Enzymes principles and applications Likhith K

Enzymes: principles and
applications (short notes)
By,
LIKHITHK
BiSE P -2021
Dept of biotechnology
St Aloysius College,
Mangaluru, Karnataka,

CONTENTS
 Introduction
 The nature and class of enzyme
 Enzymes are potent catalyst.
 Enzymes are specific catalyst
 Enzyme names and classification
 Enzyme structure and substrate binding
 Enzymes and reaction equilibrium
 Enzyme substrate complex
 Properties and mechanism of enzyme action
 Enzyme purification
 Enzyme immoblization
 Industrial enzymology
 Enzyme in analysis
 Conclusion
 Reference

INTRODUCTION
 Enzymes are biological catalysts (also known as biocatalysts) that speed up
biochemical reactions in living organisms, and which can be extracted from
cells and then used to catalyse a wide range of commercially important
processes.
 This power point covers the basic principles of enzymology, such as
classification, structure, kinetics and inhibition, and also provides an overview
of industrial applications.
THE NATURE AND CLASSIFICATION OF ENZYMES
 Enzymes are biological catalysts (also known as biocatalysts) that speed up
biochemical reactions in living organisms. They can also be extracted from cells
and then used to catalyse a wide range of commercially important processes.

 For example, they have important roles in the production of sweetening
agents and the modification of antibiotics, they are used in washing
powders and various cleaning products, and they play a key role in
analytical devices and assays that have clinical, forensic and
environmental applications.
 The word ‘enzyme’ was first used by the German physiologist Wilhelm
Kühne in 1878, when he was describing the ability of yeast to produce
alcohol from sugars, and it is derived from the Greek words en (meaning
‘within’) and zume (meaning ‘yeast’).
 In the late nineteenth century and early twentieth century, significant
advances were made in the extraction, characterization and commercial
exploitation of many enzymes, but it was not until the 1920s that
enzymes were crystallized, revealing that catalytic activity is associated
with protein molecules.
 For the next 60 years or so it was believed that all enzymes were
proteins, but in the 1980s it was found that some ribonucleic acid (RNA)
molecules are also able to exert catalytic effects.

 These RNAs, which are called ribozymes, play an important role in gene
expression.
 In the same decade, biochemists also developed the technology to
generate antibodies that possess catalytic properties. These so-called
‘abzymes’ have significant potential both as novel industrial catalysts
and in therapeutics.
 Notwithstanding these notable exceptions, much of classical enzymology,
and the remainder of this essay, is focused on the proteins that possess
catalytic activity.
 As catalysts, enzymes are only required in very low concentrations, and
they speed up reactions without themselves being consumed during the
reaction.
 We usually describe enzymes as being capable of catalysing the
conversion of substrate molecules into product molecules as follows:
Substrate Enzyme Product

General enzyme subtract reaction

Wilhelm Friedrich Kühne
(28 March 1837 – 10 June 1900)

ENZYMES ARE POTENT CATALYSTS
 The enormous catalytic activity of enzymes can perhaps best be
expressed by a constant, kcat, that is variously referred to as the turnover
rate, turnover frequency or turnover number.
 This constant represents the number of substrate molecules that can be
converted to product by a single enzyme molecule per unit time (usually
per minute or per second).
 For example, a single molecule of carbonic anhydrase can catalyse the
conversion of over half a million molecules of its substrates, carbon
dioxide (CO2) and water (H2O), into the product, bicarbonate (HCO3
−),
every second—a truly remarkable achievement.

ENZYMES ARE SPECIFIC CATALYSTS
 As well as being highly potent catalysts, enzymes also possess
remarkable specificity in that they generally catalyse the conversion of
only one type (or at most a range of similar types) of substrate molecule
into product molecules.
 Some enzymes demonstrate group specificity. For example, alkaline
phosphatase (an enzyme that is commonly encountered in first-year
laboratory sessions on enzyme kinetics) can remove a phosphate group
from a variety of substrates.
 Other enzymes demonstrate much higher specificity, which is described
as absolute specificity. For example, glucose oxidase shows almost total
specificity for its substrate, β-D-glucose, and virtually no activity with
any other mono saccharides.
 As we shall see later, this specificity is of paramount importance in
many analytical assays and devices (biosensors) that measure a specific
substrate (e.g. glucose) in a complex mixture (e.g. a blood or urine
sample).

ENZYME NAMES AND CLASSIFICATION
 Enzymes typically have common names (often called ‘trivial names’) which
refer to the reaction that they catalyse, with the suffix -ase (e.g. oxidase,
dehydrogenase, carboxylase), although individual proteolytic enzymes generally
have the suffix -in (e.g. trypsin, chymotrypsin, papain).
 Often the trivial name also indicates the substrate on which the enzyme acts (e.g.
glucose oxidase, alcohol dehydrogenase, pyruvate decarboxylase). However,
some trivial names (e.g. invertase, diastase, catalase) provide little information
about the substrate, the product or the reaction involved.
 Due to the growing complexity of and inconsistency in the naming of enzymes,
the International Union of Biochemistry set up the Enzyme Commission to
address this issue. The first Enzyme Commission Report was published in 1961,
and provided a systematic approach to the naming of enzymes.
 The sixth edition, published in 1992, contained details of nearly 3 200 different
enzymes, and supplements published annually have now extended this number to
over 5 000.

 Within this system, all enzymes are described by a four-part Enzyme
Commission (EC) number. For example, the enzyme with the trivial name
lactate dehydrogenase has the EC number 1.1.1.27, and is more correctly
called L–lactate: NAD+ oxidoreductase.
 The first part of the EC number refers to the reaction that the enzyme catalyses .
The remaining digits have different meanings according to the nature of the
reaction identified by the first digit.
 For example, within the oxidoreductase category, the second digit denotes the
hydrogen donor and the third digit denotes the hydrogen acceptor.

ENZYME CLASSIFICATION: SECONDARY CLASSES OF
OXIDOREDUCTASE ENYMES IN EC SYSTEM

ENZYME CLASSIFICATION: TERTIARY CLASSES OF
OXIDOREDUCTASE ENZYMES IN EC SYSTEM

 Thus lactate dehydrogenase with the EC number 1.1.1.27 is an oxidoreductase
(indicated by the first digit) with the alcohol group of the lactate molecule as the
hydrogen donor (second digit) and NAD+ as the hydrogen acceptor (third digit),
and is the 27th enzyme to be categorized within this group (fourth digit).
 Fortunately, it is now very easy to find this information for any individual
enzyme using the Enzyme Nomenclature Database

ENZYME STRUCTURE AND SUBSTRATE BINDING
 Amino acid-based enzymes are globular proteins that range in size from less
than 100 to more than 2 000 amino acid residues.
 These amino acids can be arranged as one or more polypeptide chains that are
folded and bent to form a specific three-dimensional structure, incorporating a
small area known as the active site, where the substrate actually binds.
 The active site may well involve only a small number (less than 10) of the
constituent amino acids.
 It is the shape and charge properties of the active site that enable it to bind to a
single type of substrate molecule, so that the enzyme is able to demonstrate
considerable specificity in its catalytic activity.
 The hypothesis that enzyme specificity results from the complementary nature
of the substrate and its active site was first proposed by the German chemist
Emil Fischer in 1894, and became known as Fischer's ‘lock and key
hypothesis’, whereby only a key of the correct size and shape (the substrate) fits
into the keyhole (the active site) of the lock (the enzyme).

Fischer's ‘lock and key hypothesis

 It is astounding that this theory was proposed at a time when it was not
even established that enzymes were proteins. As more was learned about
enzyme structure through techniques such as X-ray crystallography, it
became clear that enzymes are not rigid structures, but are in fact quite
flexible in shape.
 In the light of this finding, in 1958 Daniel Koshland extended Fischer's
ideas and presented the ‘induced-fit model’ of substrate and enzyme
binding, in which the enzyme molecule changes its shape slightly to
accommodate the binding of the substrate.
 The analogy that is commonly used is the ‘hand-in-glove model’, where
the hand and glove are broadly complementary in shape, but the glove is
moulded around the hand as it is inserted in order to provide a perfect
match.
 Since it is the active site alone that binds to the substrate, it is logical to
ask what is the role of the rest of the protein molecule. The simple
answer is that it acts to stabilize the active site and provide an
appropriate environment for interaction of the site with the substrate
molecule.
 Therefore the active site cannot be separated out from the rest of the
protein without loss of catalytic activity, although laboratory-based
directed (or forced) evolution studies have shown that it is sometimes
possible to generate smaller enzymes that do retain activity.

Daniel Koshland 's‘induced-fit model

 It should be noted that although a large number of enzymes consist solely of
protein, many also contain a non-protein component, known as a cofactor, that
is necessary for the enzyme's catalytic activity.
 A cofactor may be another organic molecule, in which case it is called a
coenzyme, or it may be an inorganic molecule, typically a metal ion such as
iron, manganese, cobalt, copper or zinc.
 A coenzyme that binds tightly and permanently to the protein is generally
referred to as the prosthetic group of the enzyme.
 When an enzyme requires a cofactor for its activity, the inactive protein
component is generally referred to as an apoenzyme, and the apoenzyme plus
the cofactor (i.e. the active enzyme) is called a holoenzyme.
 The need for minerals and vitamins in the human diet is partly attributable to
their roles within metabolism as cofactors and coenzymes.

