This course as you know, deals with one of
the largest group of biomolecules what we
know as enzymes.
You are familiar with number of biomolecules
like carbohydrates, lipids, proteins.
This enzyme falls into the category of proteins
and the course will cover various aspects
of enzymes, particularly in relation to their
application as a biocatalyst in the process
industries.
I think one of the very basic issues which
come to mind is that why do we study enzymes
as a separate class?
In addition to your study of various biomolecules
in other courses like Biochemistry, we have
separate course on enzymes and some of these
aspects I will be dealing in today’s lecture,
which will basically be of introductive nature
and also give you the scope of this course.
The first issue as to why we study enzymes
opens up many features of enzyme molecules
and the first and probably the most important
is their role in a living cell or their role
in the realm of biotechnology as a whole.
If you look at the basic definition or a very
broad operational definition of biotechnology
it deals with the application of scientific
and engineering principles to the processing
of materials by biological agents to provide
goods and services.
Here I like you to mark the term biological
agents and these biological agents cover microbial
cells, cellular organelles, enzymes, may be
other parts of the cells like membranes in
a variety of biological material that can
be used as a catalyst in process industries.
That is the upcoming feature of biotechnology
and among these groups of biocatalyst enzymes
play a very significant role.
If you go a little deeper into the various
activities of a living cell besides looking
at the industrial application, a living cell
is a very complex entity.
It carries out all functions of any chemical
process plant that you have known or you can
conceive.
Most complex chemical processing plant say
for example petrochemical complex where the
petroleum is fractionated into various products
and I like to give you a very simplified analogy
of a living cell to that of a chemical process
plant.
The cell does a variety of functions that
is crushing and grinding, transport of materials,
power generation, control of information,
transmission of information and then release
of finish products to the user client.
All these functions are also basically carried
out by a living cell.
Now the key issue is that all these functions
in the living cell, may it be a microbial
cell, may it be a plant cell or it might be
an animal cell, they are mediated by the catalyst
known as enzymes.
Therefore the enzymes play a very central
role in the function of living cells or in
other words you can say that they are one
of the most important executives in a living
cell.
Even disturbance in one of the enzyme catalysed
reactions can lead to some abnormality in
the cell function.
We are familiar with many of the diseases
which are seen when one of the enzymes stops
functioning.
Similarly in case of a living cell if the
enzyme system fails many of the processes
can be disturbed ultimately leading to the
life cycle of the cell itself.
It is such an important component it becomes
important to understand how they perform those
reactions and how they manage the integrity
of whole cell.
The most significant part is that they regulate
the various functions of the cell in such
an accurate manner probably no other control
system in any chemical process plant can think
of.
There is a perfect control, fine tuning of
the enzyme activities as per the desired requirements
and we also know that the cells are energetical,
in terms of material consumption are very
efficient systems and wastage is minimum.
The energy is generated to the required level;
materials are stored in case they are not
needed.
All those functions are a key part of the
living cell and all these functions are attributed
to the function of enzymes and that is where
we get to understand enzymes in a more detail
fashion.
Also we must appreciate that 
the cells, making these functions possible,
carry out a large variety of reactions.
They can range from hydrolysis to polymerization.
We are all familiar of how the different components
or molecules or intermediates in a living
cell are synthesized.
They all take place through a series of chemical
reactions.
These reactions could be hydrolysis could
be polymerization, could be epoxidation, could
be reduction and so on.
A whole lot of chemical reactions do take
place in a living cell.
The same scenario is that even a chemical
process industry depends on a whole lot of
chemical reactions which are catalyzed either
spontaneously or with the use of catalyst.
The analogy lies that the reactions that are
catalyzed by the enzymes in a living cell
if they are carried out in an industrial atmosphere
with perfect energetically economical situation,
environmentally friendly conditions we have
a good friend in enzymes and that is again
another basis of use of enzymes 
in the process industries and also the possibility
of studying them in such a great detail.
If you look at some of the aspect of enzymes
and the process via catalyst, a very broad
picture which refers to the role of enzymes
as the bio catalyst, we must be aware that
all these enzymes functions at the ambient
conditions at the conditions at which living
cells are known to live.
There might be some exceptions.
The organisms that are found under very extreme
conditions are exceptions but most of the
organisms that live under ambient conditions
produce or they provide us enzymes which can
function under mild reaction conditions.
That is the basis of their energetic efficiency.
That means the energy requirement of those
reactions they catalyze could be very meager.
They have high turn over number.
When we discuss the reaction mechanism we
will see that they are very efficient catalyst
and when I say very efficient catalyst our
counter part to compare is the chemical catalyst.
