organisms.
Genetic engineering refers to laboratory technologies for
manipulating genes within living cells.
The main goal of this technique is to splice a foreign gene
for a desired product into circular forms of DNA (plasmids)
and then insert them into an organism so that the foreign
gene may be expressed and the organism can create the
product.
Cell Fusion
Cell fusion is the process of combining the beneficial properties
of two separate types of cells to generate a single hybrid cell
with nuclei and cytoplasm.
Example
Specialized immune system cells can generate helpful antibodies.
However, cultivating such cells is difficult due to their modest
development rate.
Tumor cells, exhibit longevity and fast growth.
By fusing the two cells, a hybridoma with both characteristics
can be generated.
The hybridoma cells' monoclonal antibodies (MAbs) can be
employed for diagnosis, Disease treatemt and protein purification
Expensive and rare pharmaceuticals such as
 Insulin
 Human growth hormone
 Interferon
 Vaccines
 Monoclonal Antibodies
Can be produced from genetically modified cells or
hybridoma cells cheaply and in large quantities
It is possible to create
disease-free seed stocks or healthier,
higher-yielding food animals.
Important agricultural species can be genetically modified to
have stress, Herbicide and insect resistance features.
Furthermore, recombinant DNA technology can be used to create
genetically engineered microbes that can manufacture more
chemical compounds than unmodified microorganisms
Biochemical Engineering
Pure scientists initiated and developed recombinant
DNA or cell fusion technologies, the end result of
which can be the development of a new breed of
cells in minute amounts capable of producing a
product
The successful commercialization of this method
necessitates the development of a large-scale
process that is both technologically and
commercially viable
In order to design an effective bioreactor to cultivate
the cells in the most optimum conditions. As a result,
biochemical engineering is one of the most significant
fields of biotechnology for commercialization.
Consider a common biological process (bioprocess) that
involves microbial cells.
Raw materials, typically biomass, are processed and
combined with ingredients required for cell growth.
sterilized to remove all other living bacteria before being
placed into bioreactor or fermenter, which is often
outfitted with agitators, baffles, air spargers, and
different sensing devices to manage fermentation
conditions.
A pure strain of microorganisms is introduced into the
vessel
The fermentation process will stop after the number
of cells reach's its maximum concentration and the
medium depleted.
The mixture will further transported to product
recovery and purification process
Biochemical engineers must collaborate with
biological scientists to carry out a bioprocess on a
wide scale:
 To choose the best biological catalyst
 Workable environment for catalyst
 Separate the desired products from the reaction
mixture in the most economical way
The previous activities require process design and
development
Similar strategies that have been used effectively in
chemical processes can be used with minor adjustments.
The following are the basic questions that must be asked
for process development and design:
A. What Change can be expected to occur?
B. How fast will the process take place?
C. Howthe system can be operated and controlled for the
maximum yield?
D. How the downstream process is managed?
Biological Process
Living cells or their components are used in industrial
applications to generate desired physical or chemical
changes.
Biological processes offer advantages and limitations when
compared to Traditional processes.
The following are the primary benefits:
I. Mild reaction condition
II. Specificity
III. Effectiveness
IV. Renewable Resources
V. Recombinant DNA Technology
However the biological process have the following
disadvantages
1. Complex Product Mixture
 The product mixture contains cell mass many metabolic
by-products and unconsumed nutrients
2. Dilute Aqueous Environment
 In an aqueous media, the components of economic
importance are only produced in trace amounts. As a
result, separation is prohibitively expensive.
3. Contamination
 Because numerous environmental bacteria and molds grow
well in most media, the fermenter system is quickly
contaminated.
 The cultivation of plant or animal cells becomes more
challenging since their growth rates are substantially slower
than those of ambient bacteria or molds.
4. Variability
 Cells tend to mutate and may lose some features that are
critical to the process's success.
 Enzymes are relatively sensitive or unstable substances that
must be handled with caution.
 Chapter Two
Enzyme Kinetics
Outline
Enzyme Kinetics
 Introduction

