BATANGAS STATE UNIVERSITY

College of Engineering, Architecture and Fine Arts

Gov. Pablo Borbon Campus II,
Alangilan, Batangas City, Philippines 4200

ChE526
BIOCHEMICAL ENGINEERING

BIOCHEMICAL ENGINEERING
& ENZYME KINETICS

Valencia, Jenna Mariz

Victorio, Shenjin
Villamin, Princess
Yaba, Tricia Elena
Yurong, Jackie Meileen
ChE-5202

Dr. Sicily B. Tiu

January 23, 2017

BIOCHEMICAL ENGINEERING
Definition
Biochemical engineering is a branch of chemical engineering that mainly deals
with the design and construction of unit processes that involve biological organisms or
molecules, such as bioreactors. Its applications are in the petrochemical industry, food,
pharmaceutical, biotechnology, and water treatment industries.
It is concerned with conducting biological processes on an industrial scale,
providing a link between biology with chemical engineering. The role of biochemical
engineers has become more important in recent years due to the dramatic
developments of biotechnology.

Biochemistry vs. Biochemical engineering

Biochemistry is the branch of science concerned with the chemical and
physicochemical processes that occur within living organisms.
Biochemical Engineering is concerned with design, development, implementation
and operation of processes for handling biological materials and biocatalysts.
Biochemistry is more about discovering how life works. Biochemists figure out how
different proteins interact with each other, and how those interactions are important in
the processes that keep us alive. Biochemical engineering is more about rearranging
these interactions to make life work better. Biochemical engineers often make modified
proteins, or devices, which help regulate human health when it's out of balance.

Core concepts of biochemical engineering

Introduction
BIOTECHNOLOGY

Biotechnology is the art and science of converting reactants into useful products
by the action of microorganisms or enzymes.

BIO-PROCESSING

Any process in which microorganisms play an essential role in getting

transformation of feed into useful products is called as bio- processing.

Biochemical Engineering

o It is the extension of chemical engineering principles to systems using a

biological catalyst to bring about desired chemical transformations.
o It mainly deals with the design and construction of unit processes that
involve biological organisms or molecules, such as bioreactors.
o It is usually divided into biochemical reaction engineering and bio
separations.
o It is an important area in modern biotechnology.
o Cell culture can be scaled up, biological products separated, purified and
prepared on a large scale.
 o It is the key for biotechnology development to intensify the researches
into biological reactors and the separation, purification technologies for biological
products.

BIOCHEMICAL INDUSTRIES

1. Food Industries
2. Pharmaceutical Industries
3. Brewery and Distilling Industries
4. Waste Treatment

The basic questions which need to be asked for the process development and
design are as follows:

1. What change can be expected to occur?

To answer this question, one must have an understanding of the basic sciences
for the process involved.
2. How fast will the process take place?
If a certain process can produce a product, it is important to know how fast the
process can take place.
3. How can the system be operated and controlled for the maximum yield?
For the optimum operation and control, reliable on-line sensing devices need to
be developed.
4. How can the products be separated with maximum purity and minimum costs?
For this step, the downstream processing (or bioseparation), a biochemical
engineer can utilize various separation techniques developed in chemical
processes such as distillation, absorption, extraction, adsorption, drying, filtration,
precipitation, and leaching.

Roles of Biochemical Engineers

The main role of a biochemical engineer is to optimize the growth of microbes

under aerobic conditions in a reaction mixture with volume of around a thousand
liter
They design economic processes in which maximum biomass yield is obtained
with lesser input of raw material and at cheap operating costs
They ensure the maximum efficiency, safety operation and quality of product
output
Biochemical engineers arent limited to a lab alone, they can also include the
overall supervision of a plant, and providing technical management services
Chemical engineer who has immense knowledge about the basics of life science
and biotechnology

TRIVIA:
Average Biochemical Engineer Hourly Wage in the US:
Biochemical Engineers earn a median hourly wage of $46.11. Hourly
wages typically start from $25.01 and go up to $72.52.

Average Biochemical Engineer Yearly in the US:

Biochemical Engineers earn a median salary of $95,900 per year. Salaries
typically start from $52,010 and go up to $150,830.
 ENZYMES
- are chemicals that arent consumed in a reaction but can speed up a reaction.
- are biological catalysts that are protein molecules in nature.
- Produced by living cells (animal, plant, and microorganism) and are absolutely
essential as catalysts in biochemical reactions.

