Enzyme Technology Challenge - Biotechnology Engineering Video Lecture - Biotechnology Engineering (BT)

Today we shall discuss about some of the issues
and the future directions of enzyme technology
that we are likely to face in near future.
So far we have been talking about the status
of enzyme technology as is being practiced
in the industries as for the application of
enzymes are concerned in different sectors
of their application.
The issues that I will be talking today are
ones which are still posing challenge and
are restricted to laboratory investigations
so far and it is likely that some of them
in a very short time will come out as industrial
processing concepts.
First issue that I want to draw your attention
is the bio processing with co-factor linked
enzymes.
As I mentioned earlier that a large number
of enzymes that are known are mediated through
co-enzymes or co-factors as you know and most
of the applications that we talked earlier,
they related to only hydrolytic enzymes which
do not require co-factors with the sole exception
of glucose isomerase which is an isomerase
and it also does not require co-factor.
The large number of cofactor linked enzymes
therefore opens a big challenge to the industry
or to the scientist who are concerned with
the diversification of application of enzymes.
The basic motivation or rational for the use
of co-factor linked enzymes comes from the
function of living cell in which a number
of co-factor linked enzymes are operational
in various metabolic path ways which are used
by cells for carrying out their various catabolic
as well as anabolic reactions.
The basic concept in a living cell, when it
comes to cofactor linked enzymes, relates
to cycling of the cofactor between two enzyme
catalyzed reactions.
The two enzyme catalyzed reactions are coupled
in a cell in such a way that the cofactor
which is used in one direction is recycled
back through another reaction with which it
is linked and the total equilibrium is in
favor of the desired intermediate the direct
part to be synthesized by the cell.
As far as their industrial application is
concerned the issue is restricted to mainly
three issues: one is that the coenzyme and
the enzyme must be retained in the reactor.
We have seen in our earlier deliberations
where we have talked about immobilized enzymes
that the enzyme can be retained in the reactor
and used over a long period of time.
A similar concept has to be developed for
co-enzyme retention in the reactor.
Although it is little more difficult in the
sense that while enzymes provide you a large
molecular size as well as a number of functional
groups on the molecule which can be used for
coupling to matrix where the required enzyme
is a small molecular weight compound not such
a large molecule and also the number of functional
groups available on the molecule are very
restricted.
So exactly the same concept may not be applied,
but still one needs to retain the co-enzyme
so that it is not consumed during the reaction.
The basic concept to retain the enzyme as
listed here is the use of the water soluble
polymer bound coenzyme.
For retention of the coenzyme we use instead
of a small molecular weight coenzyme, a water
soluble polymer bound coenzyme.
Because of binding purposes we add another
functional group which can be used for coupling
to a water soluble polymer which may not be
present on the original coenzyme as it is
and therefore you need an analogue.
When I say analogue I am referring to some
chemical and structural modifications but
the role of coenzyme is retained by the molecule.
The second issue is regeneration of the coenzyme.
The regeneration of the co-enzyme like in
a living cell can be carried out by coupling
to enzyme catalyzed reaction, as you see here.
In a membrane reactor in which a reaction
has to be carried out, I illustrate a general
redox reaction, AH plus B going to A plus
BH.
There is an oxidation of AH and reduction
of B and this can be done by two enzymes.
One of them oxidizes AH and produces A, where
as the other one reduces B using the same
co-factor in the reverse direction.
If this co-enzyme is NAD, this has to be coupled
to a polymer so that the coenzyme is not lost
during use.
Another important feature is that the product
needs to be separated to drive the coupled
reaction and the separation of product is
mediated by use of membrane reactor in which
the high molecular weight enzyme as well as
the coenzyme, which is now made to be high
molecular weight, is retained in the reactor
and thereby the product can be separated from
the reaction mixture and the reaction can
be driven in the desired direction.
I will illustrate the point with one of the
examples.
There is lot of chemistry involved because
different people have proposed different kind
of chemical reactions.
I am not interested to give you detailed chemistry.
Just to explain the concept for example you
take polyacrylamide, which is a water soluble
polymer.