THE COMPONENTS OF A HOLOENZYME

ENZYMES AND REACTION EQUILIBRIUM
How do enzymes work?
 The broad answer to this question is that they do not alter the
equilibrium (i.e. the thermodynamics) of a reaction. This is because
enzymes do not fundamentally change the structure and energetics of the
products and reagents, but rather they simply allow the reaction
equilibrium to be attained more rapidly.
 Let us therefore begin by clarifying the concept of chemical equilibrium.
 In many cases the equilibrium of a reaction is far ‘to the right’—that is,
virtually all of the substrate (S) is converted into product (P). For this
reason, reactions are often written as follows:
S → P
 This is a simplification, as in all cases it is more correct to write this
reaction as follows:
S ⇌ P
 This indicates the presence of an equilibrium.
 To understand this concept it is perhaps most helpful to look at a reaction
where the equilibrium point is quite central.

 In this reaction, if we start with a solution of 1 mol l−1 glucose and add
the enzyme, then upon completion we will have a mixture of
approximately 0.5 mol l−1 glucose and 0.5 mol l−1 fructose.
 This is the equilibrium point of this particular reaction, and although it
may only take a couple of seconds to reach this end point with the
enzyme present, we would in fact come to the same point if we put
glucose into solution and waited many months for the reaction to occur
in the absence of the enzyme. Interestingly, we could also have started
this reaction with a 1 mol l−1 fructose solution, and it would have
proceeded in the opposite direction until the same equilibrium point had
been reached.
 The equilibrium point for this reaction is expressed by the equilibrium
constant Keq as follows:
Keq = Substrate concentration at end point/Substrate concentration at end point
= 0.5/0.5=1

 Thus for a reaction with central equilibrium, Keq = 1, for an equilibrium ‘to the
right’ Keq is >1, and for an equilibrium ‘to the left’ Keq is <1.
 Therefore if a reaction has a Keq value of 106, the equilibrium is very far to the
right and can be simplified by denoting it as a single arrow. We may often
describe this type of reaction as ‘going to completion’.
 Conversely, if a reaction has a Keq value of 10−6, the equilibrium is very far to
the left, and for all practical purposes it would not really be considered to
proceed at all.
 It should be noted that although the concentration of reactants has no effect on
the equilibrium point, environmental factors such as pH and temperature can
and do affect the position of the equilibrium.
 It should also be noted that any biochemical reaction which occurs in vivo in a
living system does not occur in isolation, but as part of a metabolic pathway,
which makes it more difficult to conceptualize the relationship between
reactants and reactions. In vivo reactions are not allowed to proceed to their
equilibrium position.
 If they did, the reaction would essentially stop (i.e. the forward and reverse
reactions would balance each other), and there would be no net flux through the
pathway.
 However, in many complex biochemical pathways some of the individual
reaction steps are close to equilibrium, whereas others are far from equilibrium,
the latter (catalysed by regulatory enzymes) having the greatest capacity to
control the overall flux of materials through the pathway.

ENZYMES FORM COMPLEXES WITH THEIR
SUBSTRATES
 We often describe an enzyme-catalysed reaction as proceeding through three
stages as follows:
E + S → ES complex → E + P
 The ES complex represents a position where the substrate (S) is bound to the
enzyme (E) such that the reaction is made more favourable. As soon as the
reaction has occurred, the product molecule (P) dissociates from the enzyme,
which is then free to bind to another substrate molecule.
 At some point during this process the substrate is converted into an intermediate
form (often called the transition state) and then into the product.
 The exact mechanism whereby the enzyme acts to increase the rate of the
reaction differs from one system to another. However, the general principle is
that by binding of the substrate to the enzyme, the reaction involving the
substrate is made more favourable by lowering the activation energy of the
reaction.
 In terms of energetics, reactions can be either exergonic (releasing energy) or
endergonic (consuming energy). However, even in an exergonic reaction a small
amount of energy, termed the activation energy, is needed to give the reaction a
‘kick start.’

 A good analogy is that of a match, the head of which contains a mixture of
energy-rich chemicals (phosphorus sesquisulfide and potassium chlorate).
 When a match burns it releases substantial amounts of light and heat energy
(exergonically reacting with O2 in the air). However, and perhaps fortunately, a
match will not spontaneously ignite, but rather a small input of energy in the
form of heat generated through friction (i.e. striking of the match) is needed to
initiate the reaction.
 Of course once the match has been struck the amount of energy released is
considerable, and greatly exceeds the small energy input during the striking
process.
 As shown in Figure, enzymes are considered to lower the activation energy of a
system by making it energetically easier for the transition state to form.
 In the presence of an enzyme catalyst, the formation of the transition state is
energetically more favourable (i.e. it requires less energy for the ‘kick start’),
thereby accelerating the rate at which the reaction will proceed, but not
fundamentally changing the energy levels of either the reactant or the product.

Effect of an enzyme on reducing the activation energy required to
start a reaction where (a) is uncatalysed and (b) is enzyme-
catalysed reaction.

PROPERTIES AND MECHANISMS OF ENZYME
ACTION
1. Enzyme kinetics
 Enzyme kinetics is the study of factors that determine the speed of
enzyme-catalysed reactions. It utilizes some mathematical equations .
 However, the theory of kinetics is both logical and simple, and it is
essential to develop an understanding of this subject in order to be able
to appreciate the role of enzymes both in metabolism and in
biotechnology.
 Assays of enzyme activity can be performed in either a discontinuous or
continuous fashion.
 Discontinuous methods involve mixing the substrate and enzyme
together and measuring the product formed after a set period of time, so
these methods are generally easy and quick to perform.
 In general we would use such discontinuous assays when we know little
about the system, or alternatively when we know a great deal about the
system and are certain that the time interval we are choosing is
appropriate.

 In continuous enzyme assays we would generally study the rate of an enzyme-
catalysed reaction by mixing the enzyme with the substrate and continuously
measuring the appearance of product over time.
 Of course we could equally well measure the rate of the reaction by measuring
the disappearance of substrate over time.
 Apart from the actual direction (one increasing and one decreasing), the two
values would be identical.
 In enzyme kinetics experiments, for convenience we very often use an artificial
substrate called a chromogen that yields a brightly coloured product, making
the reaction easy to follow using a colorimeter or a spectrophotometer.
 However, we could in fact use any available analytical equipment that has the
capacity to measure the concentration of either the product or the substrate.

 In almost all cases we would also add a buffer solution to the mixture. As we
shall see, enzyme activity is strongly influenced by pH, so it is important to set
the pH at a specific value and keep it constant throughout the experiment.
 Our first enzyme kinetics experiment may therefore involve mixing a substrate
solution (chromogen) with a buffer solution and adding the enzyme.
 This mixture would then be placed in a spectrophotometer and the appearance
of the coloured product would be measured.
 This would enable us to follow a rapid reaction which, after a few seconds or
minutes, might start to slow down, as shown in Figure.

FORMATION OF PRODUCT IN AN ENZYME-CATALYSED
REACTION, PLOTTED AGAINST TIME

 A common reason for this slowing down of the speed (rate) of the reaction is
that the substrate within the mixture is being used up and thus becoming
limiting.
 Alternatively, it may be that the enzyme is unstable and is denaturing over the
course of the experiment, or it could be that the pH of the mixture is changing,
as many reactions either consume or release protons.
 For these reasons, when we are asked to specify the rate of a reaction we do so
early on, as soon as the enzyme has been added, and when none of the above-
mentioned limitations apply.
 We refer to this initial rapid rate as the initial velocity (v0). Measurement of the
reaction rate at this early stage is also quite straightforward, as the rate is
effectively linear, so we can simply draw a straight line and measure the
gradient (by dividing the concentration change by the time interval) in order to
evaluate the reaction rate over this period.
 We may now perform a range of similar enzyme assays to evaluate how the
initial velocity changes when the substrate or enzyme concentration is altered,
or when the pH is changed. These studies will help us to characterize the
properties of the enzyme under study.
 The relationship between enzyme concentration and the rate of the reaction is
usually a simple one. If we repeat the experiment just described, but add 10%
more enzyme, the reaction will be 10% faster, and if we double the enzyme
concentration the reaction will proceed twice as fast.
 Thus there is a simple linear relationship between the reaction rate and the
amount of enzyme available to catalyse the reaction as in Figure below.