 Simple Enzyme Kinetics and Enzyme Reactor with

Simple Kinetics
 Inhibition of Enzyme Reactions
 Influences on Enzyme Reactions
Introduction
Enzymes
 Are protein molecules that act as biological catalysts.
 They are synthesized by living cells (animal, plant, and
microbe) and
 Are critical as catalysts in biological reactions.
 Almost every cell reaction necessitates the presence of
a unique enzyme.
Enzymes play an important role in the formation
and breakdown of chemical bonds in living systems.
Enzyme reactions are different from chemical reactions, as
follows:
1. An enzyme catalyst is highly specific
2. The rate of an enzyme-catalyzed is much faster than
nonbiological catalyst
3. Operational condition are very mild
4. Very sensitive or unstable molecules
Commercial Application of Enzymes
Enzymes have been employed since the dawn of
time.
 Used for making sweets from starch
 Clotting of milk cheese
 Brewing soy sauces
Have been used commercially since 1890s
Fungal cell extracts, amylase taka diastase, were
first added to brewing bats to facilitate the
breakdown of starch into sugar
Since an enzyme is a protein,
Its function depends on the sequences of amino acids
Its has a complicated tertiary structure
Commercially produced enzymes are
 Industrial enzymes (amylase, protease, isomerase)
 Analytical enzymes ( Glucose oxidase, galactose
oxidase)
 Medical enzymes ( Asparaginase, proteases, lipases)
Example
 − amylase, glucoamylase and glucose isomerase
serve mainly to convert starch into high fructose corn
syrup (HFCS)
It is Sweeter than glucose and can be used in a place of
table sugar (sucrose) in soft drinks

 Alkaline protease is added in laundry detergents

Simple Enzyme Kinetics
The study of the rate of enzyme response and how
it is impacted by various chemical and physical
circumstances is often referred to as enzyme
kinetics.
Kinetic studies of enzymatic reactions reveal
information about
 The basic mechanism of the enzyme reaction
 Factors that characterize the enzyme's
features.
The rate equations derived from the kinetic studies
can be used to calculate
 Reaction time,
 Yields, and
 The optimum economic condition,
All of which are critical in the design of an effective
bioreactor.
Assume a substrate s change to product p with the
help of Enzyme

The rate of reaction expressed in terms of either

the change of substrate Cs or the product
concentration Cp
In order to understand the effectiveness and
characteristics of an enzyme reaction
It is important to know the reaction rate is
influenced by reaction conditions
 Substrate
 Product and
 Enzyme concentration
 The reaction rate is proportional to substrate conc. (first
order reaction)when substrate concentration is in low
range
 The reaction rate does not depend on the substrate
concentration when the substrate concentration is high,
since the reaction rate changes gradually from first order
to zero order as the substrate concentration is increased.
 The maximum reaction rate rmax is proportional to the
enzyme concentration within the range of the enzyme
tested.
Brown (1902) proposed that an enzyme forms a
complex with its substrate
The complex then breaks down to the product and
regenerates free enzymes
Reaction rate equation can be derived from the
above mechanism based on the following
assumption
I. Total enzyme conc. is constant
II. The amount of Enzyme is very small compared
to substrate
III. Product inhibition is negligible since product
conc. is low
Enzyme Reactor with Simple Kinetics
A bioreactor is a device that uses enzymes or living
cells to generate biochemical changes.
A bioreactor is frequently called fermenter
Batch or Steady-state Plug-flow reactor
The simplest reactor configuration for any enzyme
reaction is batch mode
 Agitator
 pH
Ideal batch reactor is assumed to be well mixed
Assumed that an enzyme reaction is initiated at t=0
The equation expressing the change of the substrate
conc. with respect to time can be obtained by
integration.

This equation shows the change Cs with respect to

time in batch reactor
Where Km and rmax are known
Plug-flow reactor
 Substrate enters in one end of the tube which is
packed with immobilized enzyme and the
product leaves at the other end
 There is no mixing, the properties of the flowing
stream will vary in longitudinal and radial
direction.
 At steady state the properties are constant with
respect to time
 At ideal steady state plug-flow react the time t
replace with residence time
 A continuous stirred-tank reactor (CSTR) is an ideal
reactor that assumes the reactor contents are well
mixed.
 The concentrations of the individual components of
the exit stream are considered to be the same as
their concentrations in the reactor.
 By minimizing downtime, continuous running of the
enzyme reactor can considerably boost reactor
production.
 It is also simple to automate to reduce operational
cost
The Substrate balance of a CSTR
Inhibition of Enzyme Reaction