There are a large number of reactions which
can be catalyzed both by enzymes as well as
by chemical catalyst and the subtle mechanisms
that are involved in the enzymatic catalysis
make them very efficient catalyst meaning
a high turn over number or a significant reduction
in the energy of activation of reaction.
The very characteristic feature of enzyme
is their specificity.
That 
means for any given reaction you need a specific
enzyme and such specificity has both advantages
as well as disadvantages.
It is advantageous in the sense that specificity
will bring in lack of any byproducts because
it will not act on any of the other substrate
molecule which are present as additions or
contaminant in the …… stream and you will
only be reacting the desired substrate and
converting it to product.
On the other hand the disadvantage is from
industrial point of view.
You cannot produce them in very large quantity
because you require small quantities of catalyst
for specific reactions.
If one catalyst works on a variety of reactions
the life is simple as happens in the case
of chemical catalyst.
For example vanadium pentoxide or colloidal
platinum can act as oxidation catalyst for
n number of reactions or Raney nickel for
hydrogenation.
Whatever has to be hydrogenated it can be
used.
But the same scenario doesn’t happen.
You need a very specific enzyme and that brings
in some sort of disadvantage as far as their
role as industrial catalyst is concerned.
Two of the definitely undesirable features
of an enzyme as catalyst is they are relatively
fragile in nature and with very mild deviations
from their normal operating conditions they
can be deactivated.
The whole function is dependent upon a very
subtle conformation.
This conformation is result of not only covalent
interactions but of a large number of non
covalent interactions which can be disturbed
very easily and that is why the deactivation
of enzymes can occur and deactivation from
industrial point of view is some kind of a
loss.
The other factor is cost which is again an
undesirable feature which probably applies
to any commodity we want to use for industrial
purposes.
We will always like to choose a commodity
which is less expensive and does the job and
because the enzymes are produced in small
amounts by the living cell and the only source
of enzyme to us are living cells and they
are synthesized in very small quantities.
So obviously their cost per unit quantity
is comparably very high and coupled with their
stability it becomes an expensive proposition.
But we are also aware of the some of recent
developments in biotechnology, molecular biology
and other sciences which involve isolation,
purification, as well as stabilization.
The costs have been brought down, the concentration
of the enzyme protein which can be synthesized
has gone up and this situation is changing.
Another very significant equation as far as
enzymes as processed via catalysts are concerned
is between availability and applications.
We saw that they carry out lot a large number
of reactions.
But although in a living cell various enzymes,
which carry out these reactions are available
their availability in large quantities for
commercial applications are limited.
As matter of fact only those enzymes which
are required in bulk, when I say bulk I am
referring to at least, if not tons, quintals
many of them in tons really, they cannot be
produced on a mass scale and their availability
for commercial applications is limited.
There are certain classical examples of amylases,
glucoisomerase, cellulase, lipase, proteases,
which are the enzymes that are produced in
bulk and are available in large quantity.
The other part is because of their bulk production
the cost has also come down and become reasonable
for commercial applications.
The issue of availability is very intrinsically
linked with the applications of enzymes.
Unless applications emerge one will not like
to produce them in large quantities.
The paradox remains.
The applications do not emerge till they are
available in large quantity.
There are a large number of examples which
will illustrate my point.
Take for example a candidate like lipase - the
enzyme that hydrolyses lipids into the consequent
fatty acids and glycerol.
Obviously the enzyme is not available in the
quantities that many of the other enzymes,
like amylases and proteases, are available.
But there is a potential that it can be produced
provided the applications emerge so that the
scale of operations which is required for
bulk operation can be easily maintained and
so the reversible interaction between availability
and application is a very intrinsic feature
and probably will illustrate the story of
many of the enzymes which have become commercially
viable today.
As far as availability is concerned there
are two major factors.
One is that we have to synthesize them.
Theoretically one can produce enzymes from
any living source microbial, plant or animals
but the trend in the reason past has been
that the microorganism has been the preferred
choice for variety of reasons.
Some of you may be familiar with them and
most important one is diversity.
Secondly you can choose micro organism which
will be able to act on a particular substrate,
synthesize required enzyme.
On the other hand you also have a very clear
understanding of the genetic machinery of
the cell, the microbial cell that controls
the enzyme synthesis, which you will manipulate
leading to hyper production of the enzyme.
Their bulk productions, their bulk growth
is very simple.
The parameters, the equipment are all standardized
thanks to many other products which are produced
microbially that gives an edge for microbial
biosynthesis of enzymes.
