BIOCHEMICAL ENGINEERING

BIOCHEMICAL ENGINEERING
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’).
Enzymes are potent catalysts
 As catalysts, enzymes are only required in very low concentrations, and they speed up reactions without
themselves being consumed during the reaction.

 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 catalyze
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 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 monosaccharides.
Enzyme Names and classification

 Enzymes typically have common names (often called ‘trivial names’) which refer to the reaction that they catalyze,
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.
 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 catalyzes.

 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.
• 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).
 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 If it is the active site alone that binds to the
chains that are folded and bent to form a specific three- substrate, it is logical to ask what is the role
dimensional structure, incorporating a small area known as the of the rest of the protein molecule?
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.
 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.
 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.
Enzymes and Reaction Equilibria
 Enzymes Enhance Reaction Rates.
 How do they work?

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.
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
  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.

Enzymes form complexes with the substrate

We often describe an enzyme-catalysed reaction as proceeding through three stages as follows:

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.’

• 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.
Properties and mechanism of enzyme action
 Enzyme kinetics is the study of factors that determine the speed of enzyme-catalysed reactions.
 Assays (measurements) 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 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.
 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.
 A common reason for 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.
 • When we perform a series of enzyme assays using the same
 The relationship between enzyme concentration
enzyme concentration, but with a range of different
and the rate of the reaction is usually a simple
substrate concentrations, a slightly more complex
one.
relationship emerges, as shown in Figure.
 If we repeat the experiment just described, but • Initially, when the substrate concentration is increased, the
add 10% more enzyme, the reaction will be 10% rate of reaction increases considerably.
faster, and if we double the enzyme concentration • However, as the substrate concentration is increased further
the reaction will proceed twice as fast. the effects on the reaction rate start to decline, until a stage
is reached where increasing the substrate concentration has
 This relationship applies both to enzymes in vivo
little further effect on the reaction rate.
and to those used in biotechnological
• At this point the enzyme is considered to be coming close
applications, where regulation of the amount of
to saturation with substrate, and demonstrating its maximal
enzyme present may control reaction rates.
velocity (Vmax).
 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).
 Infact 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.
 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:
 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:
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 = Rate of ES complex breakdown

 Rate of ES complex formation = Rate of ES complex breakdown

Hence, at steady state:

Rearranging

The Michaelis constant Km can be

defined as

The term k2[E]T in fact represents Vmax, the maximal

velocity. Thus Michaelis and Menten were able to
derive their final equation as:
Since the concentration of substrate greatly exceeds the concentration
of enzyme (i.e. [S] ≫ [E]), the concentration of uncombined substrate
[S] is almost equal to the total concentration of substrate. The
concentration of uncombined enzyme [E] is equal to the total enzyme
concentration [E]T minus that combined with substrate [ES].
Introducing these terms to into the equation above and solving for ES
gives us the following:
 Michaelis-Menten approach (Michaelis and
Menten, 1913):
Briggs-Haldane approach (Briggs and Haldane, 1925):
 It is assumed that the product-releasing step, is much ▪ The change of the intermediate concentration with
slower than the reversible reaction, and the slow step respect to time is assumed to be negligible, that is,
determines the rate, while the other is at equilibrium. d(CES)/dt = 0.
 This is an assumption which is often employed in ▪ This is also known as the pseudosteady-state (or quasi-
heterogeneous catalytic reactions in chemical kinetics. steady-state) assumption in chemical kinetics and is often
 Even though the enzyme is soluble in water, the used in developing rate expressions in homogeneous
enzyme molecules have large and complicated three- catalytic reactions
dimensional structures.
 Therefore, enzymes can be analogous to solid
catalysts in chemical reactions.
 Furthermore, the first step for an enzyme reaction
also involves the formation of an enzyme-substrate
complex, which is based on a very weak interaction.
 Therefore, it is reasonable to assume that the
enzyme-substrate complex formation step is much
faster than the product releasing step which involves
chemical changes.
Michaelis-Menten Approach

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

Unless otherwise specified, the concentration is expressed as molar unit, such as kmol/m3 or mol/L. The
concentration of the enzyme-substrate complex CES in, can be related to the substrate concentration CS and
the free-enzyme concentration CE from the assumption that the first reversible reaction is in equilibrium. Then,
the forward reaction is equal to the reverse reaction so that

If we assume that the total enzyme contents are conserved, the free-enzyme concentration CE can be related
to the initial enzyme concentration CE0