This is derivatised by hydrogen hydrate to
polyacrylamide hydrazide and this polyacrylamide
hydrazide then is coupled to a co-enzyme NAD
which is derivatised in the form of carboxymethyl
NAD.
So therefore a carboxymethyl group is generated
here and carboxymethyl NAD is then activated
by carbodiimide, the reaction which you are
familiar, with the carboxylic group containing
enzymes by activation by carbodiimide and
therefore you get an activated co-enzyme molecule.
A carboxymethyl which is NAD analogue is activated
using carbodiimide under acidic conditions
to give you an activated NAD.
You can call it NAD activated.
This activated NAD is coupled with the polyacrlyamide
hydrazide and this on coupling gives you a
water soluble large molecular weight coenzyme
analogue which is functionally compatible
with the dehydrogenase which you want to use
and similar systems can be designed for use.
This describes a typical laboratory setup
in which a stirred cell type of ultra filtration
system has been used.
A is the feed reservoir; it is passed through
a valve which can direct the reaction vessel
either to the reservoir or to the cylinder
for pressure.
It can have both the directions and the reaction
can be carried out high pressure and the reactor
contains the polymer bound co-enzyme, the
substrate and the two enzymes.
We are carrying out reaction in CSTR and when
the reaction goes on, the product formed is
sent out as the permeate in the ultra filtration
cell and the enzyme and the polymer bound
coenzyme is retained in the reactor and the
substrate can be fed continuously by pressure
gradient in the reactor vessel and the reactor
can be continued over a long period of time.
Two of the reactions which have been carried
in such a reactor is one is continuous oxidation
of ethanol using permeabilized yeast cells.
The yeast cells when they are permeabilized,
the lipid on the membrane is dissolved by
a solvent and it becomes freely permeable
to various substrates.
That means it has lost its permeability barriers
for substrate and product exchange.
In fact it has shown to give some kind of
enzymatic activity which can be called as
NADH oxidase; it oxidizes the reduced NAD
into oxygen like an oxidase activity and it
generates hydrogen peroxide and this hydrogen
peroxide to break down requires peroxidase
or else it will inactivate the original enzyme
alcohol dehydrogenase.
In the same reactor system if you use ethanol,
polyacrylamide hydrazide coupled NAD and peroxidase
you can get a net conversion oxidation of
ethanol to acetaldehyde and the profiles shown
here give you the kind of reaction progress.
This is reaction progress in hours.
In the first profile there is no peroxidase
added which means as soon as the reaction
starts the hydrogen peroxide produced inactivates
the alcohol dehydrogenase and no further reaction
takes place.
Only when you add hydrogen peroxide, here
hydrogen peroxide or peroxidase has been added,
you get the reaction going on to a sizable
extent until the time again because of some
reasons either the enzyme has leaked out and
the alcohol concentration again builds up
and there is no reduction.
In third case the system was improved by treatment
of the alcohol dehydrogenase containing cells
by glutaraldehyde which is aimed to fix the
alcohol dehydrogenase on to some cellular
organelles and there by making it almost in
the form of an immobilized enzyme so that
it doesn’t leak out and you can see the
clear advantage that the reaction is extended
over a large period of time comparatively
but again still it goes and although this
system can be demonstrated for eight hours
or ten hours but still there are some problems
which are still so far not understood as to
what happens in the case of cofactors cycling.
The concentration of NAD here is very, very
small and therefore the cycling is taking
place; there is no doubt.
But because you are ultimately here reducing
the concentration let us say from hundred
milli molar to an extent of about seventy
milli molar which means thirty milli molar
concentration has been reduced which means
that in a NAD concentration of let us say
ten milli molar, seven hundred cycles have
taken place.
But ultimately this cycling is not continuous
over a long period of time and that is still
inherent problem.
A similar profile is observed in the case
of conversion of pyruvate to alanine using
same polyacrylamide hydrazide coupled NAD
and formate dehydrogenase and alanine dehydrogenase.
As I mentioned the use of format dehydrogenase
has an advantages that the reaction products
are carbon dioxide and water which can be
easily stripped off but you have to provide
formate as a substrate along with it and the
circles indicate no glutaraldehyde treatment
and the triangles is with glutaraldehyde which
very clearly indicates that the down fall
is prevented and the enzyme is available over
a longer period of time and reaction continues.