The other important part is isolation, purification
and stability.
In isolation and purification one of the very
characteristic features is that most of the
enzymes are purified by a series of steps.
An electrophoretically homogeneous protein
means it is a perfectly pure enzyme.
For most of the industrial applications except
for some of the medical or diagnostic applications
you will not require a highly purified enzyme.
For bulk industrial processing we have to
stop at purity level somewhere in between.
Compared to this crude enzyme where this specific
activity is very low to a level where the
contaminating enzyme that can disturb the
process are revoked.
But note that all the contaminants are revoked
which may not be required because any order
of purification will mean additional cost,
enhancement in the value of the product.
As far as the applications are concerned a
lot of engineering inputs are required particularly
for the extended use of the catalyst and we
don’t want to use it just once.
As a matter of fact I think one can take a
clue again here from a living cell.
The cell maintains a very perfect economy
in terms of its energy as well as materials
to synthesize proteins.
It doesn’t use the proteins only once.
It uses in a cycle and over a period of time.
There is always a maintenance part where it
has to maintain the protein synthesis but
it is being used repeatedly and in many of
the organs of the cell, the enzymes are supported
on certain organelles so that they behave
almost in a fashion of insolublised enzymes.
On the basis of the same concept one can immobilize
the enzymes on a support, insoluble support
what we call as immobilize enzyme and use
them for processes that can be used in continuous
mode like any other chemical process, the
term very commonly used in the chemical industry
is heterogeneous catalysis.
Then a very important aspect is bulk environment
design.
So far we have been limited in our mind regarding
the application of enzymes that they can work
only in aqueous environment.
This today is probably not a very perfect
statement because a number of enzymes have
been shown to function even in a non aqueous
environment or in other words more accurately
micro aqueous environment where you require
a very small concentration of water, rest
of the bulk environment will be replaced by
an organic solvent and that can give us a
whole new range of chemical reactions which
can be carried out using the enzymes and biocatalyst
particularly a transition from hydrolysis
to synthesis and many of these reactions have
become commercially viable today.
Ultimately for any process application of
the enzymes you need to engineer the systems
even after you have done immobilization, even
after you have studied the optimum reaction
parameter, the whole systems has to be engineered.
I mean to say that you have to develop particular
hardware reactor, an enzyme reactor in which
the whole reactions can be carried out with
maximum productivity because ultimately the
whole concept or the whole interest between
will be on the productivity and which can
be tailored by doing the proper process engineering.
Then you may need to monitor certain parameters,
the instrumentation control, down stream processing
of the product, all has to be integrated so
as to have a complete application route.
In summary the broad objective of this course
as I mentioned so far will be to study various
scientific, technological and engineering
aspects associated with the application of
enzymes as a bio catalyst in process industry.
This in brief terms can be the broad definition
of or broad objective of this course.
I like you to be very critical about the scientific,
technological and engineering aspects.
Almost an analogy with the biotechnology in
general even the enzyme technology also has
a very multidisciplinary nature.
It is not that one discipline for example
understanding only biochemistry can lead to
understanding of the applications of enzymes
in the process industries.
A whole range of basic and engineering sciences
are required to be understood.
For example if you start let us say a basic
science like chemistry.
Many aspects of chemistry have to be understood
in relation to enzymes or in other words you
can add the term biochemistry.
The functional aspect of the enzymes like
the kind of chemical reactions they catalyze,
the thermodynamic limits, the equilibrium
convergence, the free energy change, whether
the reaction is feasible, the coupling of
reactions so that they become feasible in
terms of free energy change and all those
aspects of the enzymes protein will come under
realm of bio chemistry.
A very important role is played by physics.
Today a large variety of developments that
have taken place in the area of biotechnology
in general and enzymes in particular can be
attributed to the use of a number of physical
techniques which have made us to understand
a variety of systems and develop and improve
upon them for various applications and physics
becomes a very important component to understand
the enzyme role.
You are familiar that the result of these
applications and developments is the science
of biophysics as you know it today.
After chemistry and physics I think the major
scientific component will be biology.
For any enzyme to be produced in large quantity
you need to identify a biological system which
can produce the enzyme.
It can be microorganism, it can be a plant
or it can be animal.
The bulk of the applications or in the bulk
of the cases of enzymes we will look at microbial
systems.
That is the component which biology will make
us understand.
We will have to understand its morphology,
its physiology so as to be able to design
an appropriate enzyme production system.
The role of protein biosynthesis were their
organism which will regulate the synthesis
of enzymes.
The regulatory mechanisms involved in the
bio synthesis of those enzyme proteins will
also be understood through biology.