So, now we have three equations from which we can eliminate CE and CES to express the rate expression as the
function of substrate concentration and the initial enzyme concentration.
 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.
 Briggs-Haldane Approach

 The rates of product formation and of substrate consumption are

Assume that the change of CES with time, dCES/dt, is negligible compared to that of CP or CS.
 which is the same as the Michaelis-Menten equation, except that the meaning of KM is different. In the
Michaelis-Menten approach, KM is equal to the dissociation constant k2/k1, while in the Briggs-Haldane
approach, it is equal to ( k2 + k3)/k1.
 The product-releasing step is much slower than the enzyme-substrate complex dissociation step. This
is true with many enzyme reactions. Since the formation of the complex involves only weak
interactions, it is likely that the rate of dissociation of the complex will be rapid.
 The breakdown of the complex to yield products will involve the making and breaking of chemical
bonds, which is much slower than the enzyme-substrate complex dissociation step
 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.
 Example problem 1

 When glucose is converted to fructose by glucose isomerase, the slow product formation step is also reversible as:

 Derive the rate equation by employing (a) the Michaelis-Menten and (b) the Briggs-Haldane approach. Explain
when the rate equation derived by the Briggs-Haldane approach can be simplified to that derived by the Michaelis
Menten approach
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,
we would have difficulty estimating Vmax from a direct plot as
shown in Figure 1.
If these seven points are plotted on a graph of 1/velocity against
1/substrate concentration (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.
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 catalyzed reaction is maximal, and above and
below which the velocity declines
• 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
Enzymes are sensitive to inhibitions
 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).
Competitive Inhibition

 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.
 Competitive Inhibition:
In the case of competitive inhibition, the inhibitor binds to the active site and prevents the substrate from
binding. Reaction equation are as follows

Note that we are assuming that formation of both enzyme complexes is in equilibrium with the respective
substrate/inhibitor, and that KS and KI are dissociation constants. The final form of the equation will be the same if we
don’t assume equilibrium and instead use the quasi-steady state assumption, except that KM=KS in this case. The rate
equation is then obtained from a mass balance on product formation and the enzyme species:
 The impact of a competitive inhibitor is to alter the
Michaelis constant KM such that the enzyme would
appear to have a lower affinity for the substrate
(higher KM = lower affinity).

 This makes sense, since the inhibitor is binding to

the same site as the substrate.

 So, as is the case with high KM, it is necessary to have

Competitive inhibition – at low S, the reaction rate is reduced
more substrate to achieve a higher reaction rate,
since the substrate can outcompete for the binding by the (1+[I]/KI) term, but at high S, the intrinsic maximum

sites. velocity can be reached. Therefore, competitive inhibition can

be overcome by assuring that S >> I, which makes sense.
Non-Competitive Inhibition
 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 noncompetitive 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.
Non-competitive Inhibition:
In this case, the inhibitor can bind to either free enzyme or enzyme-substrate complex, and likewise, the substrate
can bind to free enzyme or the enzyme-inhibitor complex.
Binding of one does not prohibit binding of the other; however, the E-I and E-S-I complexes are both dead-end
(meaning product cannot be formed if I is bound). The reaction equations are then as follows:

The assumption is that binding of substrate doesn’t affect the equilibrium of

binding to inhibitor and vice versa.

Let’s now get everything in terms of [E], [I], and [S] –

• So, in this case, we again get KS=KM, and see that the Vmax term is
altered by a term that includes the inhibitor concentration.
• So the affinity of the substrate appears unaltered, while the
maximum reaction rate is changed.
• Note that as in the case of uncompetitive inhibition, you have E-
S-I being formed from E-S, but you also have E-I being formed
from free E.
• So the effect that was apparent in the case of uncompetitive
inhibition is not present here because of the balanced drain on
free enzyme.
• Instead, consider that the circular nature of the binding of
enzyme, substrate and inhibitor has the net effect of reducing the
amount of free enzyme available. So, you can think of this as
reducing Vmax by reducing E0.
• Note that the assumption of binding of substrate not affecting
the dissociation for binding of inhibitor and vice versa is usually
not true.
• In this case, the rate expression becomes more complicated
Noncompetitive inhibition – at low S or high S, the
since there are now four different dissociation constants. The
effect is on Vmax, so the net effect will always be a
inhibition is then considered mixed.
reduction in the reaction rate.
Un-Competitive Inhibition
• 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 favoring 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.
Uncompetitive Inhibition:
In the case of uncompetitive inhibition, the inhibitor binds to the E-S complex and prevents conversion to product.