But the data beyond sixteen hours have not
been shown here but within a couple of hours
definitely not more than twenty four hours
it is able to say and the reactor performance
drops down.
The second issue was to recall bio processing
of unnatural substrate.
Here we had highlighted that there are a number
of enzyme reactions which although give you
lot of benefits, very favorable concepts,
in terms of their specificity, in terms of
the desired nature of the product they produce
in the right stereoisomeric form but still
the problem is also in relation to the never
specificity gives you a much more problem.
If you look at the two reactions that I referred
to earlier that is conversion of fumaric acid
to malic acids and also maleic acid to aspartic
acid.
Both reactions involve addition of water and
ammonia respectively on to a carbon, carbon
double bond and give you the corresponding
products malic acid and aspartic acid.
Both the products find application in the
food or related industry.
Malic acid is directly used in most of the
beverages as the acidulant and aspartic acid,
the bulk use is in synthesis of aspartane
but the quantity required is not very large.
On the other hand look at similar reactions
which are of commercial value and in large
quantity.
They are just representative reactions; there
are many more reactions which come under this
category.
You see lactic acid to acrylic acid.
Removal of water molecule from lactic acid
is reverse of what fumarase does and this
reaction is of immense value and if it can
be carried out it can serve a great purpose.
Similarly extraction of water from ethanol
to give ethylene or dehydration of ethanol.
Then again extraction of water from 1,3-butanediol
to produce 1,3-butadiene which is a substrate
or monomer for synthetic rubber.
All the three substrates which are listed
here are bulk fermentation products.
If they can transform enzymatically using
an enzyme something similar to that of fumerase
or a modified fumerase probably one can have
very significant impact on the enzyme technology.
Such efforts are drawing attention of people
so that we can design enzymes reactions to
meet the requirements.
In general although the enzymes catalyse very
desirable chemical transformations like fumaric
acid to malic acid or maleic acid to aspartic
acid but often they convert only substrate
that have limited commercial interest.
In fact if these enzymes can be used for those
conversions which have very varied and very
large commercial interest the impact will
be much greater and one can be really fascinated
with the industrial application of enzymes.
Other feature was people tend to accept the
enzyme names very literally and in absolute
sense.
When we name glucose oxidase we interpret
that glucose is a substrate with oxygen as
the ultimate lactone acceptor for oxidation
of glucose.
Similarly galactose oxidase also is in the
same concept but in practice these enzymes
are not so highly specific.
In the first slide we saw that some of the
enzymes which are of industrial value are
very specific.
They have very narrow specificity and we need
to broaden their specificity.
On the other hand we have certain enzymes
which are broadly specific which are not very
narrowly specific but we are not able to use
them for variety of reactions.
So both the things have to be really tackled
so as to be able to apply enzymes on a large
perspective like for example glucose oxidase
can use large number of electron acceptor
other than oxygen which can be then reduced
at the expense of glucose.
Glucose will get oxidized and the corresponding
electron acceptor will get reduced and one
can carry out a number of organic chemical
transformations.
Galactose oxidase is another enzyme which
is known to catalyse the classical reaction
of galactose to the corresponding aldehyde
by oxygen.
But this galactose oxidase is a very broad
…… applicable enzyme and it really acts
on a variety of aliphatic and aromatic alcohols
to give rise to corresponding aldehydes and
these aldehydes, the conversion of alcohol
to aldehydes oxidation reactions, are of great
significance in perfumery and cosmetic industry
and a number of reactions can be catalyzed.
One thing you must be sure is that the rates
on different substrates might be different.
Today with an understanding of the site directed
mutagenesis, it is possible to change the
substrate specificity and also improve the
catalytic functional rates.
So the message is that in addition to the
natural reactions catalyzed by enzymes there
is always a range of non-natural reactions
that are of commercial interest.
The challenge is to unfold and link these
non-natural reactions for the enzyme available
in bulk quantities.
Another feature is first we must pay attention
to those enzymes which are already available
in bulk quantities.
Because one would like to look at a particular
bio transformation but if the enzyme is not
available in bulk quantity at a low cost the
application may be inhibitory; may not be
feasible.