A major function of enzymes in a living system is to catalyze the making and

breaking of chemical bonds. Therefore, like any other catalysts, they increase the
rate of reaction without themselves undergoing permanent chemical changes.
Catalase one of the most common enzymes that is found in almost all living cells,
especially eukaryotic cells.
- Breaks down hydrogen peroxide

Hydrogen Peroxide produced naturally in chemical reactions

How enzymes work

For two molecules to react they must collide with one another. They must collide
in the right direction (orientation) and with sufficient energy. Sufficient energy means
that between them they have enough energy to overcome the energy barrier to reaction.
This is called the activation energy.

Enzymes have an active site. This is part of the molecule that has just the right
shape and functional groups to bind to one of the reacting molecules. The reacting
molecule that binds to the enzyme is called the substrate.

Enzymes can be regulated by other molecules that either increase or reduce

their activity. Molecules that increase the activity of an enzymes are called activators,
while molecule that decrease activity of an enzyme are called inhibitors.
There are many kinds of molecules that block or promote enzyme function, and
that affect enzyme function by different routes.
Competitive vs. noncompetitive
In many well-studied cases, an activator or inhibitor's binding is reversible,
meaning that the molecule doesn't permanently attach to the enzyme. Some important
types of drugs act as reversible inhibitors. For example, the drug tipranivir, which is
used to treat HIV, is a reversible inhibitor. It blocks activity of a viral enzyme that helps
the virus make more copies of itself.
Reversible inhibitors are divided into groups based on their binding behavior. We
won't discuss all of the types here, but we will look at two important groups: competitive
and noncompetitive inhibitors.
An inhibitor may bind to an enzyme and block binding of the substrate, for
example, by attaching to the active site. This is called competitive inhibition, because
the inhibitor competes with the substrate for the enzyme. That is, only the inhibitor or
the substrate can be bound at a given moment.
 In noncompetitive inhibition, the inhibitor doesn't block the substrate from
binding to the active site. Instead, it attaches at another site and blocks the enzyme
from doing its job. This inhibition is said to be "noncompetitive" because the inhibitor
and substrate can both be bound at the same time.

Diagram illustrating competitive and noncompetitive inhibition.

The competitive inhibitor binds to the active site and prevents the substrate from
binding there. The noncompetitive inhibitor binds to a different site on the enzyme; it
doesn't block substrate binding, but it causes other changes in the enzyme so that it can
no longer catalyze the reaction efficiently.

Allosteric regulation

Allosteric regulation, broadly speaking, is just any form of regulation where the
regulatory molecule (an activator or inhibitor) binds to an enzyme someplace other than
the active site. The place where the regulator binds is called the allosteric site.
 The left part of this diagram shows allosteric inhibition. The allosteric inhibitor
binds to an enzyme at a site other than the active site. The shape of the active site is
altered so that the enzyme can no longer bind to its substrate.
The right part of this diagram shows allosteric activation. The allosteric activator
binds to an enzyme at a site other than the active site. The shape of the active site is
changed, allowing substrate to bind at a higher affinity.
Pretty much all cases of noncompetitive inhibition (along with some cases of
competitive inhibition, the ones where the inhibitor binds elsewhere than the active site)
are forms of allosteric regulation.
However, some enzymes that are allosterically regulated have a set of unique
properties that set them apart. These enzymes, which include some of our key
metabolic regulators, are often given the name of allosteric enzymes. Allosteric
enzymes typically have multiple active sites located on different protein subunits. When
an allosteric inhibitor binds to an enzyme, all active sites on the protein subunits are
changed slightly so that they work less well.
There are also allosteric activators. Some allosteric activators bind to locations
on an enzyme other than the active site, causing an increase in the function of the
active site. Also, in a process called cooperativity, the substrate itself can serve as an
allosteric activator: when it binds to one active site, the activity of the other active sites
goes up.^{3}3start superscript, 3, end superscript This is considered allosteric regulation
because the substrate affects active sites far from its binding site.