Assuming that this binding is in equilibrium and can be represented with a dissociation constant. So, the rate
equation can be derived as follows:
 From this expression, we can see that both Vmax and KM are altered
by a term that includes the inhibitor concentration and dissociation
constant. For both parameters, the values decrease as the inhibitor
concentration increases.
 This means the maximum velocity decreases, but the affinity for
substrate appears to increase (KM is decreasing). This is NOT
intuitive. It doesn’t make much sense for the presence of an
inhibitor to increase the affinity for the substrate, and in fact, it is
important that you remember that the inhibitor does not change
the intrinsic properties of the enzyme with respect to a particular
substrate.
 However, this makes a little more sense if we remember what a
rapid equilibrium assumption implies and how one would drive an
equilibrium reaction towards product.

Uncompetitive inhibition – at low S, the effect of the inhibitor

cancels out. The way to think about this is that if S is present in
very small amounts, there’s not enough of E-S around to form
the E-S-I complex, so the effect of the inhibitor is not seen. At
high S, then the effect is seen with the maximum velocity,Vmax.
Irreversible Inhibitor
 If an inhibitor binds permanently to an enzyme it is known as an irreversible inhibitor.
 Many irreversible inhibitors covalently attach to the enzymes that they inhibit causing their structure to be
permanently altered.
 Thus, many irreversible inhibitors are therefore potent toxins.

Organophosphorus compounds such as diisopropyl 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 cause muscle convulsions and lead to death. DFP was originally evaluated by the
British as a chemical warfare agent during World War II, and modified versions of this compound are now widely used as
organophosphate pesticides, including parathione and malathione.
Allosteric Regulators and the Control of Enzyme Activity

 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, 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).
 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 hemoglobin (Hb) 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
Comparison of Myoglobin (A) and
inhibitors, whereas molecules that move the protein to its R-state will act as Hemoglobin (B) structures and
allosteric activators or promoters. binding kinetics with oxygen (C)
 The Concerted Model, also known as the symmetry model, of
hemoglobin is used to explain the cooperativity in oxygen binding as
well as the transitions of proteins which are made up of identical
subunits.
 It focuses on the two states of the Hemoglobin; the T and R states.
 The T state of the hemoglobin is more tense as it is in the
deoxyhemoglobin form while the R state of the hemoglobin is more
relaxed as it is in the oxyhemglobin form.
 T state is constrained due to the subunit-subunit interactions while
the R state is more flexible due to the ability of oxygen binding.
 The difference in deoxyhemoglobin and oxyhemoglobin is that the
“deoxy” form doesn’t have oxygen and the “oxy” form is highly
oxygen bound.
 The binding of oxygen at one site increases or facilitates the binding
affinity in other active sites. Thus, a sigmoidal curve is observed for the
oxygen binding kinetics of hemoglobin.
 In the concerted model of the hemoglobin, it shows that the one As shown in (A) above, the tense-state (T-state) of hemoglobin is
oxygen binding to an active site will increase the probability of other favored when oxygen binding is low. The tetramer shifts from the T-
oxygen binding to the other active sites in the hemoglobin. This ability state to the relaxed-state (R-state) with oxygen binding. In fact, the
is known as cooperativity or cooperative binding. binding of one oxygen with one subunit increases the liklihood of
oxygen binding with other subunits, known as cooperativity. (B)
shows the graphic representation of Hemoglobin (Hb) binding to
oxygen which transitions between the T-state and R-state.
Origin, Purification, and Uses of Enzymes

