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

Tenorio, Rizza
Torres, Jean Adriane
Sulthan, Ahmed
Soriano, Joyce Ann
Silva, Kreza
Sandoval, Christine
ChE-5201

Dr. Sicily B. Tiu

January 23, 2017

INTRODUCTION
Biochemical engineering 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.
Biotechnology can be broadly defined as "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 ..."
(Congress of the United States, 1984). If biotechnology is defined in this general sense, the area
cannot be considered new. Since ancient days, people knew how to utilize microorganisms to
ferment beverage and food, though they did not know what was responsible for those
biological changes. People also knew how to crossbreed plants and animals for better yields.
Successful commercialization of this process requires the development of a large-scale process
that is technologically viable and economically efficient.

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.
DEFINITION OF FERMENTATION
Traditionally, fermentation was defined as the process for the production of alcohol or lactic
acid from glucose (C6H1206).

A broader definition of fermentation is "an enzymatically controlled transformation of an

organic compound" according to Webster's New College Dictionary (A Merriam-Webster, 1977)
that we adopt in this text.

Enzyme Kinetics
Enzymes are biological catalysts that are protein molecules in nature. They are produced
by living cells (animal, plant, and microorganism) and are absolutely essential as catalysts in
biochemical reactions. Almost every reaction in a cell requires the presence of a specific
enzyme. 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.

The catalytic ability of enzymes is due to its particular protein structure. A specific
chemical reaction is catalyzed at a small portion of the surface of an enzyme, which is known as
the active site. Some physical and chemical interactions occur at this site to catalyze a certain
chemical reaction for a certain enzyme.

Enzyme reactions are different from chemical reactions, as follows:

1. An enzyme catalyst is highly specific, and catalyzes only one or a small number of chemical
reactions. A great variety of enzymes exist, which can catalyze a very wide range of reactions.
2. The rate of an enzyme-catalyzed reaction is usually much faster than that of the same
reaction when directed by nonbiological catalysts. Only a small amount of enzyme is required
to produce a desired effect.
3. The reaction conditions (temperature, pressure, pH, and so on) for the enzyme reactions are
very mild.
4. Enzymes are comparatively sensitive or unstable molecules and require care in their use.

Nomenclature and Classification of Enzyme

Substrate - the reactant in an enzyme-catalyzed reaction ; the substance upon which the
enzyme 'acts‘
1. The suffix -ase identifies a substance as an enzyme e.g. urease, sucrase, and lipase but some
still ends in -in like trypsin, chymotrypsin, and pepsin
Nomenclature and Classification of Enzyme
"pepsis" - means digestion
“tyrein" - to wear down
2. The type of reaction catalyzed by an enzyme is often noted with a prefix. An oxidase enzyme
catalyzes an oxidation reaction
3. The identity of the substrate is often noted in addition to the type of reaction. Enzyme names
of this type include glucose oxidase, pyruvate carboxilase, and succinate dehydrogenase.
Infrequently, the substrate but not the reaction type is given, as in the names of urease and
lactase. In such names, the reaction involved is hydrolysis.

Classification by the Reactions they Catalyze

The International Union of Biochemistry and Molecular Biology have developed a
nomenclature for enzymes, the EC numbers; each enzyme is described by a sequence of four
numbers preceded by "EC". The first number broadly classifies the enzyme based on its
mechanism.

EC 1.0 Oxidoreductase
- Is an enzyme that catalyzes an oxidation-reduction reaction.
- Requires a coenzyme
Sections
EC 1.1.1 With NAD+ or NADP+ as acceptor
EC 1.1.2 With a cytochrome as acceptor
EC 1.1.3 With oxygen as acceptor
EC 1.1.4 With a disulfide as acceptor
EC 1.1.5 With a quinone or similar compound as acceptor
EC 1.1.99 With other acceptors

EC 1.1.1.24 quinate dehydrogenase

EC 1.1.1.25 shikimate dehydrogenase
EC 1.1.1.26 glyoxylate reductase
EC 1.1.1.27 L-lactate dehydrogenase
EC 1.1.1.28 D-lactate dehydrogenase
EC 1.1.1.29 glycerate dehydrogenase
EC 1.1.1.30 3-hydroxybutyrate dehydrogenase

EC 2.0 – Transferase
- An enzyme that catalyzes the transfer of a functional group from one molecule to
another
- EC 2.1 Transferring one-carbon groups
- EC 2.2 Transferring aldehyde or ketonic groups
- EC 2.3 Acyltransferases
- EC 2.4 Glycosyltransferases
- EC 2.5 Transferring alkyl or aryl groups, other than methyl groups
- EC 2.6 Transferring nitrogenous groups (transaminase)
- EC 2.7 Transferring phosphorus-containing groups (kinases)
- EC 2.8 Transferring sulfur-containing groups
- EC 2.9 Transferring selenium-containing groups
- EC 2.10 Transferring molybdenum- or tungsten-containing groups

EC 3.0 Hydrolase
- An enzyme that catalyzes a hydrolysis reaction in which the addition of a water
molecule to a bond causes the bond to break.