RELATIONSHIP BETWEEN ENZYME CONCENTRATION AND THE
RATE OF AN ENZYME-CATALYSED REACTION

 This relationship applies both to enzymes in vivo and to those used in
biotechnological applications, where regulation of the amount of enzyme
present may control reaction rates.
 When we perform a series of enzyme assays using the same enzyme
concentration, but with a range of different substrate concentrations, a slightly
more complex relationship emerges, as shown in Figure below. Initially, when
the substrate concentration is increased, the rate of reaction increases
considerably.
 However, as the substrate concentration is increased further the effects on the
reaction rate start to decline, until a stage is reached where increasing the
substrate concentration has little further effect on the reaction rate.
 At this point the enzyme is considered to be coming close to saturation with
substrate, and demonstrating its maximal velocity (Vmax). Note that this
maximal velocity is in fact a theoretical limit that will not be truly achieved in
any experiment, although we might come very close to it.

RELATIONSHIP BETWEEN SUBSTRATE
CONCENTRATION AND THE RATE OF AN ENZYME-
CATALYSED REACTION

 The relationship described here is a fairly common one, which a mathematician
would immediately identify as a rectangular hyperbola. The equation that
describes such a relationship is as follows:
y=a.x/x+b
 The two constants a and b thus allow us to describe this hyperbolic
relationship, just as with a linear relationship (y = mx + c), which can be
expressed by the two constants m (the slope) and c (the intercept).
 We have in fact already defined the constant a — it is Vmax. The constant b is a
little more complex, as it is the value on the x-axis that gives half of the
maximal value of y. In enzymology we refer to this as the Michaelis constant
(Km), which is defined as the substrate concentration that gives half-maximal
velocity.
 Our final equation, usually called the Michaelis–Menten equation, therefore
becomes
Initial rate of reaction(v0) = Vmax×Substrate concentration/Substrate
concentration+Km

 In 1913, Leonor Michaelis and Maud Menten first showed that it was in fact
possible to derive this equation mathematically from first principles, with some
simple assumptions about the way in which an enzyme reacts with a substrate to
form a product.
 Central to their derivation is the concept that the reaction takes place via the
formation of an ES complex which, once formed, can either dissociate
(productively) to release product, or else dissociate in the reverse direction
without any formation of product.
 Thus the reaction can be represented as follows, with k1, k−1 and k2 being the
rate constants of the three individual reaction steps:
E+S⇌k−1k1ES⟶k2E+P
 The Michaelis–Menten derivation requires two important assumptions.
 The first assumption is that we are considering the initial velocity of the reaction
(v0), when the product concentration will be negligibly small (i.e. [S] ≫ [P]),
such that we can ignore the possibility of any product reverting to substrate.
 The second assumption is that the concentration of substrate greatly exceeds the
concentration of enzyme (i.e. [S]≫[E]).

 The derivation begins with an equation for the expression of the initial rate, the
rate of formation of product, as the rate at which the ES complex dissociates to
form product.
 This is based upon the rate constant k2 and the concentration of the ES complex,
as follows:
v0=d[P]/dt=k2×[ES].............1
 Since ES is an intermediate, its concentration is unknown, but we can express it
in terms of known values. In a steady-state approximation we can assume that
although the concentration of substrate and product changes, the concentration
of the ES complex itself remains constant.
 The rate of formation of the ES complex and the rate of its breakdown must
therefore balance, where:
 Rate of ES complex formation = k1[E][S]
and
 Rate of ES complex breakdown = (k−1 + k2)[ES]
 Hence, at steady state:
k1[E][S] = k−1 + k2[ES]

 This equation can be rearranged to yield [ES] as follows:
[ES]=k1[E][S]/k−1+k2...........2
 The Michaelis constant Km can be defined as follows:
Km=k−1+k2/k1
 Equation 2 may thus be simplified to:
[ES]=[E][S]/Km......................3
 Since the concentration of substrate greatly exceeds the concentration of enzyme (i.e.
[S] ≫ [E]), the concentration of un combined substrate [S] is almost equal to the total
concentration of substrate.
 The concentration of un combined enzyme [E] is equal to the total enzyme
concentration [E]T minus that combined with substrate [ES].
 Introducing these terms to Equation 3 and solving for ES gives us the following:
[ES]=[E]T[S]/[S]+Km..............4
 We can then introduce this term into Equation 1 to give:
v0=k2[E]T [S]/[S]+Km.............5

 The term k2[E]T in fact represents Vmax, the maximal velocity.
 Thus Michaelis and Menten were able to derive their final equation as:
v0=Vmax[S]/[S]+Km
 Michaelis constants have been determined for many commonly used enzymes,
and are typically in the lower millimolar range.

 It should be noted that enzymes which catalyse the same reaction, but
which are derived from different organisms, can have widely
differing Km values.
 Furthermore, an enzyme with multiple substrates can have quite
different Km values for each substrate.
 A low Km value indicates that the enzyme requires only a small amount
of substrate in order to become saturated. Therefore the maximum
velocity is reached at relatively low substrate concentrations.
 A high Km value indicates the need for high substrate concentrations in
order to achieve maximum reaction velocity.
 Thus we generally refer to Km as a measure of the affinity of the enzyme
for its substrate—in fact it is an inverse measure, where a
high Km indicates a low affinity, and vice versa. The Km value tells us
several important things about a particular enzyme.

 An enzyme with a low Km value relative to the physiological concentration of
substrate will probably always be saturated with substrate, and will therefore act
at a constant rate, regardless of variations in the concentration of substrate
within the physiological range.
 An enzyme with a high Km value relative to the physiological concentration of
substrate will not be saturated with substrate, and its activity will therefore vary
according to the concentration of substrate, so the rate of formation of product
will depend on the availability of substrate.
 If an enzyme acts on several substrates, the substrate with the lowest Km value is
frequently assumed to be that enzyme's ‘natural’ substrate, although this may
not be true in all cases.
 If two enzymes (with similar Vmax) in different metabolic pathways compete for
the same substrate, then if we know the Km values for the two enzymes we can
predict the relative activity of the two pathways.
 Essentially the pathway that has the enzyme with the lower Km value is likely to
be the ‘preferred pathway’, and more substrate will flow through that pathway
under most conditions.

 For example, phosphofructokinase (PFK) is the enzyme that catalyses the first
committed step in the glycolytic pathway, which generates energy in the form of
ATP for the cell, whereas glucose-1-phosphate uridylyltransferase (GUT) is an
enzyme early in the pathway leading to the synthesis of glycogen (an energy
storage molecule).
 Both enzymes use hexose monophosphates as substrates, but the Km of PFK for
its substrate is lower than that of GUT for its substrate. Thus at lower cellular
hexose phosphate concentrations, PFK will be active and GUT will be largely
inactive.
 At higher hexose phosphate concentrations both pathways will be active. This
means that the cells only store glycogen in times of plenty, and always give
preference to the pathway of ATP production, which is the more essential
function.

 Very often it is not possible to estimate Km values from a direct plot of velocity
against substrate concentration because we have not used high enough substrate
concentrations to come even close to estimating maximal velocity, and therefore
we cannot evaluate half-maximal velocity and thus Km.
 Fortunately, we can plot our experimental data in a slightly different way in
order to obtain these values. The most commonly used alternative is the
Lineweaver–Burk plot (often called the double-reciprocal plot).
 This plot linearizes the hyperbolic curved relationship, and the line produced is
easy to extrapolate, allowing evaluation of Vmax and Km. For example, if we
obtained only the first seven data points in Figure (Relationship between
substrate concentration and the rate of an enzyme-catalysed reaction), we would
have difficulty estimating Vmax from a direct plot as shown in Figure a (Direct
plot).
 However, as shown in Figure b (Lineweaver–Burk plot of the same kinetic
data.), if these seven points are plotted on a graph of 1/velocity (1/V) against
1/substrate concentration (1/[S]) (i.e. a double-reciprocal plot), the data are
linearized, and the line can be easily extrapolated to the left to provide intercepts
on both the y-axis and the x-axis, from which Vmax and Km, respectively, can be
evaluated

(a) Direct plot. (b) Lineweaver–Burk plot of the same kinetic
data

 One significant practical drawback of using the Lineweaver–Burk plot is the
excessive influence that it gives to measurements made at the lowest substrate
concentrations.
 These concentrations might well be the most prone to error (due to difficulties in
making multiple dilutions), and result in reaction rates that, because they are
slow, might also be most prone to measurement error.
 Often, as shown in Figure below, such points when transformed on the
Lineweaver–Burk plot have a significant impact on the line of best fit estimated
from the data, and therefore on the extrapolated values of both Vmax and Km.
 The two sets of points shown in Figure are identical except for the single point
at the top right, which reflects (because of the plot's double-reciprocal nature) a
single point derived from a very low substrate concentration and a low reaction
rate.
 However, this single point can have an enormous impact on the line of best fit
and the accompanying estimates of kinetic constants.

