PTT 202:
ORGANIC CHEMISTRY
FOR BIOTECHNOLOGY
PREPARED BY:
NOR HELYA IMAN KAMALUDIN
helya@unimap.edu.my
Introduction of enzymes
Enzymes occupy an important place in analytical
biochemistry and many investigations require their
detection and quantitation.
Enzyme assay provide convenient methods for
assessing the quality of foodstuffs and checking the
efficiency of sterilization and pasteurization processes.
The increasing availability of highly purified
enzyme preparations, both in solution and in
immobilized forms, it permits the development
of a wide range methods.
Enzyme in biological reactions
Enzymes are "biological catalysts." "Biological" means the substance
that is produced or is derived from some living organism. "Catalyst"
denotes a substance that has the ability to increase the rate of a
chemical reaction, and is not changed or destroyed by the chemical
reaction that it accelerates.
Catalysts are specific in nature as to the type of reaction they can
catalyze. Enzymes, as a subclass of catalysts, are very specific in
nature. Each enzyme can act to catalyze only very select chemical
reactions and only with very select substances.
Many chemical reactions do proceed but at such a slow rate that
their progress would seem to be imperceptible at normally
encountered environmental temperature
Enzymes often increase the rate of a chemical reaction between 10
and 20 million times what the speed of reaction would be when left
uncatalyzed (at a given temperature).
Enzyme in biological reactions (cont)
The oxidation of glucose or other sugars to useable energy by
animals and plants.
• In conjunction with a series of enzymes created by the living organism,
however, the oxidation reaction of sugar does proceed quite rapidly at
temperatures up to 100°F (38°C).
• Therefore, enzymes allow the living organism to make use of the potential
energy contained in sugar and other food substances.
Nutrients (complex macromolecules) locked in certain organics
or in hard-to-digest matrices may be released or predigested by
a high degree of heat or concentrated acid treatment
• In an alternative manner, specific enzymes can promote the pre-digestion of
certain complex nutrients and facilitate the release of highly digestible
nutrients in organics during processing without the need of excessive heat or
rigorous chemical treatment.
Catalytic antibodies
Catalytic antibodies is the ability of the highly evolved machinery of
immune system to produce structurally and functionally complex
molecules like antibodies
The idea of using antibodies to catalyze chemical
reactions can be traced back to the catalytic concept of
Linus Pauling.
According to Pauling, if the structure of the antigen binding site of
antibodies were to be produced in a random manner, the antigen
binding site of some of the antibodies may resemble the active site of
enzymes and such molecules could have the ability to transform
substrates to products.
Catalytic antibodies
The use of
antibodies to
stabilize negatively
and positively
charged transition
states
The generation of
antibodies with
catalytic groups and
cofactors in their
combining sites
The use of
antibodies as
entropic traps
Strategies for
Generating
Catalytic
Antibodies
The use of cofactors
by catalytic
antibodies.
Artificial enzymes
Artificial enzymes
Characteristics:
Definition:
Is a synthetic, organic
molecule prepared to
recreate the active site
of an enzyme.
Made based on a hostmolecule such as a
cyclodextrin, crown
ethers or calixarene etc
Catalysing various
reactions with rate
increases up to
103;substantially lower
than natural enzymes
that typically causes
rate increases above 106
Example:
Scaffolded histidine
residues can be used as
mimics of certain
metalloproteins and enzymes.
Enzyme assay methods
Direct
assays
Coupled
assays
• Enzyme assays in which either
the substrate or the product of
the test enzymes is measured.
• The reaction mixture which
includes the substrates for the
initial or test enzyme and also
the additional enzyme and
reagents necessary to convert
the product of the final reaction.
Coupled assay method
Example of coupled assay reactions
Auxiliary enzyme
Indicator enzyme
Coupled assay method
Indicator
enzyme
• The enzyme that catalyses
the final measurable reaction
in a coupled enzyme assay
Auxiliary
enzyme
• The enzyme that catalyses
the intermediary reactions in
a coupled enzyme assay.
Optimized assays
pH
Activators
Parameters
need to be
optimized
Km values
Inhibitors
Optimized assays
pH
Activator/
Inhibitor
Km values
• pH optimum of an enzyme will often vary from
one substrate to another
• The buffer system used may alter the optimum pH
of an enzyme and affect overall enzyme activity
• It is essential to identify the need for any
activators and to determine their optimal
concentrations.
• The optimum concentration of all activators can
be achieved by careful control of concentrations
and by increasing the amount of any inhibited
supplementary enzymes.
• To achieve maximum velocity (higher reaction
rate) a substrate concentration should be at least
ten times greater than the Km value for the enzyme
should be used.
Measurement of enzyme activity
In order to follow the progress of an enzyme-catalysed reaction,
it is necessary to measure either the depletion of the substrate or
the accumulation of the product.
The rate at which the substrate is converted to the product by the
action of an enzyme is dependent upon the concentrations of
both the enzyme and the substrate.
Figure 1:
The initial rate of such a reaction is maximal and it is the initial
rate of reaction that reflects to the enzyme activity.
Measurement of enzyme activity

The rate of reaction
declines from a
maximal initial
velocity, V0, which can
be represented by the
tangent to the curve at
zero time. The rate falls
least during the first
15-20% of the total
reaction change.
Figure 1:Characteristics progress curve of an enzyme-catalysed reaction
Measurement of enzyme activity

The major factors contribute to the declines in the
reaction rate:
1. Depletion of the substrate and increasing amount of
product competing for the enzyme.
2. Inactivation of the enzyme, particularly if an
appreciable reaction time is involved.
Measurement of enzyme activity

