BCH 4211 Lecture Note

ADVANCE ENZYMOLOGY
LECTURE NOTE
BCH 4211
PREPARED BY
ABUBAKAR MUSA
ENZYME ASSAY TECHNIQUES
 Assay simply means measurement of the enzymatic activity that is
based on the determination of either the rate of formation of the
product or the rate of utilization or disappearance of reactant or
substrate under control conditions.
 Most assays are carried out at 30°C-37°C. Adequate buffering capacity
is always beingensured. Apparatus used must be clean.
 Analytical assays may be classified as (1) continous (2) discontinuous.
.
 Kcat: is defined as the amount of enzyme that catalyze I mole of substrate per
second.
 Turnover number: is defined as the number of moles of substrate transformed
per minute per second.
 There exist different enzyme assay techniques, however, irrespective of the principle
of the chosen method, the enzyme assay requires the use of excess substrate (zero
order kinetics at least equal to 10Km) and an appropriate control is required.
 This control is in all respect the same as the test assay but lacks either the enzyme or
substrate. Both the test and the control must be subjected to the same experimental
condition.
 In order to standardize the report on enzyme activities, the commission of
enzyme of the international union of biochemistry defined a standard unit i.e
International unit (I.U) of enzyme as the amount of enzyme that catalysed the
formation of 1 micro mole of product per minute under defined condition.
 The concentration of enzyme in an impure preparation is expressed in terms
of units/ml while the specificity activity is expressed as units/mg protein.
 In most cases, as the enzyme is being purified, its specific activity is expected
to increase to maximum
TYPES OF ENZYME ASSAY TECHNIQUES
 VISIBLE AND
UV
SPECTROPHOTOMETRIC METHOD
SPECTROFLUORIMETRY
RADIOISOTOPE
IMMUNOCHEMICAL METHOD
MACRO CALORIMETRIC METHOD
MANOMETRIC METHOD
COUPLE ASSAY
METHOD/TECHNIQUES
ENZYME PURIFICATION
Enzymes are purified by employing successive

chemical or physical fractionation procedures.
However, it should be noted that there is no fast
and hard rule in respect of the protocol to be
controlled.
It is usually by a trial and error study based on a
pilot study.
CONT….
The utmost idea of each step is to get rid of
the contaminants as possible and retain much
desired enzyme. The efficiency of each step
is given by
(1) The yield or recovery (i.e percentage of
the total enzyme activity originally present
that is retained.
(2) The purification factor (the factor by
which the specific activity of the preparation
has increased).
CONT
 The preparation of the cell free extract may be prepared by several
means depending on the nature of the starting material (tissue, or cell,
or organism) and the size of the preparation.
 For instance, cell breakage method may be by autolysis freeze thaw,
mechanical grinding and holistic homogenization e.t.c. the resulting
homogenate is usually centrifuged to remove unbroken cells and large
debris.
 At times, differential centrifugation for mitochondria, chloroplast,
microsome, ribosome, or nucleic acids could be carried out.

CONT..
 The purity of the final preparation should be checked by several
methods before one can conclude if the final preparation is
homogenous.
 Some of the criteria are
 (1) Homogenous enzyme preparation should elute from an ion-
exchange or gel filtration column as a single symmetrical activity and
protein peak with a constant specific activity throughout.
(2) Probably give a single bond (if not a dimer or polymer without
artifacts or background noise).

REGULATION OF ENZYME ACTIVITY
In every metabolic pathway, the activity of at

least one enzyme is subject to regulation so
that the flux of material through the pathway
can be controlled.
There are at least 4 means of control namely:
Allosteric control
Feedback inhibition
Control by reversible covalent modification
Control by irreversible covalent modification
ALLOSTERIC CONTROL
 Some enzymes are reversibly inhibited or activated by the
presence of metabolites that are not their substrate or product.
 These metabolites, if inhibitory are normally distant products
of the pathway, thereby providing negative feedback for the
activity of the pathway.
 The enzymes controlled in this way usually have additional
binding site other than the active or substrate binding site.
CONT..
 The binding of inhibitor or activators at distant site from
active site often brought about conformational changes in
the enzyme molecule which may decrease or increase its
catalytic activity.
 Thus, allosteric enzymes usually compose of subunits and
use to have multiple interacting active centres and often
shows sigmoidal graph of initial rate versus [S] and
therefore do not obey Michaelis Menten kinetics.
FEEDBACK INHIBITION
 This is a means by which biosynthetic pathways are
regulated and involves the process whereby and products or
near end products control the metabolic flux by inhibiting
one or more of the enzyme at the early part of the pathway.
 Often maximum feedback inhibition is attained only by the
combined actions of multiple end products.
 Feedback inhibition could be (1) sequential (2) concerted
(3)cumulative (4) co-operative
CONTROL BY REVERSIBLE
COVALENT MODIFICATION
 This regulation is often the response to a signal coming from outside the
cells such as response to hormone. In this case, the enzyme is itself the
substrate of other enzymes.
 One of these modifies the enzyme making it active while the other
reverses the modification thereby is inactive.
A typical example is the control of glycogen phosphorylase through
phosphorylation and dephosphorylation of specific serine residue.
 The enzyme exist in two forms i.e glycogen phosphorylase a (active)
and glycogen phosphorylase b (inactive).