Lineweaver–Burk plot of similar kinetic data, which differ only
in a single. (Final data point (a) 1/v 0.03 at 1/S of 0.2 and (b)
1/v 0.031 at 1/S of 0.18)

2. Enzymes are affected by pH and temperature
 Various environmental factors are able to affect the rate of enzyme-catalysed
reactions through reversible or irreversible changes in the protein structure. The
effects of pH and temperature are generally well understood.
 Most enzymes have a characteristic optimum pH at which the velocity of the
catalysed reaction is maximal, and above and below which the velocity declines.
 The pH profile is dependent on a number of factors. As the pH changes, the
ionization of groups both at the enzyme's active site and on the substrate can
alter, influencing the rate of binding of the substrate to the active site.
 These effects are often reversible. For example, if we take an enzyme with an
optimal pH (pHopt) of 7.0 and place it in an environment at pH 6.0 or 8.0, the
charge properties of the enzyme and the substrate may be suboptimal, such that
binding and hence the reaction rate are lowered.

The pH profile of β-glucosidase.

 If we then readjust the pH to 7.0, the optimal charge properties and hence the
maximal activity of the enzyme are often restored. However, if we place the
enzyme in a more extreme acidic or alkaline environment (e.g. at pH 1 or 14),
although these conditions may not actually lead to changes in the very stable
covalent structure of the protein (i.e. its configuration), they may well produce
changes in the conformation (shape) of the protein such that, when it is returned
to pH 7.0, the original conformation and hence the enzyme's full catalytic
activity are not restored.
 It should be noted that the optimum pH of an enzyme may not be identical to
that of its normal intracellular surroundings. This indicates that the local pH can
exert a controlling influence on enzyme activity.

 The effects of temperature on enzyme activity are quite complex, and can
be regarded as two forces acting simultaneously but in opposite
directions. As the temperature is raised, the rate of molecular movement
and hence the rate of reaction increases, but at the same time there is a
progressive inactivation caused by denaturation of the enzyme protein.
 This becomes more pronounced as the temperature increases, so that an
apparent temperature optimum (Topt) is observed.
 Thermal denaturation is time dependent, and for an enzyme the term
‘optimum temperature’ has little real meaning unless the duration of
exposure to that temperature is recorded.
 The thermal stability of an enzyme can be determined by first exposing
the protein to a range of temperatures for a fixed period of time, and
subsequently measuring its activity at one favourable temperature (e.g.
25°C).

The effect of temperature on enzyme activity

 The temperature at which denaturation becomes important varies from one
enzyme to another. Normally it is negligible below 30°C, and starts to become
appreciable above 40°C.
 Typically, enzymes derived from microbial sources show much higher thermal
stability than do those from mammalian sources, and enzymes derived from
extremely thermophilic microorganisms, such as thermolysin (a protease
from Bacillus thermoproteolyticus) and Taq polymerase (a DNA polymerase
from Thermus aquaticus), might be completely thermostable at 70°C and still
retain substantial levels of activity even at 100°C.

3. Enzymes are sensitive to inhibitors
 Substances that reduce the activity of an enzyme-catalysed reaction are known
as inhibitors. They act by either directly or indirectly influencing the catalytic
properties of the active site. Inhibitors can be foreign to the cell or natural
components of it.
 Those in the latter category can represent an important element of the
regulation of cell metabolism. Many toxins and also many pharmacologically
active agents (both illegal drugs and prescription and over-the-counter
medicines) act by inhibiting specific enzyme-catalysed processes.
• Reversible inhibition
 Inhibitors are classified as reversible inhibitors when they bind reversibly to an
enzyme. A molecule that is structurally similar to the normal substrate may be
able to bind reversibly to the enzyme's active site and therefore act as a
competitive inhibitor.
 For example, malonate is a competitive inhibitor of the enzyme succinate
dehydrogenase, as it is capable of binding to the enzyme's active site due to its
close structural similarity to the enzyme's natural substrate, succinate.
 When malonate occupies the active site of succinate dehydrogenase it prevents
the natural substrate, succinate, from binding, thereby slowing down the rate of
oxidation of succinate to fumarate (i.e. inhibiting the reaction).

 One of the characteristics of competitive inhibitors is that they can be
displaced from the active site if high concentrations of substrate are
used, thereby restoring enzyme activity.
 Thus competitive inhibitors increase the Km of a reaction because they
increase the concentration of substrate required to saturate the enzyme.
However, they do not change Vmax itself.
 In the case of certain enzymes, high concentrations of either the
substrate or the product can be inhibitory. For example, invertase activity
is considerably reduced in the presence of high concentrations of sucrose
(its substrate), whereas the β-galactosidase of Aspergillus niger is
strongly inhibited by galactose (its product). Products of an enzyme
reaction are some of the most commonly encountered competitive
inhibitors.
 Other types of reversible inhibitor also exist. Non-competitive inhibitors
react with the enzyme at a site distinct from the active site. Therefore the
binding of the inhibitor does not physically block the substrate–binding
site, but it does prevent subsequent reaction.
 Most non-competitive inhibitors are chemically unrelated to the
substrate, and their inhibition cannot be overcome by increasing the
substrate concentration. Such inhibitors in effect reduce the
concentration of the active enzyme in solution, thereby reducing
the Vmax of the reaction. However, they do not change the value of Km.

 Uncompetitive inhibition is rather rare, occurring when the inhibitor is only able
to bind to the enzyme once a substrate molecule has itself bound. As such,
inhibition is most significant at high substrate concentrations, and results in a
reduction in the Vmax of the reaction.
 Uncompetitive inhibition also causes a reduction in Km, which seems somewhat
counterintuitive as this means that the affinity of the enzyme for its substrate is
actually increased when the inhibitor is present.
 This effect occurs because the binding of the inhibitor to the ES complex
effectively removes ES complex and thereby affects the overall equilibrium of
the reaction favouring ES complex formation.
 It is noteworthy however that since both Vmax and Km are reduced the observed
reaction rates with inhibitor present are always lower than those in the absence
of the uncompetitive inhibitor.
• Irreversible inhibitors and poisons
 If an inhibitor binds permanently to an enzyme it is known as an irreversible
inhibitor. Many irreversible inhibitors are therefore potent toxins.
 Organophosphorus compounds such as di isopropyl fluorophosphate (DFP)
inhibit acetylcholinesterase activity by reacting covalently with an important
serine residue found within the active site of the enzyme.

 The physiological effect of this inactivation is interference with neurotransmitter
inactivation at the synapses of nerves, resulting in the constant propagation of nerve
impulses, which can lead to death. DFP was originally evaluated by the British as a
chemical warfare agent during World War Two, and modified versions of this
compound are now widely used as organophosphate pesticides (e.g. parathione,
malathione).
4. Allosteric regulators and the control of enzyme activity
 Having spent time learning about enzyme kinetics and the Michaelis–Menten
relationship, it is often quite disconcerting to find that some of the most important
enzymes do not in fact display such properties.
 Allosteric enzymes are key regulatory enzymes that control the activities of
metabolic pathways by responding to inhibitors and activators. These enzymes in
fact show a sigmoidal (S-shaped) relationship between reaction rate and substrate
concentration (Figure below Activity/substrate profiles of allosteric (a) and non-
allosteric (b) enzymes with the same affinity and maximal velocity.), rather than the
usual hyperbolic relationship.
 Thus for allosteric enzymes there is an area where activity is lower than that of an
equivalent ‘normal’ enzyme, and also an area where activity is higher than that of an
equivalent ‘normal’ enzyme, with a rapid transition between these two phases. This
is rather like a switch that can quickly be changed from ‘off’ (low activity) to ‘on’
(full activity).

Activity/substrate profiles of allosteric (a) and non-allosteric
(b) enzymes with the same affinity and maximal velocity

 Most allosteric enzymes are polymeric—that is, they are composed of at least
two (and often many more) individual polypeptide chains. They also have
multiple active sites where the substrate can bind.
 Much of our understanding of the function of allosteric enzymes comes from
studies of haemoglobin which, although it is not an enzyme, binds oxygen in a
similarly co-operative way and thus also demonstrates this sigmoidal
relationship.
 Allosteric enzymes have an initially low affinity for the substrate, but when a
single substrate molecule binds, this may break some bonds within the enzyme
and thereby change the shape of the protein such that the remaining active sites
are able to bind with a higher affinity.
 Therefore allosteric enzymes are often described as moving from a tensed state
or T-state (low affinity) in which no substrate is bound, to a relaxed state or R-
state (high affinity) as substrate binds.
 Other molecules can also bind to allosteric enzymes, at additional regulatory
sites (i.e. not at the active site). Molecules that stabilize the protein in its T-state
therefore act as allosteric inhibitors, whereas molecules that move the protein to
its R-state will act as allosteric activators or promoters.
 A good example of an allosteric enzyme is aspartate transcarbamoylase
(ATCase), a key regulatory enzyme that catalyses the first committed step in the
sequence of reactions that produce the pyrimidine nucleotides which are
essential components of DNA and RNA. The reaction is as follows:

 The end product in the pathway, the pyrimidine nucleotide cytidine triphosphate
(CTP), is an active allosteric inhibitor of the enzyme ATCase.
 Therefore when there is a high concentration of CTP in the cell, this feeds back
and inhibits the ATCase enzyme, reducing its activity and thus lowering the rate
of production of further pyrimidine nucleotides.
 As the concentration of CTP in the cell decreases then so does the inhibition of
ATCase, and the resulting increase in enzyme activity leads to the production of
more pyrimidine nucleotides.
 This negative feedback inhibition is an important element of biochemical
homeostasis within the cell. However, in order to synthesize DNA and RNA, the
cell requires not only pyrimidine nucleotides but also purine nucleotides, and
these are needed in roughly equal proportions.
 Purine synthesis occurs through a different pathway, but interestingly the final
product, the purine nucleotide adenosine triphosphate (ATP), is a potent
activator of the enzyme ATCase.
 This is logical, since when the cell contains high concentrations of purine
nucleotides it will require equally high concentrations of pyrimidine nucleotides
in order for these two types of nucleotide to combine to form the polymers DNA
and RNA.
 Thus ATCase is able to regulate the production of pyrimidine nucleotides within
the cell according to cellular demand, and also to ensure that pyrimidine
nucleotide synthesis is synchronized with purine nucleotide synthesis—an
elegant biochemical mechanism for the regulation of an extremely important
metabolic process.