In the recent years an added dimension is
molecular biology where we tend to understand
the structure function relationship of various
biological molecules at a molecular level.
How nucleic acids take part in information
transformation from one cell to next generation,
what are the mechanism of cell-cell signaling,
communication between different cells or how
does the protein molecules behave as a defense
molecules?
All variety of those functions at a molecular
level has become an added dimension which
has been at the center of the bio technology
in general.
Similar applications will come here for biology
also.
A very important scientific development has
been in the computational science.
Most of the industrial applications will require
the application of computational science in
those systems.
Today even design of an enzyme protein, modifications
for tailoring it to give some desirable property
is a job which can be taken up only with the
help of computational science.
Experimental sciences will take years together
to come out with those conclusions which can
be brought in by many of the computational
programs.
You will need lot of data available to you
before you can apply them but once the data
are available one can easily compute and predict
many of the behavior which otherwise would
have taken a very long time.
For example site directed mutagenesis is what
happens if an amino acid is removed and another
amino acid is inserted in the polymeric chain
that is in the protein chain.
Its structural behavior can be very easily
understood with the computer graphics which
otherwise doing experimentally probably is
a Herculean task.
These are some of the basic sciences which
are important.
On the other hand we have a whole range of
engineering sciences which are important as
far as enzyme technology is concerned.
You need to design enzyme reactors which can
take care of transfer of materials; transport
of energy, transport of momentum and all transport
processes can be taken care.
You need instrumentation for monitoring and
control of the reactor, you need the concept
of electronics particularly in some applications
like biosensors, enzyme will require transducers
and the ultimate is biochips.
We are far from the reality but still as the
concept it has probably attracted the attention
of many scientists.
That means can be used in the protein molecule
or enzyme molecules as biochips in the computers.
The whole computation becomes based on biological
molecules.
So a large number of engineering operations
including mass transfer, heat transfer, material
balance, energy balance, product separation,
kinetics, thermodynamics is also involved
in understanding of enzyme science and therefore
one of the characteristic feature is that
the whole enzyme science interfaces with a
large number of basic engineering sciences
and it is this interface which makes it so
attractive to study, exciting to study and
important to study.
Today it has become impossible probably for
a bio technologist to leave aside an enzyme
molecule, understanding of enzyme molecule
in detail before he can call himself to be
a professional in the area.
Another important aspect which I think I will
like to touch upon in today’s lecture will
be on enzyme classification.
Now you will be surprised to know that so
far globally about three thousand enzymes
have been isolated, characterized and reported
in literature.
It’s not a very large number compared to
the feasibility of combinations that are feasible.
If we look at enzymes as a polymer consisting
of nineteen different amino acids with an
average length, let us say 150 or 200 amino
acids linked together.
With nineteen different amino acids probably
the possibility of, variety of, diversity
of proteins that are feasible is enormous
almost approaching infinity.
We have been able to isolate and characterize
only about three thousand.
Now this three thousand, just to understand
and bring it into a systematic feature, have
been classified into six different classes.
In fact this job was done way back in early
seventies by international union of pure and
applied chemistry, what you know as IUPAC.
They gave a classification system which is
acceptable globally today and it classifies
all the enzymes into six major classes’:
oxidoreductases, transferases, hydrolases,
lyases, isomerases and ligases.
The whole range of chemical reactions or bio
chemical reactions that take place in a living
cell are mediated by these different classes.
We must know that some of the reactions are
not independent.
They are coupled there by producing effect
which could be a polymerization reaction.
Directly a polymerization doesn’t take place
as it is.
It is mediated by series of transferases and
then ultimately the transferases and ligases.
Ultimately you end up with the polymer of
different monomers.
But basic enzymes can be classified and each
of this class is also characterized in sub
classes.
Sub classes refer to the type of substrate
or the type of the product that is produced
by those enzymes.
So basically the whole range of each of the
class here oxidoreductases a bulk of the oxidases,
reductases and the hydrolases fall under this
category.
Transferases involve group transfer reactions
where one of the functional group with a substrate
molecule is phosphorylated.
Usually they are bisubstrate reactions involving
two substrates.
In hydrolases by and large the most commonly
employed industrial catalyst is used mainly
because of simplicity.
Because the co-substrate is water which is
present in large quantity and one can use
them for hydrolytic reactions.
By changing the bulk environment hydrolysis
can be tailored to be used for synthetic reactions
as well.
In lyases an additive enzyme which catalyses
the elimination reactions that result in the
double bond formation is used.