A modulator (or effector) is a chemical that can

bind to enzymes and change their catalytic activity.
An inhibitor is a type of modulator that reduces
enzyme activity.
It can reduce the rate of reaction in
 Competitive,
 Noncompetitive, or
 Partially competitive situations.
Competitive inhibition
Because a competitive inhibitor has a high structural
resemblance to the substrate, both the inhibitor and the
substrate compete for an enzyme's active site.
The development of an enzyme-inhibitor complex limits
the quantity of enzyme available for interaction with the
substrate, lowering the rate of reaction.
Noncompetitive inhibition
Bind to enzymes in a variety of ways.
They can bind to the enzymes in a reversible or
irreversible manner at the active site or in another area.
In any event, the resulting complex is dormant.
Other Influences on Enzyme Reaction
Various chemical and physical variables influence
the rate of an enzyme reaction.
The concentration of various components
(substrate, product, enzyme, cofactor, and so on),
pH, temperature, and shear are all crucial.
The effect of pH
The pH of the reaction solution has a considerable
influence on the rate of an enzyme reaction both in
vivo and in vitro.
Effect of Temperature
The Arrhenius equation can describe the temperature
dependency of various enzyme-catalyzed processes.
Because the atoms in the enzyme molecule have higher
energies and a greater tendency to move as the
temperature rises, the rate of reaction increases.
=
As the temperature rises, denaturation processes progressively
destroy the activity of enzyme molecules.
Because of weak hydrogen bond unfolding
Effect of shear
One of the mechanism to denature enzyme is
mechanical force (Fluid shear)
The influence of shear on enzyme stability is
significant for considering enzyme reactor design
since the contents of the reactor must be agitated
or shaken for better mas-transfer
 Chapter Three

Immobilized Enzyme
Introduction
Because most enzymes are globular proteins, they
are water soluble.
Separating enzyme for reuse in batch procedure is
difficult or impracticable
Enzymes can be chemically or physically immobilized
on the surface or inside of an insoluble matrix.
In addition to the above it can be immobilized in
soluble form by enclosing them in a semipermeable
membrane.
 Immobilized enzyme has the benefit of being
reusable
 Since it can be easily removed from the reaction
solution and retained in a continuous flow reactor
 Immobilized enzyme may exhibit selectively altered
chemical or physical properties
 Mimic the realistic natural environment from where
the enzyme originated the cell
Immobilization Techniques
Immobilization techniques can be classified in two
methods
 Chemical method (Covalent bond)
 Physical Methods (Non Covalent bond)
Chemical Methods
The most common approach for immobilizing enzymes is
the covalent attachment of enzyme molecules to water
insoluble, functionalized substrates via nonessential
amino acid residues (that is, amino acids minus water).
Functional groups of nonessential amino acid
residues that are suitable for the immobilization
process are
 Free , , carboxyl groups
 , amino groups
 Phenyl, hydroxyl, sulfhydryl or imidazole groups
Another variation of immobilization by covalent
attachment is the copolymerization of the enzyme
with a reaction monomer
Where MNE may have the following structure