 There are some rare, although important, cases of monomeric enzymes
that have only one substrate-binding site but are capable of
demonstrating the sigmoidal reaction kinetics characteristic of allosteric
enzymes.
 Particularly noteworthy in this context is the monomeric enzyme
glucokinase (also called hexokinase IV), which catalyses the
phosphorylation of glucose to glucose-6-phosphate (which may then
either be metabolized by the glycolytic pathway or be used in glycogen
synthesis).
 It has been postulated that this kinetic behaviour is a result of individual
glucokinase molecules existing in one of two forms—a low-affinity form
and a high-affinity form. The low-affinity form of the enzyme reacts
with its substrate (glucose), is then turned into the high-affinity form,
and remains in that state for a short time before slowly returning to its
original low-affinity form (demonstrating a so-called slow transition).
 Therefore at high substrate concentrations the enzyme is likely to react
with a second substrate molecule soon after the first one (i.e. while still
in its high-affinity form), whereas at lower substrate concentrations the
enzyme may transition back to its low-affinity form before it reacts with
subsequent substrate molecules. This results in its characteristic
sigmoidal reaction kinetics.

5. Enzymes may be intracellular or extracellular
 Although many enzymes are retained within the cell, and may be located in
specific subcellular compartments, others are released into the surrounding
environment.
 The majority of enzymes in industrial use are extracellular proteins from either
fungal sources (e.g. Aspergillus species) or bacterial sources
(e.g. Bacillus species). Examples of these include α-amylase, cellulase,
dextranase, proteases and amyloglucosidase.
 Many other enzymes for non-industrial use are intracellular and are produced in
much smaller amounts by the cell. Examples of these include asparaginase,
catalase, cholesterol oxidase, glucose oxidase and glucose-6-phosphate
dehydrogenase.
ENZYME PURIFICATION
 Within the cell, enzymes are generally found along with other proteins, nucleic
acids, polysaccharides and lipids. The activity of the enzyme in relation to the
total protein present (i.e. the specific activity) can be determined and used as a
measure of enzyme purity.
 A variety of methods can be used to remove contaminating material in order to
purify the enzyme and increase its specific activity. Enzymes that are used as
diagnostic reagents and in clinical therapeutics are normally prepared to a high
degree of purity, because great emphasis is placed on the specificity of the
reaction that is being catalysed.

 Clearly the higher the level of purification, the greater the cost of enzyme
production. In the case of many bulk industrial enzymes the degree of
purification is less important, and such enzymes may often be sold as very crude
preparations of culture broth containing the growth medium, organisms (whole
or fragmented) and enzymes of interest.
 However, even when the cheapest bulk enzymes are utilized (e.g. proteases for
use in washing powders), the enzyme cost can contribute around 5–10% of the
final product value.
1. Pretreatment
 At the end of a fermentation in which a microorganism rich in the required
enzyme has been cultured, the broth may be cooled rapidly to 5°C to prevent
further microbial growth and stabilize the enzyme product.
 The pH may also be adjusted to optimize enzyme stability. If the enzyme-
producing organism is a fungus, this may be removed by centrifugation at low
speed.
 If the enzyme source is bacterial, the bacteria are often flocculated with
aluminum sulfate or calcium chloride, which negate the charge on the bacterial
membranes, causing them to clump and thus come out of suspension.

2. Treatment
 Extracellular enzymes are found in the liquid component of the pretreatment
process. However, intracellular enzymes require more extensive treatment.
 The biomass may be concentrated by centrifugation and washed to remove
medium components.
 The cellular component must then be ruptured to release the enzyme content.
This can be done using one or more of the following processes:
• ball milling (using glass beads)
• enzymic removal of the cell wall
• freeze–thaw cycles
• liquid shearing through a small orifice at high pressure (e.g. within a French
press)
• osmotic shock
• sonication.
 Separation of enzymes from the resulting solution may then involve a variety of
separation processes, which are often employed in a sequential fashion.

 The first step in an enzyme purification procedure commonly involves
separation of the proteins from the non-protein components by a process of
salting out.
 Proteins remain in aqueous solution because of interactions between the
hydrophilic (water-loving) amino acids and the surrounding water molecules
(the solvent). If the ionic strength of the solvent is increased by adding an
agent such as ammonium sulfate, some of the water molecules will interact
with the salt ions, thereby decreasing the number of water molecules available
to interact with the protein.
 Under such conditions, when protein molecules cannot interact with the
solvent, they interact with each other, coagulating and coming out of solution
in the form of a precipitate. This precipitate (containing the enzyme of interest
and other proteins) can then be filtered or centrifuged, and separated from the
supernatant.
 Since different proteins vary in the extent to which they interact with water, it
is possible to perform this process using a series of additions of ammonium
sulfate, increasing the ionic strength in a stepwise fashion and removing the
precipitate at each stage. Thus such fractional precipitation is not only capable
of separating protein from non-protein components, but can also enable
separation of the enzyme of interest from some of the other protein
components.

 Subsequently a wide variety of techniques may be used for further purification, and
steps involving chromatography are standard practice.
 Ion-exchange chromatography is often effective during the early stages of the
purification process. The protein solution is added to a column containing an
insoluble polymer (e.g. cellulose) that has been modified so that its ionic
characteristics will determine the type of mobile ion (i.e. cation or anion) it attracts.
Proteins whose net charge is opposite to that of the ion-exchange material will bind
to it, whereas all other proteins will pass through the column. A subsequent change
in pH or the introduction of a salt solution will alter the electrostatic forces, allowing
the retained protein to be released into solution again.
 Gel filtration can be utilized in the later stages of a purification protocol to separate
molecules on the basis of molecular size. Columns containing a bed of cross-linked
gel particles such as Sephadex are used. These gel particles exclude large protein
molecules while allowing the entry of smaller molecules. Separation occurs because
the larger protein molecules follow a path down the column between the Sephadex
particles (occupying a smaller fraction of the column volume). Larger molecules
therefore have a shorter elution time and are recovered first from the gel filtration
column.

 Affinity chromatography procedures can often enable purification protocols to
be substantially simplified. Typically, with respect to enzyme purification, a
column would be packed with a particulate stationary phase to which a ligand
molecule such as a substrate analogue, inhibitor or cofactor of the enzyme of
interest would be firmly bound. As the sample mixture is passed through the
column, the enzyme interacts with, and binds, to the immobilised ligand, being
retained within the column as all of the other components of the mixture pass
through the column unrewarded. Subsequently a solution of the ligand is
introduced to the column to release (elute) and thereby recover the bound
enzyme from the column in a highly purified form.
 Nowadays numerous alternative affinity chromatography procedures exist that
are able to separate enzymes by binding to areas of the molecule away form
their active site. Advances in molecular biology enable us to purify recombinant
proteins, including enzymes, through affinity tagging. In a typical approach the
gene for the enzyme of interest would be modified to code for a further short
amino acid sequence at either the N- or C- terminal. For example, a range of
polyhistidine tagging procedures are available to yield protein products with six
or more consecutive histidine residues at their N- or C- terminal end.

 When a mixture containing the tagged protein of interest is subsequently passed
through a column containing a nickel-nitrilotriacetic acid (Ni-NTA) agarose
resin, the histidine residues on the recombinant protein bind to the nickel ions
attached to the support resin, retaining the protein, whilst other protein and non-
protein components pass through the column. Elution of the bound protein can
then be accomplished by adding imidazole to the column, or by reducing the pH
to 5-6 to displace the His-tagged protein from the nickel ions.
 Such techniques are therefore capable of rapidly and highly effectively isolating
an enzyme from a complex mixture in only one step, and typically provide
protein purities of up to 95%. If more highly purified enzyme products are
required, other supplemental options are also available, including various forms
of preparative electrophoresis e.g. disc-gel electrophoresis and isoelectric
focusing.
3. Finishing of enzymes
 Enzymes are antigenic, and since problems occurred in the late 1960s when
manufacturing workers exhibited severe allergic responses after breathing
enzyme dusts, procedures have now been implemented to reduce dust
formation.