 Enzymes are essential components of animals, plants and microorganisms, due to the fact that they catalyze
and co-ordinate the complex reactions of cellular metabolism.
 Up until the 1970s, most of the commercial application of enzymes involved animal and plant sources.
 At that time, bulk enzymes were generally only used within the food-processing industry, and enzymes
from animals and plants were preferred, as they were considered to be free from the problems of toxicity
and contamination that were associated with enzymes of microbial origin.
 However, as demand grew and as fermentation technology developed, the competitive cost of microbial
enzymes was recognized and they became more widely used.
 Compared with enzymes from plant and animal sources, microbial enzymes have economic, technical and
ethical advantages.
Economic Advantages
 The sheer quantity of enzyme that can be produced within a short time, and in a small production facility, greatly favors
the use of microorganisms. For example, during the production of rennin (a milk-coagulating enzyme used in cheese
manufacture) the traditional approach is to use the enzyme extracted from the stomach of a calf (a young cow still
feeding on its mother’s milk).
 The average quantity of rennet extracted from a calf ’s stomach is 10 kg, and it takes several months of intensive
farming to produce a calf.
 In comparison, a 1000-litre fermentor of recombinant Bacillus subtilis can produce 20 kg of enzyme within 12 h.
 Thus the microbial product is clearly preferable economically, and is free from the ethical issues that surround the use
of animals.
 Indeed, most of the cheese now sold in supermarkets is made from milk coagulated with microbial enzymes (so is
suitable for vegetarians).
 A further advantage of using microbial enzymes is their ease of extraction.
 Many of the microbial enzymes used in biotechnological processes are secreted extracellularly, which greatly simplifies
their extraction and purification.
 Microbial intracellular enzymes are also often easier to obtain than the equivalent animal or plant enzymes, as they
generally require fewer extraction and purification steps.
 Animal and plant sources usually need to be transported to the extraction facility, whereas when microorganisms are
used the same facility can generally be employed for production and extraction.
 In addition, commercially important animal and plant enzymes are often located within only one organ or tissue, so the
remaining material is essentially a waste product, disposal of which is required.
 Finally, enzymes from plant and animal sources show wide variation in yield, and may only be available at certain times
of year, whereas none of these problems are associated with microbial enzymes.
Technical Advantages

 Microbial enzymes often have properties that make them more suitable for commercial exploitation.
 In comparison with enzymes from animal and plant sources, the stability of microbial enzymes is usually high.
 For example, the high temperature stability of enzymes from thermophilic microorganisms is often useful when
the process must operate at high temperatures (e.g. during starch processing).
 Microorganisms are also very amenable to genetic modification to produce novel or altered enzymes, using
relatively simple methods such as plasmid insertion.
 The genetic manipulation of animals and plants is technically much more difficult, is more expensive and is still
the subject of significant ethical concern.
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
 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.
 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 catalyzed.
 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.
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.
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
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).
PROTEIN PURIFICATION AND CHARACTERIZATION
EXTRACTING PURE PROTEINS FROM CELLS

 Purification techniques focus mainly on size & charge

 The first step is homogenization (grinding, Potter–Elvejhem homogenizer, sonication, freezing and thawing,
detergents)
 Differential centrifugation (600 g: unbroken cells & nuclei; 15,000 g: mitochondria; 100,000 g: ribosomes
and membrane fragments)