So best is to initially look at those enzymes
which are available in large quantities and
one can only diversify their application to
a large range of non-natural reactions which
are of commercial value and this will require
very clear understanding of their reaction
mechanism because presently those reactions
are being carried out by chemical transformations
and making use of the reaction mechanism one
can probably think of certain unconventional
reactions.
The third interesting area which I wanted
to talk was bio-organic synthesis using enzymes
in micro-aqueous medium.
Normally we understand that enzymes are operable
in aqueous medium and the basic reason is
that a large number of non-covalent interactions
that we are familiar with enzyme structure
are through interaction of the enzyme protein
with the water molecule and water is a requisite
specie in the case of maintenance of a confirmation
of an enzyme molecule and to carry out the
chemical or bio chemical reactions.
Such a perception in the earlier days was
based on a very limited understanding of the
enzyme structure and function and the limited
understanding was that people had looked at
the stability of the enzyme in water miscible
solvents say for example acetone which is
still used for precipitation of enzyme proteins;
it makes aggregates and precipitates; dioxanes
which are water miscible solvents and beyond
certain concentration of these solvents in
the bulk medium inactivates the enzyme.
Now people understood that enzymes cannot
be used in the non-polar environment which
ultimately proved wrong in atleast for the
last twenty years and people have looked at
a number of water immiscible solvents which
are known to keep enzyme in a very stable
confirmation along with the maintenance of
enzyme confirmation because the quantity of
water required for interaction with the enzyme
protein is a very small bound water molecule
around the external surface of the molecule
which is retained and which is not very easy
to be removed by normal dehydration methods.
Unless you subject it to chemical treatments
you cannot really remove it by conventional
drying methods the bound water present in
the enzyme molecule.
In the recent years number of applications
have already emerged but on a limited scale
and probably the scope it offers is much,
much larger than it is being practiced today.
Some of the advantages of organic media for
bio-catalysis are: one is many of the reactions
which require non-polar substrates lipids
mainly are very difficult to carry out in
aqueous medium because they are not soluble
or the concentration that will be soluble
will be very small.
So if you want to carry out those reactions
in high concentration you need a non-polar
bulk medium and the substrate can be dissolved
and then they can be used for enzymatic catalysis.
Another major advantage is the hydrolysis
can catalyse synthetic reactions.
That means in the absence of water for hydrolysis
which require water as a second substrate
or for hydrolysis reaction water is the reactant.
If the water is stripped off or is not present
then the reaction can go in the reverse direction
and instead of hydrolysis, synthesis can be
used and in fact it is in this area a very
significant application have emerged where
the ester synthesis has been carried out even
commercially in fact for some of the very
high value products.
Particularly, more importantly for resolution
of racemic mixtures which we know that the
enzymes are able to recognize one of the isomers
the resolution can be carried out in the case
of chemically synthesized racemic mixture.
Then many of these can be used for modification
of enzyme specificity as far as the reaction
is concerned in the organic medium.
It has been also seen that when you subject
enzyme molecule into anhydrous organic solvent
the enzyme becomes very rigid.
If you put an enzyme molecule into aqueous
media, in buffer the molecule in highly mobile.
It interacts with the water molecule freely
and it is very mobile but when you put in
organic non-polar medium its mobility is drastically
reduced.
In fact one of the major Scientist working
in this area he has called these as rocks
in the organic solvent.
The molecule becomes so rigid that the structure
is very rigid and deactivation is impossible.
The molecule becomes rigid; it doesn’t deactivate
it retains its activity and that is one of
the advantage and during this rigidification
lot of modification in the substrate specificity
also occurs and this has been noted in many
cases and coincidentally bulk of the studies
in use of enzymes in the non-polar medium
has been in the area of hydrolases mainly
lipase and proteases.
They are the two classes of enzymes lipases
and proteases which have been extensively
studied for variety of reactions.
I will illustrate with some of the examples.
Then reduction of side reactions catalyzed
by water.
When water is not present any reaction catalyzed
by water is inhibited.
Then higher stability both thermal stability
and no problems with microbial contamination
because the solvent itself will act as a preservative
and not allow any microbial contamination
to take place and the enzyme can be stored
over a long period of time.