 These involve supplying enzymes as liquids wherever possible, or
increasing the particle size of dry powders from 10 μm to 200–500 μm
by either prilling (mixing the enzyme with polyethylene glycol and
preparing small spheres by atomization) or marumerizing (mixing the
enzyme with a binder and water, extruding long filaments, converting
them into spheres in a marumerizer, drying them and covering them with
a waxy coat).
ENZYME IMMOBILIZATION
 During the production of commercially important products via enzymatic
catalysis, soluble enzymes have traditionally been used in batch processes that
employ some form of stirred-tank reactor (STR).
 In these processes, at the end of the batch run the product must be separated
from any unused substrate, and also from the enzyme catalyst.
 Removal of the enzyme at this stage can be achieved by thermal denaturation
(only if the product is thermostable) or by ammonium sulfate precipitation or
ultrafiltration.
 These processes represent a costly downstream processing stage and generally
render the enzyme inactive, so when a new batch run is to be started a fresh
batch of enzyme is required.

 Immobilized enzyme systems, in contrast, ‘fix’ the enzyme so that it can be
reused many times, which has a significant impact on production costs. As a
very simple example, if an enzyme is mixed with a solution of warm (but not
too hot) agar and this is allowed to set, the enzyme will be entrapped (for the
purposes of this example let us ignore the fact that the enzyme will gradually
leak out of this gel).
 The agar can then be cut up into cubes and these can be placed in a STR,
together with substrate.
 Again the reaction would be allowed to proceed (and it might actually be
slower due to diffusional constraints and other effects described later). At the
end of the batch run the catalyst can now be easily separated from the product
by passing the reactor contents through a coarse mesh.
 Immediately an important downstream processing step has been carried out
and, just as importantly, the active enzyme has been recovered so that it can be
reused for the next batch run. This ease of separation of enzyme from product is
a major advantage of all immobilized systems over their counterparts that use
free (i.e. soluble) enzyme.

 This physical advantage of ease of reuse of immobilized biocatalysts is
one of the main reasons why such systems are favoured commercially.
However, immobilization may also produce biochemical changes that
lead to enhanced biocatalyst stability, which may be manifested as:
• an increased rate of catalysis
• prolonged duration of catalysis
• greater operational stability to extremes of pH, temperature, etc.
 The particular advantages conferred by immobilization will therefore
differ from one system to another. It should be noted that often there
may be no biochemical advantage at all, and the simple physical
advantage of ease of separation of the biocatalyst from the product may
be sufficient to favour the commercial development of an immobilized
process.
 The basic principle of immobilization is to partition the biocatalyst into a
distinct phase, rather than mix it homogeneously with the substrate.
 Will this not cause reaction rates to be low?
 The answer to this question is yes, and the relationship between the
activity of an immobilized system and a non-immobilized system can be
expressed as the effectiveness factor (η), where:

Effectiveness factor=Activity of immobilized biocatalyst/Activity of non -
immobilized biocatalyst
 Thus an immobilized system with an effectiveness factor of 0.1 would show
only 10% of the activity of a non-immobilized system with the same amount of
enzyme and operating under the same conditions. At first sight this might
appear to be a major problem.
 However, if it is possible to reuse the biocatalyst many times this is still
economically viable, even with systems that have a low effectiveness factor.
 In principle, therefore, for economic viability:
Effectiveness factor × Number of times of reuse ≥ 1
 Thus if an immobilized system has an effectiveness factor of 0.1 (i.e. 10%) and
we can reuse the biocatalyst 10 times, we essentially achieve the same overall
catalytic activity with both the non-immobilized system and the immobilized
one. However, if we are able to reuse the biocatalyst 100 times we in fact
obtain 10 times more total activity from the immobilized system than from the
equivalent non-immobilized system, so the immobilized system may be
economically preferable.
 Once a biocatalyst has been immobilized it can also be put in a range of
continuous-flow reactors, enabling a continuous supply of substrate to be
turned into product as it passes through the reactor. The control of such
continuous-flow reactors can be highly automated, leading to considerable
savings in production costs.

 For example, a STR can be easily modified to produce a continuous-flow stirred-
tank reactor (CSTR), in which the enzyme is held within the reactor by a coarse
mesh, and the product continuously flows out of the reactor as substrate is pumped
in.
 It is also possible to produce a packed-bed reactor (PBR), in which the agar cubes
are packed into a column and the substrate is pumped through the bed without any
need for stirring.
 CSTRs and PBRs enable the enzyme to be reused many times before it needs to be
replaced. For example, in the production of high-fructose syrups, the immobilized
glucose isomerase enzyme would typically be used continuously for between 2 and
4 months, and only after this time (when its activity would have dropped to 25% of
the original level) would it need to be replaced.
 The overall operating costs of continuous-flow reactors are often significantly lower
than those of equivalent batch processes. Batch reactors need to be emptied and
refilled frequently at regular intervals.
 Not only is this procedure expensive, but it also means that there are
considerable periods of time when such reactors are not productive (so-called
‘downtime’). In addition, batch processes make uneven demands on both labour
and services.
 They may also result in pronounced batch-to-batch variations, as the reaction
conditions change with time, and they may be difficult to scale up, due to the
changing power requirements for efficient mixing.

 Due to their higher overall process efficiency, continuous processes
using immobilized enzymes may be undertaken in production facilities
that are around 10 to 100 times smaller than those required for
equivalent batch processes using soluble enzymes.
 Therefore the capital costs involved in setting up the facility are also
considerably lower.
Immobilization techniques
 It should be noted that although the agar entrapment method described
here has provided a useful example, it is not a particularly effective form
of immobilization.
 The high temperature required to prevent the agar from setting may lead
to thermal inactivation of the enzyme, and the agar gel itself is very
porous and will allow the enzyme to leak out into the surrounding
solution.
 There are in fact thousands of different techniques of immobilization, all
of which are much more effective than our example. In general these
techniques can be classified as belonging to one of three categories:

• Adsorption
• Covalent bonding
• Entrapment

1. Adsorption
 The physical adsorption of an enzyme to a supporting matrix is the oldest
method of immobilization. As early as 1916, J.M. Nelson and Edward G. Griffin
described the adsorption of yeast invertase on to activated charcoal, and the
subsequent use of this preparation for sucrose hydrolysis.
 Over the years a variety of adsorbents have been used, including cellulose,
Sephadex, polystyrene, kaolinite, collagen, alumina, silica gel and glass. Such
immobilization procedures are extremely easy to perform, as the adsorbent and
enzyme are simply stirred together for a time (typically minutes to hours).
 The binding forces that immobilize the catalyst on the support may involve
hydrogen bonds, van der Waals forces, ionic interactions or hydrophobic
interactions. Such forces are generally weak in comparison with covalent
bonds—for example, a hydrogen bond has an energy content of about 20 kJ
mol−1, compared with 200–500 kJ mol−1 for a covalent bond.
 Thus, when using such methods, yields (i.e. the amount of enzyme bound per
unit of adsorbent) are generally low. In addition, adsorption is generally easily
reversed, and can lead to desorption of the enzyme at a critical time.

 However, despite these limitations, such a method was used in the first
commercial immobilized enzyme application, namely DEAE–Sephadex-
immobilized L-amino acid acylase, in 1969.
 DEAE–Sephadex is an ion-exchange resin that consists of an inert dextran
particle activated by the addition of numerous diethyl aminoethyl groups.
Particles of this material remain positively charged at pH 6–8 and thus bind
strongly to proteins, which are generally negatively charged in this pH range.
 If the pH is kept constant, the enzyme and support will remain ionically linked.
However, when over time the enzyme loses its activity through denaturation, the
pH can be adjusted to a more acidic value, the old enzyme will be desorbed, and
the pH can then be readjusted back to pH 6–8 and a fresh batch of enzyme
bound.
 Thus the support matrix may be used many times, giving the process significant
economic benefits.

Properties of (a) DEAE–Sephadex and (b) CM–Sephadex ion-
exchange resins

 Clearly DEAE–Sephadex immobilization is only of value for enzymes that have
a neutral-to-alkaline pH optimum. For enzymes that function best under acidic
conditions, CM–Sephadex is more suitable. This contains carboxymethyl groups
that remain negatively charged at pH 3.5–4.5 .
 Proteins at this pH are generally positively charged and will thus ionically bind
to the support. Desorption of the enzyme will occur when the pH is adjusted to a
more alkaline value.
 Due to the simplicity and controllability of this immobilization procedure,
combined with the economic benefits of reuse of the support, ion-exchange
materials are now widely used as the method of choice in many industrial
settings.
2. Covalent bonding
 Immobilization of enzymes by covalent bonding to activated polymers is a
widely used approach since, although it is often a tedious procedure, it is
capable of producing an immobilized enzyme that is firmly bound to its support.
The range of polymers and chemical coupling procedures that are used is
enormous.
.