Water-insoluble supports for the covalent attachment of

enzyme synthetic supports such as
Acrylamide-based polymer, maleic anhydride-based
polymers, methacrylic acid-based polymers, styrene-
based polymers and polypeptides
Natural supports agarose, cellulose, dextran, glass and
starch
Enzyme active site should not be involved in the attachment
Physical Methods
Adsorption is the most basic approach for
immobilizing enzymes.
By interacting an aqueous solution of enzyme with
an adsorbent, enzymes can be physically adsorbed
on a surface-active adsorbent.
Some of the Adsorbents are
Alumina, calcium carbonates, clays, collagen,
resins, diatomaceous earth etc
There are advantage and disadvantage when using
adsorption
Advantages are
 Simple procedures
 Separation and purification can be done while
immobilized
 Don’t usually deactivated enzymes
 Reversible process
Some of the disadvantages are
 Weak bonding
 The state of immobilization is very sensitives
to the solution
pH, ionic strength, and temperature
 The amount of enzyme loaded on a unit
amount of support is usually low
Entrapment
By generating a highly cross-linked network of
polymer in the presence of an enzyme, enzymes can
be entrapped within cross-linked polymers.
This approach has a significant benefit in that there
is no chemical change of the enzyme, therefore the
inherent properties of the enzyme are not altered.
The enzyme, however, may be deactivated during
gel formation.
Enzyme waste is another issue
The most commonly used cross-linked polymer is
the polyacrylamide-gel system
It has been used to immobilized
 Alcohol dehydrogenase
 Glucose-oxidase
 Amin acid –oxidase
 Hexokmase
 Glucose isomerase
 Urease and money more enzymes
Microencapsulation
Semipermeable membrane microcapsules can be used to
immobilize enzymes.
The interfacial polymerization technique can be used.
In a vessel, an organic solvent containing one component
of copolymer with surfactant is stirred, and an aqueous
enzyme solution is added.
While the aqueous phase is dispersed as minute droplets,
the polymer membrane forms at the liquid-liquid
interface.
Effect of Mass-Transfer Resistance
Immobilization of enzymes may pose a new issue
that is not present in free soluble enzymes.
It is caused by the high particle size of the
immobilized enzyme or by the inclusion of enzymes
in the polymeric matrix.
If we trace the hypothetical path of a substrate
from the liquid to the reaction site in an
immobilized enzyme, we may see three steps,
I. Transfer from the liquid to relatively unmixed
liquid layer surrounding the immobilized enzyme
II. Diffusion through the relatively unmixed liquid
layer
III. Diffusion from the surface of the particle to the
active site of the enzyme in an inert support
External Mass-Transfer Resistance
If an enzyme is immobilized on the surface of insoluble
particle, the path is only composed of the first and
second steps
The rate of mass transfer is proportional to the driving
force, the concentration difference

Where Csb and Cs are substrate conc. In the bulk of the

solution and at the immobilized enzyme surface
Ks: mass-transfer coefficient (length/time)
A: is surface area of one immobilized enzyme particle
During the Enzymatic reaction of an immobilized
enzyme the rate of substrate transfer is equal to
that of substrate consumption
If the enzyme reaction can be described Michaelis-
Menten equation
 Reading Assignment:
Internal Mass-Transfer Resistance
 Chapter Four