Even operational stability is improved; as
I mentioned that the confirmation becomes
very rigid.
It’s not possible to alter it during reaction
and the mobility is lost.
Another major important factor is better integration
with the chemical steps in synthetic sequence
where you want to use enzyme as a part of
a sequence of reactions in an organic chemical
synthesis where the substrate is coming already
dissolved in organic solvent you can always
integrate enzymatic reaction in the sequence
of steps.
Otherwise it was difficult earlier because
if the substrate is coming in the solution
form in an organic solvent you need to remove
the organic solvent put into water medium
and then carry out the reaction which is practically
not feasible but which can be made feasible
if you use the advanced enzymes in the organic
medium.
Some of the alternative ways in which enzymes
can be used in the organic solvent are shown
here.
If you look at the first one, the ‘A’,
it is a homogeneous system containing individual
enzyme molecules, water and water miscible
organic solvent.
Typical example is acetone, water mixture.
You take ethanol water mixture put the enzyme
carry out the reactions.
Such a system practically is not very, very
attractive because as I mentioned they tend
to inactivate the enzyme because water miscible
solvents often tend to extract the bound water
layer of the enzyme and thereby the confirmation
is disturbed.
The second is the use of enzyme in two phase
systems; an organic and water two phase systems
where the enzyme is present in the aqueous
phase, the substrate is present in the non-aqueous
phase; make an emulsion by agitation or use
of surfactant, bring the enzyme and the substrate
in close proximity and then carry out the
reaction.
In the third configuration.
‘C’, the enzyme is immobilized on water
containing porous particles surrounded by
organic solvents.
That means you use a hydrophilic support and
the hydrophilic support will contain the enzyme
particle immobilized along with some water
molecules dispersed around it and the whole
particle is then immersed as shown in a magnified
way here.
The enzyme particles which are hydrophilic
contain the water molecules around the enzyme
and they are suspended in the bulk organic
medium.
The shaded portion indicates the organic medium
and the blank spaces are indicated by water
medium and the enzyme particles are marked
E.
The fourth configuration is the immobilization
of the enzyme particle on a hydrophobic matrix.
There is no water present in the proximity
of the enzyme molecule and the hydrophobic
matrix and the enzyme is immobilized in a
hydrophobic patch and it is suspended then
in the organic solvent.
The fifth one is solid enzyme particles suspended
directly.
You take enzyme particle, dry it and suspend
into solvent and because they are not soluble
they will tend to form lumps, aggregates that
is what these aggregates are shown and they
can be used to carry out the reaction.
Then the sixth one is you make reverse micelle
by use of surfactant.
You take enzyme in the micro emulsion form
and inside the reverse micelle you have the
enzyme entrapped in the water cells.
That means the enzyme experiences an atmosphere
which is hydrophilic in nature but the whole
reverse micelle is surrounded by the organic
solvent.
This is the enzyme entrapped in the reverse
micelle.
Reverse micelle is just like water in oil
emulsion where by you have enzyme entrapped.
Within the reverse micelle the amphipathic
molecules make a structure where by all the
hydrophobic tails extrude outwards towards
the bulk organic solvent and the polar heads
are all made into a small network within which
the enzyme particle is trapped and some water
is also entrapped within the cell and therefore
the enzyme remains stable almost as if it
is experiencing an aqueous phase.
But the whole particle, as a micelle particle
is immersed in the organic phase.
Then you have the last configuration in which
enzyme is modified by chemical modification
so as to be made soluble in the non-aqueous
phase.
One of the well known reported methods is
modification of enzyme by coupling to polythene
glycol.
If you couple polythene glycol, the whole
enzyme molecule becomes soluble in non-aqueous
phase and then it can be put into an organic
medium and then it behaves almost like a soluble
enzyme but in non-aqueous phase.
But again doing covalent modification of this
enzyme, you loose lot of activity and there
might be some change in the conformation so
as to alter substrate specificity in the process.
If you look at the structural understanding
of the enzyme what happens when we immobilize
either on hydrophilic or hydrophobic matrix?
Most of the enzymes in organic phase are used
in immobilized form.
If you look here this is a free soluble enzyme.