 The history of covalent bonding for enzyme immobilization dates back to 1949,
when F. Michael and J. Ewers used the azide derivative of carboxy methyl
cellulose to immobilize a variety of proteins. Activated cellulose supports
continue to be popular due to their inherent advantages of high hydrophilicity,
ready availability, potential for derivatization, and the ease with which
cellulose-based polymers can be produced either as particulate powders or as
membranous films
 It is often more effective not to build the reactive group into the cellulose itself,
but instead to use a chemical ‘bridge’ between the cellulose and the enzyme
molecule. The requirements for such a bridging or linking molecule are that it
must be small, and that once it has reacted with the support it must have a
further reactive group capable of reacting with the enzyme.
 An example of such a bridging molecule is glutaraldehyde, which contains two
aldehyde groups, one at either end of its (CH2)3 moiety. At neutral pH values the
aldehyde groups will react with free amino groups. Thus one end of the
glutaraldehyde molecule may be attached to the support, and the other to the
enzyme.
 Covalently immobilized enzymes are strongly bound to their support, so when
the proteins denature they are difficult to remove (in contrast to adsorption, as
described earlier). Therefore it is usual for both the enzyme and the support to
be replaced. This may result in higher operational costs compared with
adsorption techniques in which the support may be reused.

3. Entrapment
 The entrapment of an enzyme can be achieved in a number of ways:
• inclusion within the matrix of a highly cross-linked polymer
• separation from the bulk phase by a semi-permeable ‘microcapsule’
• dissolution in a distinct non-aqueous phase.
 An important feature of entrapment techniques is that the enzyme is not
in fact attached to anything. Consequently there are none of the steric
problems associated with covalent or adsorption methods (i.e. the
possibility of the enzyme binding in such a way that its active site is
obstructed by part of the supporting polymer matrix).
 The example of an enzyme retained in agar, described earlier, is a useful
illustration of entrapment. A preferable alternative involves mixing the
catalyst with sodium alginate gel and extruding this into a solution of
calcium chloride to produce solid calcium alginate particles.
 This technique has the advantage of not requiring the use of high
temperatures. However, although it is a popular activity in teaching
laboratories, outside that setting it is generally unsuitable for the
immobilization of purified enzymes, as these are often able to leak out of
the gel.

 Entrapment techniques for purified enzymes are more likely to involve retaining
the enzyme behind some form of ultrafiltration membrane. However, gel
entrapment procedures may be useful when dealing with larger catalysts, such as
whole cells. For example, gel-immobilized living yeast cells have been used
successfully in the manufacture of champagne by Moët & Chandon.
Immobilization: changes in enzyme properties
 Earlier in this essay it was suggested that immobilization might change the
properties of an enzyme to enhance its stability. Initially it was believed that such
enhanced stability resulted from the formation of bonds between the enzyme and
the supporting matrix that physically stabilize the structure of the protein.
 Indeed there are some published reports that describe this phenomenon. With
regard to the stabilization of proteolytic enzymes, which often exhibit more
prolonged activity in the immobilized state, this is most probably explained by
the fact that such proteases in free solution are prone to auto digestion (i.e.
enzyme molecules cleave the peptide bonds of adjacent enzyme molecules), a
process that is largely prevented when they are fixed to a supporting matrix.

 However, the effects of immobilization are more often due to the supporting
matrix changing the microenvironment around the enzyme and/or introducing
diffusional constraints that modify the activity of the catalyst. Consider, for
example, immobilization of the enzyme by adsorption on to a polyanionic
(negatively charged) support such as cellulose.
 If the substrate is a cation (i.e. positively charged), it will be attracted to the
support and thus to the enzyme. In this case the enzyme might well display
higher activity, as the substrate concentration in its microenvironment would be
higher than that in the surrounding bulk phase.
 Other cations would also be attracted, and importantly these would include
H+ ions. Thus the microenvironment would also be enriched in H+ ions, so the
pH surrounding the enzyme would be lower than the pH of the bulk phase.
Consequently the enzyme would also exhibit an altered pH profile compared
with that of its soluble counterpart.
 In addition, the immobilization matrix might act as a barrier to the diffusion of
substrates, products and other molecules. For example, if a high enzyme loading
was put into a gel particle and this was then immersed in substrate solution, the
substrate would diffuse into the gel and rapidly be converted into product.
 Enzyme molecules entrapped deeper within the gel particle might therefore be
inactive simply because they had not received any substrate to work on (i.e. all
of the substrate was converted to product in the outer layers of the particle).

 Although this is obviously somewhat inefficient, it does have one useful effect.
When over time the enzyme within the system denatures, the loss of activity of
the enzyme in the outer part of the particle means that substrate will now
diffuse deeper into the particle to reach the previously unused core enzyme
molecules.
 In effect this inner reserve of enzyme will offset the loss of enzyme activity
through denaturation, so the system will show little or no overall loss of
activity. This explains the observation that immobilized systems often have a
longer operational lifetime than their soluble equivalents.
 In addition, it is of interest that enzymes bound to natural cell membranes
(phospholipid bilayers) within living cells will also probably demonstrate these
effects, and immobilized systems thus provide useful models for the study of
such membrane-bound proteins in living cells.

INDUSTRIAL ENZYMOLOGY
 Although many industrial processes, such as cheese manufacturing, have
traditionally used impure enzyme sources, often from animals or plants, the
development of much of modern industrial enzymology has gone hand in hand
with the commercial exploitation of microbial enzymes.
 These were introduced to the West in around 1890 when the Japanese scientist
Jokichi Takamine settled in the U.S.A. and set up an enzyme factory based on
Japanese technology. The principal product was Takadiastase, a mixture of
amylolytic and proteolytic enzymes prepared by cultivating the
fungus Aspergillus oryzae on rice or wheat bran.
 Takadiastase was marketed successfully in the U.S.A. as a digestive aid for the
treatment of dyspepsia, which was then believed to result from the incomplete
digestion of starch.
 Bacterial enzymes were developed in France by August Boidin and Jean
Effront, who in 1913 found that Bacillus subtilis produced a heat-stable α-
amylase when grown in a liquid medium made by extraction of malt or grain.
The enzyme was primarily used within the textile industry for the removal of the
starch that protects the warp in the manufacture of cotton.
 In around 1930 it was found that fungal pectinases could be used in the
preparation of fruit products. In subsequent years, several other hydrolases were
developed and sold commercially (e.g. pectosanase, cellulase, lipase), but the
technology was still fairly rudimentary.

 After World War 2, the fermentation industry underwent rapid development as
methods for the production of antibiotics were developed. These methods were
soon adapted for the production of enzymes.
 In the 1960s, glucoamylase was introduced as a means of hydrolysing starch,
replacing acid hydrolysis. Subsequently, in the 1960s and 1970s, proteases were
incorporated into detergents and then glucose isomerase was introduced to
produce sweetening agents in the form of high-fructose syrups.
 Since the 1990s, lipases have been incorporated into washing powders, and a
variety of immobilized enzyme processes have been developed (see section on
enzyme immobilization), many of which utilize intracellular enzymes.
 Currently, enzymes are used in four distinct fields of commerce and technology:
• as industrial catalysts
• as therapeutic agents
• as analytic reagents
• as manipulative tools (e.g. in genetics).

 Of the thousands of different types of enzymes, about 95% are available from
suppliers in quantities ranging from μg to kg, provided essentially for research
purposes. Around 40–50 enzymes are produced on an industrial scale (i.e.
ranging from multiple kilograms to tonnes per annum).
 The global enzyme market is currently dominated by the hydrolases, especially
the proteases, together with amylases, cellulases and lipases supplied either as
liquid concentrates or as powders or granules that release the soluble enzyme on
dissolution.
 Global production is dominated by two companies, which between them supply
more than two-thirds of the global enzyme market, namely the Danish company
Novozymes, with a market share of 47%, and the U.S. company DuPont (which
recently acquired Genencor), with 21%.
 The value of the world enzyme market has increased steadily from £110 million
in 1960 to £200 million in 1970, £270 million in 1980, £1 000 million in 1990
and over £2 000 million in 2010. Food and beverage enzymes represented the
largest sector of the industrial enzymes market in 2010, with a value of £750
million, and the market for enzymes for technical applications (including
diagnostic applications, research and biotechnology) accounted for a further
£700 million.
 Estimates of future demand are in the range of £4 000–5 000 million between
2015 and 2016, growing at a rate of 6–7% annually. The developing economies
of the Asia-Pacific Region, the Middle East and Africa are now seen to be
emerging as the fastest growing markets for industrial enzymes.