1) Cell disruption (which can done via a number of number of different processes of choice e.g Detergents lysis,
Osmolysis, freezethaw cycles, enzymatic lysis, ultrasonication, Homogenisation)
2) Centrifugation (at a specific speed depending on the organ, tissue, organelle or fluid).
3) Removal of supernatant (Decantation to obtain supernatant)
Protein Purification Methods
 It is through protein purification methods that we have been able to study and understand proteins in detail. These methods, or
derivatives of the methods, are used in the clinical labs to identify abnormal samples. Protein purification methods use fraction
techniques which are in a large part based on:
 solubility;
 size;
 charge;
 binding specificity.
 These properties of a protein are derived from the AA properties composing the protein. For example the molecular weight (MW) of a
protein is just the summation of the masses of the individual AAs composing the protein. MW is usually expressed in daltons (Da) or
kilodaltons (kDa). A Da is the same as an atomic mass unit which is approximately the mass of a nucleon and is equivalent to 1 g/mol.
 To begin any sort of purification it is important that an assay be available to identify where the protein of interest is after the
fractionation. Assays come in many different forms and depends in a large part on the type of protein you are trying to purify (i.e. is it an
enzyme?). Commonly used assay technologies are:
 spectroscopic (using Bradford reagent or a chromagenic substrate)
 immunological (using a antibody that can recognize the protein of interest)
 Crude Extracts
 To being any sort of purification procedure you need to obtain the material from which you
plan the isolate the material. Historically the abundance and ease of isolation dictated which
proteins were first studied (e.g. hemoglobin). Also many proteins are common to a large
number of species (e.g. metabolic enzymes) so they could be isolated in large abundance
from other sources, such as yeast or bovine.
 Once you have gathered the material containing the protein you want to study it is
necessary to generate a crude extract -- for proteins from muscle that would mean grinding
it up, for an intercellular protein that would mean breaking the cells open, etc. This is always
done in the presence of a buffer and inhibitors.
 Why? As a scientist you want to control the environment -- keeping the protein you are
interested in at a non-denaturing pH, you want to keep it from being cleaved by enzymes
that will be released in this process so general inhibitors will be important, etc.
Centrifugation
 Generally the first step after forming a crude extract is a simple
filtration or centrifugation to remove the large material.
Centrifugation is a process that involves the use of the
centrifugal force for the sedimentation of mixtures with a
centrifuge. This process is used to separate two immiscible
liquids with more-dense components of the mixture migrate
away from the axis of the centrifuge, while less-dense
components of the mixture migrate towards the axis.
 Centrifugation alters the effective gravitational force on to
tube/bottle so as to more rapidly and completely cause the
precipitate ("pellet") to gather on the bottom of the tube. The
remaining solution is properly called the "supernatant". The
supernatant liquid is quickly decanted from the tube/bottle
without disturbing the precipitate.
 Differential centrifugation, as shown in the figure, is multiple
rounds of centrifugation at increased speeds and time allows for
different cellular fractions to be separated.
Dialysis
 Dialysis is a procedure for exchanging the solvent around a protein. In general the protein
solution is placed inside a semi-permeable membrane (dialysis bag) which is suspended in a larger
volume of buffered solution. The key to this procedure working is that the membrane has to be
permeable to water and ions, but not to your protein of interest. Thus buffers & salts exchange
until an equilibrium is established between the inside & outside of the membrane.
 Naturally in medicine the types of dialysis you are likely to see are hemodialysis and peritoneal
dialysis which remove wastes and excess water from the blood in different ways. Hemodialysis
removes wastes and water by circulating blood outside the body through an external filter
containing a semipermeable membrane.
 The blood flows in one direction and the dialysate flows in the opposite. The counter-current
flow of the blood and dialysate maximizes the concentration gradient of solutes between the
blood and dialysate, which helps to remove more urea and creatinine from the blood. The
concentrations of solutes (for example potassium, phosphorus, and urea) are undesirably high in
the blood, but low or absent in the dialysis solution, and constant replacement of the dialysate
ensures that the concentration of undesired solutes is kept low on this side of the membrane.
The dialysis solution has levels of minerals like potassium and calcium that are similar to their
natural concentration in healthy blood.
 Steps involved in purification of a protein 3) size exclusion chromatography (also
or enzyme: called gel filtration or molecular sieving)
1) salt precipitation using (NH4)2SO4 4) ion exchange chromatography
2) dialysis 5) affinity chromatography

Salting out:
 Are proteins soluble? If yes, to which limit?
 Salt stabilizes the various charged groups on a protein molecule and enhance the polarity of water,
thus attracting protein into the solution and enhancing the solubility of protein
 Ammonium sulfate is the most common reagent to use at this step
 This technique is important but results are crude
Column Chromatography

Column chromatography is one of the most powerful fractionation methods. It can separate components of mixtures based upon:

 size (gel filtration/size exclusion)

 charge (ion exchange)

 affinity (a specific binding affinity)

 Commonalities between all three types of chromatography methods is that they all use a resin (solid phase) with special
chemical properties held in a glass cylinder (called a "column"). A buffered solution (mobile phase) percolates through the
column and is collected in tubes ("fractions") upon exiting the column. A protein mixture is applied in the mobile phase &
percolates through through the column as an expanding band. Different proteins migrate differently depending on their
properties and those of the resin.
COLUMN CHROMATOGRAPHY
Gel Filtration/Size Exclusion
 Gel filtration, or as it is sometimes referred to as size
exclusion, chromatography the resin are porous (see
figure to the left).
 Some molecules (blue here) can enter the resin and
as the lines try to indicate it is not a straight path
through; thus it takes longer for small molecules to
traverse the column than large molecules which
travel around the outside of the resin.
 This is highlighted in the figure to the right where big
molecules (blue) come off first and smaller molecules
(red) later.
 SIZE-EXCLUSION CHROMATOGRAPHY
GEL-FILTRATION CHROMATOGRAPHY

 Separation on the basis of size (MW)