EC 4.0 Lyase
- An enzyme that catalyzes the addition of a group to a double bond or the removal of a group
to form a double bond in a manner that does not involve hydrolysis or oxidation
Dehydratase – removal of the components of water from a double bond
Hydratase – effects the addition of the components of double bond
EC 6.0 – Ligase
- An enzyme that catalyzes bonding together of two molecules into one with the
participation of ATP

Commercial Applications of Enzymes

The enzymes produced commercially can be classified into three major categories (Crueger and
Crueger, 1984):

1.Industrial enzymes, such as amylases, proteases, glucose isomerase, lipase, catalases, and
penicillin acylases
2. Analytical enzymes, such as glucose oxidase, galactose oxidase, alcohol dehydrogenase,
hexokinase, muramidase, and cholesterol oxidase
3. Medical enzymes, such as asparaginase, proteases, lipases, and streptokinase

The scale of application of analytical and medical enzymes is in the range of milligrams to
grams while that of industrial enzymes is in tons. Analytical and medical enzymes are usually
required to be in their pure forms; therefore, their production costs are high.

SIMPLE ENZYME KINETICS

Enzyme kinetics deals with the rate of enzyme reaction and how it is affected by various
chemical and physical conditions.
Enzyme kinetics is the study of the chemical reactions that are catalyzed by enzymes.

(2.1)
If you measure the concentrations of substrate and product with respect to time, the
product concentration will increase and reach a maximum value, whereas the substrate
concentration will decrease.

(Fig. 2.1)

Saturation Curve for an Enzyme Reaction showing the Relation between the Substrate
concentration and Reaction rate
 (Fig. 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.

“Lock and Key” Theory

-One of the original theories to account for the formation of the enzyme-substrate

complex
Fig. 2.3 Lock and key theory for the enzyme-substrate complex

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, C EO = C ES +
CE
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.

3 Different Approaches to derive the rate equation:

1. Michaelis-Menten approach: 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 determines the rate, while the
other is at equilibrium.
2. Briggs-Haldane approach): 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.
3. Numerical solution: Solution of the simultaneous differential equations

ENZYME REACTOR WITH SIMPLE KINETICS

 A Bioreactor is a device within which biochemical transformations are caused by the
action of enzymes or living cells.
 The bioreactor is frequently called a fermenter 6 whether the transformation is carried
out by living cells or in vivo7 cellular components (that is, enzymes).
Batch or Steady-State Plug-Flow Reactor
The simplest reactor configuration for any enzyme reaction is batch mode. It is normally
equipped with an agitator to mix the reactant, and the pH of the reactant is maintained by
employing either a buffer solution or a pH controller.
In a plug-flow enzyme reactor (or tubular-flow enzyme reactor), the substrate enters
one end of a cylindrical tube which is packed with immobilized enzyme and the product stream
leaves at the other end. The ideal plug-flow enzyme reactor can approximate the long tube,
packed-bed, and hollow fiber, or multi-staged reactor.
Continuous Stirred-Tank Reactor.
A continuous stirred-tank reactor (CSTR) is an ideal reactor which is based on the
assumption that the reactor contents are well mixed. Therefore, the concentrations of the
various components of the outlet stream are assumed to be the same as the concentrations of
these components in the reactor.
Michaelis-Menten Kinetics (Leonor Michaelis and Maud Leonora Menten)
-serves an explanation to how an enzyme can cause kinetic rate enhancement of a reaction and
how reaction rates depends on the concentration of enzyme and substrate.
-attempts to describe ideal enzyme kinetics

Assumptions:

 The product releasing step, is much slower than the reversible reaction,

.
 The slow step determines the rate while the other is at equilibrium.
 Enzyme-substrate complex formation step is much faster than the product releasing
step which involves chemical changes.