As I mentioned earlier the enzyme molecule
is surrounded by water layer and this water
layer is essential for the enzyme conformation
and activity.
When the bulk solvent is water and the continuous
phase is water there is no disturbance on
this surrounding layer and the enzyme remains
stable for a reasonable period of time.
Due to thermo chemical reasons it might get
inactivated but for some time it remains intact.
When you immobilize the enzyme on to a solid
matrix, let us say a polymeric matrix, it
could be hydrophilic or hydrophobic matrix
you generate an interphase with the bulk medium
and the polymeric matrix is surrounding the
water layer which is maintained during the
reaction.
No.
Bulk here is organic solvent and here it was
water.
In the third case it is a reverse micelle.
Here also you have an interphace which consist
of hydrophobic tails.
The fatty acid or whatever lipid molecule
you have used for making the micelle, they
are arranged such that the hydrophobic heads
are diverted inside which form a structure
and tails are extruding outside and then you
disperse it through the use of surfactant
and you have an interface here consisting
mainly of the hydrophobic tails which remains
very stable in the organic medium.
Although this provides a very neat system
for carrying out enzymatic reaction in organic
phase, the only problem is the recovery of
the product, after the reaction is over, from
the system.
Recovery of product means you need to disrupt
the micelle, reform the micelle so the reaction
cannot be really carried out in a continuous
mode and that causes severe problem, operational
problem.
But otherwise as a system if you look at this
provides you an ideal system because it gives
you very large interphase; it gives you a
hydrophobic interface which can remain stable
in the ……. and also enzyme is very stable
because it is experiencing the aqueous environment.
The important parameters for the enzyme reactions
in organic solvents are the source and presentation
of the enzyme.
As I mentioned the variety of forms in which
the enzyme can exist either as a free enzyme,
immobilized enzyme, soluble conjugate, that
is …… enzymes or in microemulsion form
either of the way; that is one parameter and
one need to look at the kind of form of the
enzyme that gives the best results.
Then the choice of solvent is another feature.
It has been noted in most cases that the solvents
which are relatively more non-polar are better
suited for carrying out enzymatic reaction
mainly because of their lack of interaction
with the water layer on the enzyme molecule.
In fact people have defined as far as the
choice of solvent is concerned a parameter
what they call as log P where P is the partitioning
coefficient of the solvent between a particular
solvent and octenol.
Octanol is considered to be the extreme non-polar
medium and the partitioning coefficient of
that solvent between water and octenol is
the P value and higher the log P value means
it is more non-polar and higher the log P
value the better it is a suited solvent for
enzyme catalyst.
They do not interact with the essential water
layer on the enzyme molecule because of their
non-polar nature and therefore the enzyme
structure is maintained.
A very important factor is amount of water
present.
Almost all the reactions when we want to carry
out in organic solvent we also need a very
small quantity of water which plays a key
role in the catalytic function of the enzyme.
It is a question of debate whether this amount
of water required is to maintain the essential
water layer in equilibrium or there are some
other catalytic functions which require water
molecules by the enzyme because so far if
you look into reaction mechanisms of enzymes
excepting for maintaining the enzymes structure,
water has not been seen to play any role.
But once if we say that you have a very high
log P value of the solvent which has no miscibility
with the water it will not disturb the essential
water layer but still a small quantity of
water is required in the organic solvents
to maintain the catalytic function and there
are number of approaches which has been made
to provide that small quantity of water and
also to strip the water produced in the reaction.
When you talk of hydrolysis being used in
the reverse direction for synthetic purposes
water will be produced and this water needs
to be essentially removed.
On the other hand there is an optimum water
concentration which is required but when water
is produced water has to be removed rather
being added.
Then pH is another feature.
When you carry out the enzyme reaction in
an organic solvent the role of pH becomes
limited in the sense that as we saw earlier
in the effect of pH in enzyme kinetics, the
major role was ionization of the amino acid
residues in the enzyme protein.
A non-polar solvent will not really allow
ionization to take place and therefore pH
theoretically must not play any role.
But the role of pH in the case of enzyme reaction
in organic solvents has a different connotation
and the connotation is that it has been reported
by some workers that enzymes unlike many other
small molecular weight chemical compounds
possess a memory with respect to certain physical
features say for example pH.