CONTROL BY IRREVERSIBLE COVALENT
MODIFICATION
 The best known example of the enzyme that exhibit /undergo this
mechanism or modification is a protease enzyme named chymotrypsinogen.
The enzyme, when reacted with diisopropyl phosphofluoridate becomes
inactivated because of the reaction of serine-195 with chemical coupled with
the formation of covalent linkage and elimination of HF.
 Also, activation of zymogen is another example of this type of modification.
For instance, activation of trypsinogen by enteropeptidase will result in
formation of trypsin, which also acts on chymotrypsinogen and converts its
to chymotrypsin.
APPLICATON ENZYMES IN
MEDICINE
 Immobilized enzymes are used in medicine from 1990
immobilized enzymes are used for diagnosis and treatment of
diseases in the medical field.
 The inborn metabolic deficiency can be overcome by replacing
the encapsulated enzymes (i.e, enzymes encapsulated by
erythrocytes) instead of waste metabolites, the RBC acts as a
carrier for the exogenous enzyme drugs and the enzymes are
biocompatable in nature, hence there is no immune response
CONT..
Theenzyme encapsulation through the
electroporation is a easiest way of
immobilization in the biomedical field
and it is a reversible process for which
enzyme can be regenerated
Food industry Application
 In food industry, the purified enzymes are used but during
the purification the enzymes will denature.
 Hence the immobilization technique makes the enzymes
stable.
 The immobilized enzymes are used for the production of
syrups.
 Immobilized beta-galactosidase used for lactose hydrolysis
in whey for the production of bakers yeast.
Wastewater Treatment
 The increasing consumption of fresh water and water bodies are mixed up
with polluted industrial waste water and the waste water treatments are
necessary at present.
 The sources of dye effluents are textile industry, paper industry, leather
industry and the effluents are rich in dye colourants.
 These effluents are threat to the environment and even in low concentration
it is carcinogenic.
 Nowadays enzymes are used to degrade the dye stuffs. The enzymes used
in the wastewater treatments are preoxidases, laccase, azo reductases.

Textile Industry:
 The enzymes derived from microbial origin are of great

interest in textile industry.
 The enzymes such as cellulase, amylase, liccase,
pectinase, cutinase etc and these are used for various
textile applications such as scouring, biopolishing,
desizing, denim finishing, treating wools etc. Among these
enzymes cellulase has been widely used from the older
period to till now
CONT..
 The textile industries now turned to enzyme process instead of
using harsh chemical which affects the pollution and cause damage
to the fabrics.
 The processing of fabrics with enzymes requires high temperatures
and increased pH, the free enzymes does not able to withstand the
extreme conditions.
 Hence, enzyme immobilization for this process able to withstand at
extreme and able to maintains its activity for more than 5-6 cycles.
 PolyMethyl Methacrylateis linked with cellulase covalently.

Detergent Industry:
 The detergent industry also employs enzymes forremoval of stains.

 The enzymes used in detergent industry are protease which is used
to remove the stains of blood, egg, grass and human sweat.
 Amylase used to remove the starch based stains like potatoes,
gravies, chocolate.
 Lipase used to remove the stains of oil and fats and also used to
remove the stains in cuffs and collars.
 Cellulase is used for cotton based fabrics in order to improve
softening, colour brightening and to remove soil stains.
 Nowadays Biotech cleaning agents are widely used in the detergent
industries.
 When compared to synthetic detergents the biobased detergents
 have good cleaning property

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ADVANCE ENZYMOLOGY

based on the determination of either the rate of formation of the

product or the rate of utilization or disappearance of reactant or

substrate under control conditions.

 Most assays are carried out at 30°C-37°C. Adequate buffering capacity

is always beingensured. Apparatus used must be clean.

 Analytical assays may be classified as (1) continous (2) discontinuous.

 Turnover number: is defined as the number of moles of substrate transformed

per minute per second.

order kinetics at least equal to 10Km) and an appropriate control is required.

enzyme of the international union of biochemistry defined a standard unit i.e

formation of 1 micro mole of product per minute under defined condition.

 The concentration of enzyme in an impure preparation is expressed in terms

of units/ml while the specificity activity is expressed as units/mg protein.

Enzymes are purified by employing successive

means depending on the nature of the starting material (tissue, or cell,

or organism) and the size of the preparation.

 For instance, cell breakage method may be by autolysis freeze thaw,

mechanical grinding and holistic homogenization e.t.c. the resulting

homogenate is usually centrifuged to remove unbroken cells and large

 At times, differential centrifugation for mitochondria, chloroplast,

microsome, ribosome, or nucleic acids could be carried out.

methods before one can conclude if the final preparation is

 Some of the criteria are

 (1) Homogenous enzyme preparation should elute from an ion-

exchange or gel filtration column as a single symmetrical activity and

protein peak with a constant specific activity throughout.

artifacts or background noise).

In every metabolic pathway, the activity of at

substrate of other enzymes.

reverses the modification thereby is inactive.

A typical example is the control of glycogen phosphorylase through

phosphorylation and dephosphorylation of specific serine residue.

 The enzyme exist in two forms i.e glycogen phosphorylase a (active)

and glycogen phosphorylase b (inactive).

mechanism or modification is a protease enzyme named chymotrypsinogen.

The enzyme, when reacted with diisopropyl phosphofluoridate becomes

inactivated because of the reaction of serine-195 with chemical coupled with

the formation of covalent linkage and elimination of HF.

 Also, activation of zymogen is another example of this type of modification.

For instance, activation of trypsinogen by enteropeptidase will result in

formation of trypsin, which also acts on chymotrypsinogen and converts its

industry and the effluents are rich in dye colourants.

in the wastewater treatments are preoxidases, laccase, azo reductases.

 The enzymes derived from microbial origin are of great

 Hence, enzyme immobilization for this process able to withstand at

 PolyMethyl Methacrylateis linked with cellulase covalently.

 The detergent industry also employs enzymes forremoval of stains.

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