Isomerases are another important catalyst
of industrial enzymes which isomerises one
molecule into other isomerable molecule.
One of the most significant enzymes under
this class is isomerization of glucose to
fructose.
Ligases join two molecules at the expense
of energy source.
In most biochemical reactions ATP is the energy
currency which is consumed and regenerated
through catabolic processes and such reactions
are classified under ligases.
This is the classification based on the IUPAC
nomenclature more from the scientific point
of view.
But from the industrial point of view or from
the application point of view another important
type of classification which is made is in
terms of three classes what we know as bulk,
diagnostic and therapeutic.
This classification is based on the cost of
the enzymes.
In fact the cost of the enzyme is directly
linked with the concentrations in the starting
material.
It could be a fermentation broth in most cases.
In the fermentation broth if the concentration
of the product or the desired enzymes is very
low the cost is very high and second factor
of cost is the application and the degree
of purity you need for that particular application.
The bulk enzymes as you can see here they
fall in this category.
The concentration in the starting material
is of the order of about one kg per meter
cube which is significant enough to recover
and you don’t require for the most cases
a very high degree of purity and therefore
their cost is also reasonably low say for
example about thousand US dollars per kg.
The next category is diagnostic.
It includes also analytical enzymes.
This is the concentration in grams per meter
cube level which is much lower.
Degree of purification required is also larger
because you will not like any protein contaminant
present in the sample and their cost is therefore
significantly high of the order of about 106
US dollars per kg.
You should not get threatened by the amount
of cost of 106dollars.
The quantity required is also in micrograms.
They are not in kilograms like other molecules.
So they are affordable.
The third and probably most expensive classes
are therapeutic enzymes which are exclusively
required for medical applications almost like
life saving drugs.
Their cost is immaterial and you don’t make
choice on the basis of cost.
In the case of industrial operations, you
always make a choice between two alternatives
on the basis of the cost and effectiveness.
But if effectiveness is the same the cost
is the principle determining factor.
In the case of therapeutic products, the choice
is not on the basis of cost but on the basis
of its need if it is a life saving drug.
Many of the therapeutic enzymes particularly
the recombinant proteins that are available
from the enzymes like streptokinase and urokinase
are blood clot dissolving enzymes.
Within fraction of a minute they simplify
the situation of a cardiac failure problem
due to blood clot.
Similarly a number of other such therapeutic
proteins or enzymes are available like asparaginase,
for patients suffering from leukemia, which
is again life saving.
The cost is also of a very high order but
the quantity required is of less than micro
gram quantities but in a very highly purified
state.
The last phase of today’s lecture will cover
on course outline and references and the course
will broadly cover these aspects.
I have given you some idea about the introductory
remarks on the enzyme as process biocatalyst.
We will go into little more details on the
chemical nature of enzymes that means enzymes
or proteins.
We will go into functional nature of enzymes,
enzymes as the catalyst and what makes them
such an efficient catalyst?
The factors which are responsible for their
deactivation, the approaches that are available
for stabilizing the enzymes, the role of active
sites or ligand binding sites on the enzymes
will be dealt in functional nature.
Then we will go on to the kinetics of enzyme
catalyzed reactions because reaction kinetics
will play a very important role in the choice
of enzyme reactors, to understand reaction
rates and the role of associated physico-chemical
interactions that will take place once you
immobilize the enzyme or use them in bulk
environment other than water.
One of the most significant tools which have
made many of the enzyme reactions commercially
viable is immobilization of enzymes.
A large number of techniques are used and
we will discuss them in detail.
The use of enzyme reactors, the types of reactors
that are possible to be used with the mobilize
enzyme, with soluble enzyme, their characteristic
features, comparison between their performances,
how to choose the enzyme reactors, then mass
transfer and immobilize enzyme reactors.
Immobilization of enzyme will have some additional
physico chemical features like diffusion and
partitioning.
When they are placed in a reactor how do they
influence the performance of the system? Immobilize
cells very often in the enzymes are intercellular.
It has been felt that for many applications
their isolation and purification is not required.
You can immobilize the whole cell as long
as you do not inactivate other contaminating
enzymes in the cell which will interfere in
the process.
In the bio process design, some of the basic
design principles for the enzyme catalyzed
processes.
A few case studies I like to illustrate some
of the earlier issues with respective to specific
cases and ultimately I think I like to take
up enzyme based sensors.
The enzyme sensors have become an important
activity where immobilization enzymes are
used.
Both immobilized enzymes and immobilized cells
are used.
They have become very important tool both
in diagnostics as well as monitoring of information
from reactions.
This will be our course outline.