 Microbial enzymes are typically produced in batches by culturing the producing
organism within a batch fermenter. Fermentation typically lasts between 30 and
150 h, with the optimum enzyme yield for the process falling somewhere
between the optimum biomass yield and the point of maximal enzyme activity
within the cells.
 Relatively small fermenters with a volume of 10–100 m3 are generally
employed, allowing flexibility where a number of different products are being
produced.
 Many production systems are optimized by means of a fed-batch process, in
which substrates are gradually fed into the reactor over the course of the
fermentation, rather than being provided all at once at the start of the process.
 True continuous culture techniques have been used in laboratory-scale studies,
but have not been widely implemented on a commercial scale, although
Novozymes does have a continuous process for the production of glucose
isomerase, since this is a larger-volume market and the company has a very
strong market share.

ENZYMES IN ANALYSIS
 Enzymes have a wide variety of uses in analytical procedures. Their specificity
and potency allow both detection and amplification of a target analyte. ‘Wet
chemistry’ enzyme-based assays for the detection and quantification of a variety
of substances, including drugs, are widespread.
 Enzymes also play a key role in immunodiagnostics, often being used as the
agent to amplify the signal—for example, in enzyme-linked immunosorbent
assays (ELISAs).
 Within DNA-fingerprinting technology, the enzyme DNA polymerase plays a
key role in the amplification of DNA molecules in the polymerase chain
reaction.
 However, ‘wet chemistry’ analytical methods are increasingly being replaced by
the use of biosensors—that is, self-contained integrated devices which
incorporate a biological recognition component (usually an immobilized
enzyme) and an electrochemical detector (known as a transducer).

Biosensors
 Much of the technological development of biosensors has been motivated by the
need to measure blood glucose levels. In 2000, the World Health Organization
estimated that over 170 million people had diabetes, and predicted that this
figure will rise to over 360 million by 2030.
 In view of this, many companies have made significant investments in R&D
programmes that have led to the availability of a wide variety of glucose
biosensor devices.
 In 1962, Leland Clark Jr coined the term ‘enzyme electrode’ to describe a
device in which a traditional electrode could be modified to respond to other
materials by the inclusion of a nearby enzyme layer.
 Clark's ideas became a commercial reality in 1975 with the successful launch of
the Yellow Springs Instruments (YSI) model 23A glucose analyser. This device
incorporated glucose oxidase together with a peroxide-sensitive electrode to
measure the hydrogen peroxide (H2O2) produced during the following reaction:
Glucose + O2 → Gluconic acid + H2O2

 In this device, the rate of H2O2 formation is a measure of the rate of the reaction,
which depends on the concentration of glucose in solution, thus allowing the
latter to be estimated.
 As was discussed earlier, in enzyme-catalysed reactions the relationship
between substrate concentration and reaction rate is not linear, but hyperbolic
(as described by the Michaelis–Menten equation).
 This is also true for the glucose oxidase within a biosensor. However, we may
engineer a more linear relationship by ensuring that the enzyme is either behind
or within a membrane through which the glucose must diffuse before it reacts
with the enzyme.
 This means that the system becomes diffusionally, rather than kinetically,
limited, and the response is then more linearly related to the concentration of
glucose in solution.
 Over the years the YSI model 23A glucose analyser has been replaced by a
range of much more advanced models. The current YSI model 2900 Series
glucose analyser is shown in Figure .
 This instrument has a 96-sample rack that enables batches of samples to be run,
with the analysis of each sample taking less than a minute. The instrument can
measure the glucose content of whole blood, plasma or serum, and requires only
10 μl of sample per analysis.
 The membrane-bound glucose oxidase typically only needs to be replaced every
3 weeks, thereby reducing the cost of analysis. These systems also offer
advanced data-handling and data-storage facilities.

A laboratory-scale glucose analyser. Photograph supplied
courtesy of YSI (UK) Limited

 In addition, these instruments can be modified to analyse a wide variety of other
substances of biological interest, simply by incorporating other oxidase enzymes
into the membrane
 To enable diabetic patients to take their own blood glucose measurements, small
hand-held biosensors have also been developed, which are in fact
technologically more advanced because the enzyme and transducer are more
intimately linked on the sensor surface.
 The first device of this type was launched in 1986 by Medisense, and was based
on technology developed in the U.K. at Cranfield and Oxford Universities. The
ExacTech blood glucose meter was the size and shape of a pen, and used
disposable electrode strips.
 This device was followed by a credit card-style meter in 1989. Such devices
again rely on glucose oxidase as the biological component, but do not measure
the reaction rate via the production (and detection) of H2O2.
 Instead they rely on direct measurement of the rate of electron flow from glucose
to the electrode surface. The reactions that occur within this device may be
summarized as follows:

 The reactions that occur within this device may be summarized as follows:
• Glucose+GOX−FAD→Gluconicacid+GOX−FADH2
• GOX−FADH2+Ferrocenemediator(oxidized)→GOX−FAD
Ferrocene mediator(reduced)
and at the electrode surface:
 Ferrocene mediatorreduced → Ferrocene mediatoroxidized + e−
where GOx-
 FAD represents the FAD redox centre of glucose oxidase in its oxidized form,
and GOx-FADH2 represents the reduced form.
 Basically electrons are removed from the glucose molecules and passed via the
enzyme to the ferrocene mediator, which then donates them to the working
electrode surface, resulting in the generation of an electrical current that is
directly proportional to the rate of oxidation of glucose, and thus proportional to
the glucose concentration in the sample.
 Medisense, whose only product was its blood glucose meter, was bought by
Abbott Diagnostics in 1996, and Abbott-branded devices continued to use and
develop this technology for some time.

 In 1999, Therasense marketed a glucose meter that represented the next
generation of sensing technology, and integrated the enzyme even more
closely with the electrode.
 Originally developed by Adam Heller at the University of Texas in the
1990s, wired-enzyme electrodes do not rely on a soluble mediator such
as the ferrocene used in the Medisense devices.
 Instead the enzyme is immobilized in an osmium-based polyvinyl
imidazole hydrogel in which the electrons are passed from enzyme to
electrode by a series of fixed electroactive osmium centres that shuttle
the electrons onward in a process called ‘electron hopping.’
 In 2004, Abbott Diagnostics purchased Therasense, and instruments such
as the FreeStyle Freedom Lite meter range produced by Abbott Diabetes
Care now incorporate this wired-enzyme technology. Devices of this
type are highly amenable to miniaturization.

A hand-held glucose biosensor suitable for personal use.
Photograph supplied courtesy of Abbott Diabetes Care.

 Continuous measuring devices are becoming increasingly available, and may
well revolutionize the control of certain disease conditions. For example, with
regard to diabetes, devices such as the FreeStyle Navigator range from Abbott
Diabetes Care use the same wired-enzyme technology as that described earlier,
but now incorporate this into a tiny filament about the diameter of a thin
hypodermic needle.
 This is inserted approximately 5 mm under the skin to measure the glucose level
in the interstitial fluid that flows between the cells.
 The unit is designed to remain in situ for up to 5 days, during which time it can
measure the glucose concentration every minute. A wireless transmitter sends
the glucose readings to a separate receiver anywhere within a 30-metre range,
and this can then issue an early warning alarm to alert the user to a falling or
rising glucose level in time for them to take appropriate action and avoid a
hypoglycaemic or hyperglycaemic episode.
 In addition, experimental units have already been developed that link continuous
glucose biosensor measurement systems with pumps capable of gradually
dispensing insulin such that the diabetic condition is automatically and reliably
controlled, thereby avoiding the traditional peaks and troughs in glucose levels
that occur with conventional glucose measurement and the intermittent
administration of insulin.

• Therefore, looking to the future, we may confidently expect to see the
development of biosensor systems that can continuously monitor a range of
physiologically important analytes and automatically dispense the required
medication to alleviate the symptoms of a number of long-term chronic human
illnesses.

CONCLUSION
 This presentation has been limited to some of the basic principles of
enzymology, together with an overview of the biological applications of
enzymes.
 Enzymes have both biological and chemical attributes. Their sequences and
structures delineate their role in the genome and proteome of all living
organisms and their ability to catalyze chemical reactions extends their
biological function to metabolic pathways and networks.
 Many enzymes are promiscuous and perform multiple reactions and as protein
sequence evolves, enzymes can change their reaction profile. Combining bond
changes and reaction centers with structural information about the substrates,
products, and mechanisms is needed to capture the essence of enzyme chemistry
in a functional classification.
 The development of tools to navigate through reaction space (e.g., EC-BLAST)
paves a new way for an improved description of enzyme reactions, providing a
deeper perspective of biological function.

REFERENCE
 Changeux J.-P. 50 years of allosteric interactions: the twists and turns of the
models. Nat. Rev. Mol. Cell Biol. 2013;14:819–829.
 Kamata K., Mitsuya M., Nishimura T., Eiki J., Nagata Y. Structural basis for
allosteric regulation of the monomeric allosteric enzyme human
glucokinase. Structure. 2004;12:429–438.
 Palmer Enzymology

Enzymes principles and applications Likhith K

More Related Content

What's hot

Similar to Enzymes principles and applications Likhith K

More from LIKHITHK1

Recently uploaded

Enzymes principles and applications Likhith K