 Stationary (cross-linked gel particles): consist of one of two kinds of
polymers; the 1st is a carb. polymer (ex. dextran or agarose); often
referred to by Sephadex and Sepharose. The 2nd is based on
polyacrylamide (Bio-Gel)
 Extent of crosslinking & pore size (exclusion limit)
 Convenient & MW estimate
 Each gel has range of sizes that separate linearly with the log of the
molecular weight
MOLECULAR-SIEVE
CHROMATOGRAPHY
Ion Exchange
 Ion exchange chromatography is broken in to two types - anion & cation exchangers. There are many different types of
moieties that are used from weakly to very strongly charged thus allowing a huge range of molecules the ability to
interact.
 Unlike gel filtration chromatography, here proteins directly interact with the resin. So generally the column is
equilibrated in a buffer solution to establish a constant pH in the column, then the protein mixture is loaded where all
or some of the proteins interact with the resin depending upon their own charge. Buffer is continued to be applied until
all proteins not interacting with the resin have been washed off. At that point usually a gradient of increasing salt
concentration (disrupts ionic and hydrogen binding) in the buffer is applied to column allowing the most weakly
interacting proteins to release first followed by the more strongly and finally the most strongly interacting. This can also
be accomplished by changing the pH of the buffer being applied to the column.
Anion Exchanger
 Anion exchanger means that it removes anions from protein mixture so that means the resin must be decorated with
positively charged moieties. Before elution begins all positively and uncharged proteins will fall through the column.
When you start eluting, first you will knock off the weakly negative proteins (e.g. -1 charge), followed by those with a
stronger negative charge (-2), and finally the most negatively charged proteins (-3).
Cation Exchange
 It is exactly oppose with a cation exchanger -- here cations are removed from the protein solution so the resin must be
negatively charged. Again before elution begins all negatively and uncharged proteins will fall through the column. When
you start eluting, first you will knock off the weakly positive proteins (e.g. +1 charge), followed by those with a stronger
positive charge (+2), and finally the most positively charged proteins (+3).
ION-EXCHANGE CHROMATOGRAPHY

 Interaction based on net charge & is less specific

 Resin is either negatively charged (cation exchanger) or positively charged (anion
exchanger)
 Buffer equilibration, exchange resin is bound to counter-ions. A cation-exchange resin is usually
bound to Na+ or K+ ions, and an anion exchanger is usually bound to Cl– ions
 Proteins mixture loading
 Elution (higher salt concentration)
 AFFINITY CHROMATOGRAPHY

 It has specific binding properties

 The polymer (stationary) is covalently linked to a ligand
that binds specifically to the desired protein
 The bound protein can be eluted by adding high conc.
of the soluble ligand
 Protein–ligand interaction can also be disrupted with a
change in pH or ionic strength
 Convenient & products are very pure (Antigen-
antibody, His-tag, GST-Tag)
Electrophoresis
 Electrophoresis is the motion of dispersed particles relative
to a fluid under the influence of a uniform electric field. Thus
it separates components of a mixture based on their size
and/or charge.
 Visualization of proteins on paper or in a gel is an important
step in any electrophoresis. Often with DNA the gels are
soaked in ethidium bromide which intercalates into DNA
and fluoresces under UV light (left image). Proteins may be
visualized using silver stain or Coomassie Brilliant Blue dye
(right image). In some cases the gels are transferred to a
solid support (nitrocellulose) and then probed with specific
antibodies (Western Blot).
PAGE (PolyAcrylamide Gel Electrophoresis) -- Native Gel