For dispersing the enzyme into an organic
solvent you are starting from enzyme which
is already dispersed in some aqueous medium
and whatever pH was of that medium, the enzyme
retains that pH in the memory and the functional
aspect of the enzyme when it is transferred
to non-polar medium remains at the same pH.
So you can make different enzyme preparations
starting from initial pH in the water medium
which have different functional capabilities
and by changing the enzyme memory with respect
to pH.
Similarly temperature and nature of substrate
also are important parameters and they have
the similar features as we saw in the case
of water in aqueous medium.
Just to illustrate one of the reaction system
which we did some work here in IIT itself
and the enzyme reactors systems were developed
here and used for a number of reactions which
I have skipped.
It consists of stirred reactor in which silicon
tubing is dispersed into the organic phase
and silicon tubing, while it is non-porous
to most of the substrates and the products,
but it is porous to water vapors and what
we do is through silicon tubings we recirculate
a saturated salt solution which is known to
have a definite water activity.
In fact for calibration of the humidifiers
we use saturated salt solution and each salt,
when you make a saturated salt solution has
definite relative humidity and those salts
are circulated so as to maintain a constant
water activity in the reactor and even control
water activity.
That means this saturated salt solution if
more water is produced will take up water
and when water will get depleted it will act
almost like a water buffer just like a buffer
for acids and bases, this acts like a buffer
and one can control water activity in organic
reaction system and carry out the reaction
at a constant water activity.
A number of enzyme catalyzed reactions have
been reported like lipases for ester synthesis,
interesterifications, transesterifications
and resolution of racemic mixtures.
Because each enzyme has specificity towards
one of the isomers, substrates can be resolved
into respective isomers.
Proteases for peptide synthesis, oxidases
for variety of reactions for example cholesterol
to cholestenone, butanol to butanaldehyde,
p-phenylphenol to phenol-formaldehyde resins,
p-cresol to o-quinone and ethanol to hydrogen
peroxide.
Even transferase have been used in organic
medium for sucrose to levan, a polymer and
glucose to fructose isomerisation has been
shown to shift the equilibrium constant from
fifty percent equimolar in the favor of fructose
while when we use in the non-aqueous phase.
Last feature is synthetic enzymes in molecular
imprinting.
We know that the enzymatic catalytic function
has two key roles: one is binding of the substrate
and then certain catalytic function.
So what we need in the case of synthetic enzyme
is that we must have kind of a large molecule
with a cavity so that a substrate of definite
size can fit into it and mimic a binding process.
The other is in the proximity of the cavity
there must be some functional groups which
can do catalyses and we know that the enzymes
also follow the same kind of reaction mechanisms
as chemical catalysts do or acid base catalysis,
covalent catalysis, or proximity effects;
all those there are experienced in the enzymatic
reactions and the similar concepts are being
used to synthesize large molecules to mimic
the catalytic functions.
In fact one of the most successful attempts
has been to use cyclodextrins.
The cyclodextrins are cyclic molecules with
six, seven and eight glucose moieties coupled
together in a starch.
That means a-1,4-glucoside linkage.
If you degrade starch you will get dextrins
and these dextrins if they are cyclic molecules
we call them cyclodextrin and the alpha, beta,
gamma cyclodextrins are six, seven and eight
glucose molecules linked in the cyclic form.
The peculiar feature of this is that the cyclic
molecules make a cavity of definite size in
which you can retain the definite molecules
and this cyclodextrins are being used to attach
by covalent coupling some of the chemical
groups which can catalyse the reaction.
If you look at the cavity of cyclodextrin
it is almost like a paper and cylinder and
on the surface they attach a number of functional
groups, the same functional groups which are
known to be possessed by and this is for example
chymotrypsin analogue.
They have tried to mimic the activity of chymotrypsin;
they have named it also benzyme and some of
the functional groups are covalently coupled
which are functional in the case of catalysis
and such a complex is called a synthetic enzyme.
Many such molecules are likely to come out.
But so far not many synthetic enzymes are
available which can carry out diverse function
as the normal natural catalyst do.