Industrial Application of
Enzyme
Outline
1.Carbohydrates
2.Starch Conversion
3.Cellulose Conversion
Enzyme uses are divided into three categories:
 Industrial enzymes,
 Analytical enzymes, and
 Medical enzymes.
In this chapter, we will look at numerous industrial
processes that use industrial enzymes, such as
 Starch conversion and
 Enzymatic cellulose hydrolysis.
Before delving into the enzymatic breakdown of starch and
cellulose, let's take a look at the organic chemistry of
carbohydrates.
Carbohydrate
Carbohydrates, which include sugars, starches, and
celluloses, are a prominent class of naturally occurring
chemical molecules.
They are necessary for the survival of plant and animal
life.
Monosaccharides, oligosaccharides, and polysaccharides
are the three major types of carbohydrates.
The simplest carbohydrate units are monosaccharides.
Polysaccharides include hundreds or thousands of these
simple mono saccharide units, whereas oligosaccharides
have two or more of them.
Monosaccharides
The basic carbohydrates molecules are simple sugar or
monosaccharides
Which are polyhydroxy aldehyde, polyhydroxy ketone and
their derivatives
All simple monosaccharides have the general empirical
formula (CH2O)n.
n- is the whole number ranging from 3 to 8.
All monosaccharides can be grouped into two general
classes
A. Aldoses: contain a functional aldehyde grouping (-CHO)
B. Ketoses: contain a functional Ketone grouping (>CH)
Disaccharides
Two sugar can link to each other by losing water form OHs to
form disaccharides
Sucrose, lactose, maltose and cellobiose which all have the
same molecular formula C12H22O11
Disaccharides may hydrolyze to form two monosaccharide
molecule
Polysaccharides
Polysaccharides consists of many simple sugar units linked
together
One of the most important polysaccharide is starch
Which is produced by plant for food storage
A complete hydrolysis of starch yields glucose and the partial
hydrolysis gives maltose
Cellulose is one of the three major structural
components of all plant cell wall with other
components hemicellulose and lignin.
It is the most abundant organic compound of natural
origin on the earth.
Starch Conversation
In recent years the conversion of starch to fructose has
been a very important commercial process.
High Fructose Cron Syrup (HFCS) is approximately twice
as sweet as sucrose
It is used in soft drinks, lactic acid beverage, ice cream…
HFCS can be obtained from variety of cereals and
vegetables such as corn , cassava …
Corn is most important sources for HFCS because of low
cost and excellent utilities of its by-product
Wieldy used technology is Corn wet milling
Cellulose Conversion
Cellulosic waste has tremendous promise as a
feedstock for the production of fuels and chemicals.
Cellulose is a renewable resource that is affordable,
widely available, and abundant.
Commercial and agricultural processes generate a
large amount of waste cellulose products.
Furthermore, municipal institutions must handle or
dispose of massive amounts of cellulosic solid waste.
Lignocellulosic Material
Although lignocellulosic materials share a basic structure,
they differ widely in chemical content and physical
structure.
These materials typically comprise 30 to 60 percent
cellulose,10 to 30 percent hemicellulose (polyoses), and
10 to 20 percent lignin.
Lignin supports and protects cellulose from biological and
chemical attack, whereas cellulose gives strength and
flexibility.
Hemicellulose holds lignin and cellulose together.
Pretreatment
The purpose of pre-treatment is to change the
structure of lignocellulose biomass in order to improve
its ability to form sugars through hydrolysis.
This is accomplished by breaking the lignin seal,
removing lignin and hemicellulose, or increasing the
biomass's porosity.
Pretreatment should increase the yield of fermentable
sugars while minimizing carbohydrate degradation or
loss and the formation of inhibitors for subsequent
hydrolysis and fermentation processes
Physical, chemical, physicochemical and biological
treatments are the four fundamental types of
pretreatment techniques known
Hydrolysis is a process that depolymerizes cellulose and
hemicellulose polysaccharide chains into fermentable
oligosaccharide or monosaccharide forms
Following the pretreatment process, there are two
methods for hydrolyzing the feedstocks for fermentation,
the most common of which are chemical or biological
hydrolysis
Furthermore, there are some other hydrolysis
methods that do not use either of the above two
methods.
These methods include gamma-ray, electron-beam,
and microwave irradiation. However, those
processes are not commercially viable.
In chemical pretreatment
 Acid pretreatment
 Alkali treatment
Acidic treatment
Concentrated or diluted mineral acids such as sulfuric acid
are used to break down hemicelluloses into monomeric
sugars while also removing some lignin.
It necessitates a small amount of water because optimum
temperature necessitates a small amount of energy
The most commonly used technique for hemicellulose
breakdown in this process is diluted acid hydrolysis; the use
of diluted acid (1-4 %) at moderate temperatures of 120°C to
160°C has proven to be adequate for hemicellulose
hydrolysis while promoting little sugar decomposition.
Alkali Treatment
Pretreatment with alkali is more effective in dissolving
lignin and preventing inhibitor production.
which uses sodium or calcium hydroxide to improve
cellulose digestibility.
It usually involves a delignification process that affects
the solubilization of a significant number of
hemicelluloses.
The mechanism of the reaction is reinforced by the
saponification reaction of intermolecular ester bonds
crosslinking between hemicellulose and lignin.
Saponification causes lignin bonds to be cleaved,
making cellulose more accessible to hydrolysis
enzymes.
The main benefit is that residual alkali can be
recycled and reused, and the reaction temperature
and pressure are reduced for effective lignin
removal.
The incorporation of irreversible salts in the
biomass during pretreatment is the primary
disadvantage
Enzyme Hydrolysis
Biological pretreatment with lignin-degrading enzymes
is usually carried out at milder conditions and plays an
efficient role in the lignin removal.
Peroxidases and oxidases are the commonly available
lignocellulosic enzymes among which laccases are the
commercially used enzyme.
Laccases are copper-containing oxidase enzymes and are
involved in degradation of lignin for effective biomass
conversion.
The main advantage is that they do not require high
energy input and do not produce harmful by-products.
Hydrolysis of cellulose material includes the processing
steps that convert carbohydrates polymer which is
cellulose and hemicellulose into monomeric form.
These polymers can be catalyzed enzymatically by
Endoglucanases enzyme
Example
Combined alkaline pretreatment and enzymatic
hydrolysis on Napier grass gives the highest glucose
yield