 It is used in clinical chemistry to separate proteins by charge and/or size. Using an electric
field, molecules (such as DNA) can be made to move through a gel made of agar or
polyacrylamide.
 The electric field consists of a negative charge at one end which pushes the molecules
through the gel, and a positive charge at the other end that pulls the molecules through the
gel. The molecules being sorted are dispensed into a well in the gel material. The gel is placed
in an electrophoresis chamber, which is then connected to a power source. When the
electric current is applied, the larger molecules move more slowly through the gel while the
smaller molecules move faster. The different sized molecules form distinct bands on the gel.
 The term "gel" in this instance refers to the matrix used to contain, then separate the target
molecules. In most cases, the gel is a crosslinked polymer whose composition and porosity is
chosen based on the specific weight and composition of the target to be analyzed. When
separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually
composed of different concentrations of acrylamide and a cross-linker, producing different
sized mesh networks of polyacrylamide.
 "Electrophoresis" refers to the electromotive force (EMF) that is used to move the
molecules through the gel matrix. By placing the molecules in wells in the gel and applying an
electric field, the molecules will move through the matrix at different rates, determined
largely by their mass but also their charge and shape which varies widely for proteins.
Electrophoretic mobility of small molecules is greater than the mobility of large molecules
with the same charge density thus allowing separation.
Separate native proteins by size –
proteins stop moving when they
reach a certain gel density
SDS PAGE or Sodium Dodecyl Sulphate-
Polyacrylamide Gel
• Electrophoresis is a technique used for the
separation of proteins based on their molecular
weight. It is a technique widely used in forensics,
genetics, biotechnology and molecular biology to
separate the protein molecules based on their
electrophoretic mobility.
• The principle of SDS-PAGE states that a charged
molecule migrates to the electrode with the
opposite sign when placed in an electric field. The
separation of the charged molecules depends
upon the relative mobility of charged species.
• The smaller molecules migrate faster due to less
resistance during electrophoresis. The structure
and the charge of the proteins also influence the
rate of migration. Sodium dodecyl sulphate and
polyacrylamide eliminate the influence of
structure and charge of the proteins, and the
proteins are separated based on the length of the
polypeptide chain.
 Isoelectric focusing (IEF) is a technique for separating different
molecules by differences in their isoelectric point (pI). It is a type of
electrophoresis, usually performed on proteins in a gel, that takes
advantage of the fact that overall charge on the molecule of
interest is a function of the pH of its surroundings. When an IEF gel
is poured a pH gradient is established
 A protein that is in a pH region above its isoelectric point (pI) will
be negatively charged and will migrate towards the anode
(positive). As it migrates through a gradient of decreasing pH,
however, the protein's overall charge will increase until the protein
reaches the pH region that corresponds to its pI.
 At this point it has no net charge and so migration ceases (as there
is no electrical attraction towards either electrode). As a result, the
proteins become focused into sharp stationary bands with each
protein positioned at a point in the pH gradient corresponding to
its pI. The technique is capable of extremely high resolution with
proteins differing by a single charge being fractionated into
separate bands.
Western Blotting
 The term "blotting" refers to the transfer of biological samples from a gel to a
membrane and their subsequent detection on the surface of the membrane.
Western blotting (also called immunoblotting because an antibody is used to
specifically detect its antigen) is a routine technique for protein analysis. The
specificity of the antibody-antigen interaction enables a target protein to be
identified in the midst of a complex protein mixture in a semi-quantitative
manner.
 The first step is to separate the macromolecules using gel electrophoresis (native
or SDS-PAGE). After electrophoresis, the separated molecules are transferred to
a membrane (usually nitrocellulose). As the membrane will bind any protein
(including the antibody you will use to detect your protein of interest), after
transferring the sample the membrane must be blocked with a common (cheap!)
protein to prevent any nonspecific binding of antibodies to the membrane.
 Detailed procedures for detection of a protein on a Western blot vary widely.
Most laboratories use a indirect detection method, in which a primary antibody is
added first to bind to the antigen. This is followed by a labeled secondary
antibody which recognizes the primary antibody. Labels include biotin, fluorescent
probes, and enzyme conjugates that convert a substrate to a colored product
thus staining the membrane.
ELISA (Enzyme Linked ImmunoSorbent Assay)
 There are MANY forms of ELISAs! Most frequently used is the "sandwich"
form in which an antibody is bound to a well in a microtiter dish. The sample
is added, incubated, and then protein which were not captured by the
antibody washed away. A labeled secondary antibody which recognizes a
different part of the bound antigen can be used to quantify the amount of
antigen in the sample.
 ENZYME CHARACTERISATION
1. Determination of the effect of changes in temperature on enzyme’s activity and optimum temperature.
2. Determination of enzyme’s thermal stability.
3. Determination of the effect of changes in pH on enzyme’s activity and optimum pH.
4. Determination of pH stability.
5. Determination of the effect of changes in substrate concentration on enzyme’s activity and kinetic constants e.g Vmax,
Km, Kcat, Km/Kcat etc.
6. Determination of substrate specificity
7. Determination of molecular weight (Mw) of enzyme
8. Determination of the effect of metal ions, chelating agent or denaturating agents.
9. Determination of enzyme’s isoelectric point (pI)
10. Determination of the effect of duration of incubation.
11. Determination of active site fractional saturation (V/Vmax) at a particular substrate concentration. Determination of
Enzyme’s turnover number (Vmax/ E T )
12. Determination of activation energy (Ea)
13. Determination of salt tolerance