Cofactors and coenzymes

Many enzymes dont work optimally, or even at all, unless bound to other non-
protein helper molecules called cofactors. These may be attached temporarily to the
enzyme through ionic or hydrogen bonds, or permanently through stronger covalent
bonds. Common cofactors include inorganic ions such as iron \text {(Fe}^{2+})(Fe2+)left
parenthesis, F, e, start superscript, 2, plus, end superscript, right parenthesis and
magnesium (\text {Mg}^{2+})(Mg2+)left parenthesis, M, g, start superscript, 2, plus, end
superscript, right parenthesis. For example, the enzyme that builds DNA molecules,
DNA polymerase, requires magnesium ions to function.^44start superscript, 4, end
superscript
Coenzymes are a subset of cofactors that are organic (carbon-based)
molecules. The most common sources of coenzymes are dietary vitamins. Some
vitamins are precursors to coenzymes and others act directly as coenzymes. For
example, vitamin C is a coenzyme for several enzymes that take part in building the
protein collagen, a key part of connective tissue.

Nomenclature of Enzymes

Originally enzymes were given nondescriptive names such as:

rennin - curding of milk to start cheese-making processcr

pepsin - hydrolyzes proteins at acidic pH

trypsin - hydrolyzes proteins at mild alkaline pH

Name of substrate + ase

a-amylase starch glucose + maltose + oligosaccharides

lactase lactose glucose + galactose
lipase fat fatty acids + glycerol
maltase maltose glucose
urease urea + H20 2NH3 + CO2
cellobiase cellobiose glucose

The nomenclature was later improved by adding the suffix -ase to the name of
the substrate with which the enzyme functions, or to the reaction that is catalyzed. For
example:

Reaction which is catalyzed + ase

alcohol dehydrogenase ethanol + NAD+ acetaldehyde + NADH2

glucose isomerase glucose fructose
glucose oxidase D-glucose + O2 + H20 gluconic acid
lactic acid dehydrogenase lactic acid pyruvic acid

Commercial Applications of Enzymes

Due to on-going research by biotechnologists, enzymes now have a large number

of commercial applications. They carry many advantages, with one important one being
that enzymes are specific to only one catalytic reaction and so they therefore do not
produce a range of unwanted by-products. Commercial applications of enzymes:

Enzymes are widely used in the textile industry. They are used for improving
production methods and for fabric finishing. In this industry, a very common
 application is the use of the enzyme amylase in order to remove starch size.
Examples of enzymes that may be used in the textile industry:
o Cellulase for stonewashing denim, polishing of cotton
o Catalase removing hydrogen peroxide
o Pectinase for bioscouring (a way to scour fabrics)
o Alpha amylase for desizing at low temperatures

The food and drink industry has to be one of the largest markets for enzymes.
In the baking industry, enzymes are added to the dough when baking bread to
ensure that the bread is high in quality and has a better volume (that there is more
of it). Enzymes also have the ability to preserve bread; keeping it fresh for a longer
period of time and therefore increasing its shelf life.
o Fungal alpha amylase for dough improvement in the bread making
industry
o Glucoamylase used in fermentation
o Papain enzymes for fermentation in the brewing industry
o Beta glucanse for filtration
o Protease used in biscuit production

Enzymes are also used in the pulp and paper industry. Amylase is used for
modification of starch coating and xylanases to reduce the consumption of bleach
chemicals are very well known applications, but nowadays lipases for is used for
pitch control, esterases is used for stickies removal and amylases and cellulases
are used for improved deinking and cellulases for fiber modification have become
an integral part of the chemical solutions used in the pulp and paper mills.[2] In the
manufacturing of coated papers, a starch-based coating formulation is used in order
to coat the surface of the paper. Compared with the uncoated paper, the coating
provides a number of benefits, including; improved gloss, a smoother texture, and
printing properties.
o Cellulase can be used for pulp deinking and pulp refining
o Xylanase for pulp bleaching
o Alpha amylase starch modification

Enzymes are used in detergents and in personal care and hygiene. They are used in
many household and industrial detergents. This industry, in addition to the food
processing industry is currently one of the largest application areas for enzymes. This is
because the enzymes are very effective at relatively low temperatures and pH values.
They contribute to a: better overall cleaning performance; they are biodegradable so
they do not really effect the environment that much; they reduce water consumption
through more effective release of soil.
SIMPLE ENZYME KINETICS

Enzyme kinetics deals with the rate of enzyme reaction and how it is affected by various
chemical and physical conditions. 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.