The indicator reaction in many
coupled assays will often show
a demonstrable change after
the addition of the sample but
before the addition of the
substrate for the test enzyme.
This blank reaction may be
due to the presence of
endogeneous substrates in the
sample and its rate (B) must be
measured in order to be able
to calculate the activity of the
test enzyme (T-B) from the
total rate of reaction (T) which
results from adding the
substrate.
Figure 2: Typical reaction trace of a coupled assay
Measurement of enzyme activity
Assays for measurement reaction velocity
Kinetic enzyme
assay
• Designed to measure the velocity of the reaction.
Fixed time enzyme
assay
• Designed to measure the amount of substrate used or
product produced in a fixed time
Fixed change
enzyme assay
• Designed to measure the time taken for a fixed amount
of substrate to be used or product to be formed.
Monitoring Techniques
Luminescence
methods
Fluorimetric
methods
Spectrophotometric
methods
Electrochemical
methods
Monitoring
Techniques
Microcalorimetric
methods
Spectrophotometric methods
• Very convenient and popular method ; simplicity and precision of the technique.
• Advantages:
• Readily to temperature control using water-jacketed or electrically heated cell
holders.
• Readily to measurement of initial velocities by continuous monitoring
• Maxima ultraviolet region of spectrum can be shown.
• Characteristics:
• In order to measure the rate of substrate depletion and product formation, it
its necessary for these two components of the reaction show different
adsorption characteristics to permit the measurement of one without
interference from the other.
• The natural substrates most frequently used are the nucleotide coenzymes
NAD+ AND NADP+ which are reversibly reduced by many enzymes
• The reduced form can be detected at 340 nm in the presence of oxidized form.
Spectrophotometric methods
Figure 3: Adsorption spectra of
NAD+ and NADH.

The two forms of
coenzymes (oxidized
and reduced) that
have different spectral
characteristics.
Fluorimetric methods
• Useful method for monitoring reactions involving the nucleotide
coenzymes.
• Characteristics:
• The natural fluorescence of the reduced forms in the region of 460
nm can be used in kinetic assays
• But, when any oxidized forms of the coenzymes present are stable,
this fluorenscence destroyed at pH below 2.0
• Disadvantages:
• Many fluorescent compounds are unstable particularly in ultraviolet
light.
• The presence of other flourescent substances in either the sample or
the reagents will give a background fluorescence which will reduce
the sensivity of measurements.
• Small amounts of impurities in reagents and solvents can quench
the radiation emitted by flourescent substance.
Luminescence methods
• Bioluminescence provides the basis for sensitive enzymic assay
methods both for substrate assays and coupled enzyme assays.
• Firefly luciferase catalyses the production of light (540-600 nm) by the
oxidation of luciferin (D-LH2)
• Luciferase from bacterial sources cataluses the oxidation of the long
chain aliphatic aldehydes and requires the coenzyme FMN. The
wavelength of the emitted radiation in this reaction is approximately
490 nm.
Electrochemical methods
• The use of ion-selective electrodes opens up the wider use
of electrochemical methods for monitoring the formation
for particular products.
• Examples of application:
• For monitoring the formation of ammonia: simple
platinum electrodes can be used to monitor redox
reactions catalysed by the oxidoreductases or hydrolytic
enzymes using substrate analogues (artificial substrate)
which cause redox changes.
• For monitoring the exchange of oxygen: oxygen
electrode can be used as an alternative to manometry.
Microcalorimetric methods
• The use of microcalorimetric method is to monitor the
increase in temperature in an insulated container and also
to detect the small change in temperature as 0.001˚C.
• The amount of heat released during a reaction is
proportional to the amount of substance involved.
• The initial energy change can often be enhanced, giving
an increase in the sensivity of the method.
• Hydrogen ions released during a reaction, will protonate a
buffer with an evolution of more heat.
Immobilized enzymes
Immobilized Enzymes:
Enzymes physically confined or localized in a certain defined region of space with
retention of their catalytic activities, and which can be used repeatedly and
continuously.
Significantly reduce costs
Provides a convenient source
of enzymes for performing
substrate assays
Advantages of
Immobilized
Enzyme
Greater stability
Reduce inhibition effects than
do to soluble enzymes
Immobilized enzymes
General methods of enzyme immobilization
Covalent linking
• Covalently linked to a variety of polymers using nonessential amino acid residues
• Enzyme inhibition can be reduced by the presence of
competitive inhibitor
Adsorption
• Enzymes may be adsorbed on a variety of polar
materials such as charcoal, silica gel, alumina and ionexchange resin
• Disadvantage: enzyme may be desorbed as a result of
variation in pH and buffer concentration
Immobilized enzymes
General methods of enzyme immobilization (cont)
Entrapment
• The formation of a cross-linked polymer (polyacrylamide), in
the presence of the enzyme results in trapping of the enzyme
molecules between the matrix of the polymer.
• But, it still permits the access of small substrate molecules to
the enzymes.
Encapsulation
• Enzymes may be encapsulated by the production of an
emulsion of an aqueous solution of the enzyme in an organic
solvent.
• The droplets are stabilized by the addition of a suitable
polymer which after forming a membrane around the droplet,
is allowed to harden and the capsules can be washed free
from the other reagents
Immobilized enzymes
Applications of enzyme immobilization
Immobilized enzyme used in affinity chromatography
methods
• Used as a catalysts either in the production or removal of
compounds in chemical processes or as analytical tools.
• Incorporated into gel or microparticulate layers on dry strips or
slides.
Immobilized enzyme in conjugation with ion-selective
electrodes
• May be held in a gel or membrane around the electrode and the
substance to be measured diffuses into the enzyme gel.
• Advantages: simplicity of the method of analysis
• Disadvantage: the presence of interfering ions may present a
problem, particularly when using cation-sensitive electrodes.
Immobilized enzymes
Table 1: Substrate assays using enzyme electrodes
THANK YOU..
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