General Enzyme Reaction

 Rate Constant Reaction
k1 The binding of the enzyme to the substrate
forming the enzyme substrate complex.
k2 Catalytic rate; the catalysis reaction
producing the final reaction product and
regenerating the free enzyme (Rate Limiting
Step)
k3 The dissociation of the enzye substrate
complex to free enzyme and substrate.
k4 The reverse reaction of the catalysis

Substrate Complex

 The ES complex can be formed by combining enzyme E with substrate S at rate constant
k1.
 ES complex can either dissociate to form Ef and S, or form product P at rate constant k2
and k3 respectively.

Steady State Approximation

At steady state,
Reaction order note
ENZYME INHIBITION
A modulator is a substance which can combine with enzymes to alter their catalytic
activities. An inhibitor is a modulator which enzyme activity. It can decrease the rate of
reaction either competitively or noncompetitively.

 COMPETITIVE INHIBITION
Since a competitive inhibitor has a strong structural resemblance to the substrate,
both the inhibitor and substrate compete for the active site of an enzyme. The
formation of an enzyme-inhibitor complex reduces the amount of enzyme available for
interaction with the substrate and, as a result, the rate of reaction decreases. A
competitive inhibitor normally combines reversibly with enzyme. Therefore, the effect
of the inhibitor can be minimized by increasing the substrate concentration, unless the
substrate concentration is greater than the concentration at which the substrate itself
inhibits the reaction. The mechanism of competitive inhibition can be expressed as
follows:

If the slower reaction, the product formation step, determines the rate of reaction
according to the Michaelis-Menten assumption, the rate can be expressed as:
rP = k5CES
The enzyme balance gives CE0 = CE + CES +CEI
From the two equilibrium reactions,

where Ks and KI are dissociation constants which are the reciprocal of the equilibrium
constants. Combining the preceding four equations to eliminate CE, CES and CEI yields

where
Therefore, since KM1 is larger than KS, the reaction rate decreases due to the
presence of inhibitor. It is interesting to note that the maximum reaction rate is not
 affected by the presence of a competitive inhibitor. However, a larger amount of
substrate is required to reach the maximum rate.

 NONCOMPETITIVE INHIBITION
Noncompetitive inhibitors interact with enzymes in many different ways. They can
bind to the enzymes reversibly or irreversibly at the active site or at some other region.
In any case the resultant complex is inactive. The mechanism of noncompetitive
inhibition can be expressed as follows:

Since substrate and inhibitor do not compete for a same site for the formation of
enzyme-substrate or enzyme-inhibitor complex, we can assume that the dissociation
constant for the first equilibrium reaction is the same as that of the third equilibrium
reaction, as

Employing the Michelis-Menten approach:

where
Therefore, the maximum reaction rate will be decreased by the presence of a
noncompetitive inhibitor, while the Michaelis constant KS will not be affected by the
inhibitor.

EFFECT OF TEMPERATURE AND pH

The rate of an enzyme reaction is influenced by various chemical and physical conditions. Some
of the important factors are the concentration of various components (substrate, product,
enzyme, cofactor, and so on), pH, temperature, and shear.
Effect of pH
The typical relationship between the reaction velocity and pH shows a bell-shaped curve as
shown in the figure. The optimum pH is different for each enzyme. For example, pepsin from
the stomach has an optimum pH between 2 and 3.3, while the optimum pH of amylase, from
saliva, is 6.8. Chymotrypsin, from the pancreas, has an optimum pH in the mildly alkaline region
between 7 and 8.

The reason that the rate of enzyme reaction is influenced by pH can be explained as follows:
1. Enzyme is a protein which consists of ammo acid residues (that is, amino acids minus
water).

2. The amino acid residues possess basic, neutral, or acid side groups which can be
positively or negatively charged at any given pH. As an example (Wiseman and Gould,
1970), let's consider one acidic amino acid, glutamic acid, which is acidic in the lower pH
range.

As the pH is increased, glutamic acid is ionized as which can be expressed as

In equilibrium,
When CA- = CA, pH is equal to pK. For glutamic acid, pK 4.5.

3. An enzyme is catalytically active when the amino acid residues at the active site each
possess a particular charge. Therefore, the fraction of the catalytically active enzyme
depends on the pH.

Effect of Temperature

The rate of a chemical reaction depends on the temperature according to Arrhenius equation as
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 50°C. Some
enzymes are very resistant to denaturation by high temperature, especially the enzymes
isolated from thermophilic organisms found in certain hot environments.

REFERENCE:
Rajiv, Dutta (2008). Fundamentals of Biochemical Engineering