Assume that a substrate (S) is converted to a product (P) with the help of an enzyme
(E) in a reactor as

If you measure the concentrations of substrate and product with respect to time, the
product concentration will increase and reach a mmaximum value, whereas the
substrate concentration will decrease as shown in Figure 2.1

The rate of reaction can be expressed in terms of either the change of the substrate Cs
or the product concentrations C as follows:

In order to understand the effectiveness and characteristics of an enzyme reaction, it is

important to know how the reaction rate is influenced by reaction conditions such as
substrate, product, and enzyme concentrations. If we measure the initial reaction rate at
different levels of substrate and enzyme concentrations, we obtain a series of curves
like the one shown in Figure 2.2. From these curves we can conclude the following:

1. The reaction rate is proportional to the substrate concentration (that is, first-order
reaction) when the substrate concentration is in the low range.
2. 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.
3. The maximum reaction rate rmax is proportional to the enzyme concentration within
the range of the enzyme tested.
Henri observed this behavior in 1902 (Bailey and Ollis, p. 100, 1986) and proposed the
rate equation

where rmax and KM are kinetic parameters which need to be experimentally determined.
Eq. (2.4) expresses the three preceding observations fairly well. The rate is proportional
to Cs (first order) for low values of Cs, but with higher values of Cs, the rate becomes
constant (zero order) and equal to rmax. Since Eq. (2.4) describes the experimental
results well, we need to find the kinetic mechanisms which support this equation.

Brown (1902) proposed that an enzyme forms a complex with its substrate. The
complex then breaks down to the products and regenerates the free enzyme. The
mechanism of one substrate enzyme reaction can be expressed as

One of the original theories to account for the formation of the enzyme-substrate
complex is the "lock and key" theory. The main concept of this hypothesis is that there is
a topographical, structural compatibility between an enzyme and a substrate which
optimally favors the recognition of the substrate as shown in Figure 2.3.

The reaction rate equation can be derived from the preceding mechanism based on the
following assumptions:
1. The total enzyme concentration stays constant during the reaction, that is, CEO= CES +
C
2. The amount of an enzyme is very small compared to the amount of
substrate.Therefore, the formation of the enzyme substrate complex does not
significantly deplete the substrate.
3. The product concentration is so low that product inhibition may be considered
negligible.

In addition to the preceding assumptions, there are three different approaches to derive
the rate equation:
1. Michaelis-Menten approach (Michaelis and Menten, 1913): It is assumed that the
product-releasing step, Eq. (2.6), is much slower than the reversible reaction, Eq. (2.5),
and the slow step determihes the rate, while the other is at equilibrium. This is an
assumption which is often employed in heterogeneous catalytic reactions in chemical
kinetics. Even though the enzyme is soluble in water, the enzyme molecules have large
and complicated three-dimensional structures.
2. Briggs-Haldane approach (Briggs and Haldane, 1925): The change of the
intermediate concentration with respect to time is assumed to be negligible, that is,
d(CES)/dt = O. This is also known as the pseudo-steady-state (or quasi-steady-state)
assumption in chemical kinetics and is often used in developing rate expressions in
homogeneous catalytic reactions.
3. Numerical solution: Solution of the simultaneous differential equations developed
from Eqs. (2.5) and (2.6) without simplification.

MICHAELIS-MENTEN APPROACH
Assumption:
Product releasing step is slower than Reversible reaction.
Reversible reaction is in Equilibrium.

If the slower reaction, Product-releasing step, determines the overall rate of reaction,
the rate of product formation and substrate consumption is proportional to the
concentration of the enzyme-substrate complex as:

The concentration of the enzyme-substrate complex CES in Eq. (2.7), can be related to
the substrate concentration C S and the free-enzyme concentration C from the
assumption that the first reversible reaction Eq. (2.5) is in equilibrium. Then, the forward
reaction is equal to the reverse reaction so that

By substituting Eq. (2.8) into Eq. (2.7), the rate of reaction can be expressed as a
function of Cs and CE, of which CE cannot be easily determined. If we assume that the
total enzyme contents are conserved, the free-enzyme concentration CE can be related
to the initial enzyme concentration CEO

So, now we have three equation"s from which we can eliminate C E and CES to express
the rate expression as the function of substrate concentration and the initial enzyme
concentration. By substituting Eq. (2.8) into Eq. (2.9) for CE and rearranging for CES' we
obtain

Substitution of Eq. (2.10) into Eq. (2.7) results in the final rate equation

which is known as Michaelis-Menten equation and is identical to the empirical

expression Eq. (2.4). KM in Eq. (2.11) is known as the Michaelis constant. In the
Michaelis-Menten approach, KM is equal to the dissociation constant K1 or the
reciprocal of equilibrium constant Keq as
The unit of KM is the same as Cs. When KM is equal to Cs, r is equal to one half of rmax
according to Eq. (2.11). Therefore, the value of KM is equal to the substrate
concentration when the reaction rate is half of the maximum rate rmax (see Figure 2.2).
KM is an important kinetic parameter because it characterizes the interaction of an
enzyme with a given substrate.

The Michaelis-Menten equation is analogous to the Langmuir isotherm equation:

where:
is the fraction of the solid surface covered by gas molecules
K is the reciprocal of the adsorption equilibrium constant.

ENZYME INHIBITION

Inhibitors are chemicals that reduce the rate of enzymatic reactions. They block
the enzyme but they do not usually control it.

Types of Inhibition

1. Competitive Inhibition

In competitive inhibition, the inhibitor competes with the substrate for the same
binding site. The inhibitor binds only to the free enzyme, not to the ES complex.

Reaction Mechanism:

Example:
 Sulfanilamide is a competitive inhibitor of p-
aminobenzoic acid. Sulfanilamides (also known as
sulfa drugs, discovered in the 1930s) were the
first effective systemic anti-bacterial agents. Because
we do not make folic acid, sulfanilamides do not affect human
cells.

2. Noncompetitive Inhibition

In noncompetitive inhibition, the inhibitor does not interfere with substrate binding
and vice versa. The inhibitor binds enzyme regardless of whether the substrate is
bound. The inhibitor binds equally well to free enzyme and the ES complex.

Reaction Mechanism:

Example:

Fructose 1,6-biphosphatase inhibition by AMP

Fructose 1,6-biphosphatase is a key regulatory enzyme in the
gluconeogenesis pathway. High amounts of AMP signal that ATP levels are low and
gluconeogenesis should be shut down while glycolysis is turned on.

High AMP levels inhibit fructose 1,6-biphosphatase (shutting down

gluconeogenesis) and activate phosphofructokinase (turning on glycolysis).
Regulation of fructose 1,6-biphosphatase and phosphofructokinase by AMP
prevents a futile cycle in which glucose is simultaneously synthesized and broken
down.

3. Uncompetitive Inhibition

In competitive inhibition, the inhibitor binds only to the ES complex. It does not
bind to the free enzyme so there is noel complex in the reaction mechanism.

Reaction Mechanism:
 Example:

Alkaline phosphatase inhibition by phenylalanine

At alkaline pH, alkaline phospatase catalyzes the release of inorganic
phosphate from phosphate esters. It is found in a number of tissues, including
liver, bile ducts, intestine, bone, kidney, placenta, and leukocytes. Alkaline
phosphatase plays a role in the deposition of hydroxyapetite in osteoid cells
during bone formation.

The function of alkaline phosphatase in other tissues is not known. Serum

alkaline phosphatase levels are important diagnostics markers for bone and liver
disease.

4. Irreversible Inhibition

In irreversible inhibition, the inhibitor binds to the enzyme irreversibly through

formation of covalent bond with the enzyme, permanently inactivating the enzyme.
Enzyme is inactivated until all of the irreversible inhibitor is used up.

Reaction Mechanism:

Examples:

Diisopropylphosphofluoridate
- Prototype for the nerve gas sarin
- Permanently inactivates serine proteases by forming a covalent bond with the
active site serin.
Reaction Mechanism:
EFFECT OF TEMPERATURE
The rate of a chemical reaction depends on the temperature according to Arrhenius
equation as

The temperature dependence of many enzyme-catalyzed reactions can be described by

the Arrhenius equation. An increase in the temperature increases the rate of reaction,
since the atoms in the enzyme molecule have' greater energies and a greater tendency
to move. However, the temperature is limited to the usual biological range. As the
temperature rises, denaturation processes progressively destroy the activity of enzyme
molecules. This is due to the unfolding of the protein chain after' the breakage of weak
(for example, hydrogen) bonds, so that the overall reaction velocity drops.

For many proteins, denaturation begins to occur at 45 to 50C. Some enzymes are very
resistant to denaturation by high temperature, especially the enzymes isolated from
thermophilic organisms found in certain hot environments.

EFFECT of pH

The rate of an enzyme reaction is strongly influenced by the pH of the reaction solution
both in vivo and in vitro The typical relationship between the reaction velocity and pH
shows a bell-shaped curve Figure 2.13.

The optimum pH is different for each enzyme:

Pepsin from the stomach pH 2-3.3

Amylase from saliva pH 6.8
Chymotrypsin from the pancreas pH 7-8