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8/13/2019 Producing Handling and Storing Heat Sensitive Proteins in Middle East
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Debayan Ghosh
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EPYGEN IS A BIOTECHNOLOGY
RESEARCH AND MANUFACTURINGORGANIZATION
Epygen Vision is to create a world class Life Science
organization which will emphasise on strong research
combined with an applied focus on Industrial, Pharmaceutical
and Agriculture Biotechnology.
The company explicitly fosters individual creativity and
initiative among employees, encouraging scientists
and technologists to keep product pipeline full with productsand solutions for tomorrow.
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Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
Average
Maximum
Temperature C
24.0 25.4 28.2 32.9 37.6 39.5 40.8 41.3 38.9 35.4 30.5 26.2
Average MinimumTemperature C 14.3 15.4 17.6 20.8 24.6 27.2 29.9 30.2 27.5 23.9 19.9 16.3
Mean Rainfall
(mm)
15.6 25.0 21.0 7.0 0.4 0.0 0.8 0.0 0.0 1.2 2.7 14.9
Mean No. of Days
with Rain
5.4 4.9 5.9 2.6 0.3 0.0 0.5 0.6 0.1 0.2 1.3 3.6
Sunshine Hours /
day
8.2 8.5 8.6 10.2 11.3 11.5 10.7 10.5 10.4 9.9 9.3 8.2
Mean Sea
Temperature C
20.9 20.7 22.3 25.0 28.5 31.2 32.2 32.9 31.9 29.9 27.0 23.4
Dubai Temperature Chart
Source : Met Department Web Site
For Enzyme Protein: Months To Watch: May-October
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CLIMATE CHALLENGE FOR A BIOTECH
COMPANY:
1.HOW TO MAKE PRODUCTS THAT ARE
MORE STABLE.
2.HOW TO MAKE STABLE FORMULATIONS.
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Denaturation of Proteins means...
Disruption and destruction of both the secondary and tertiary
structures.
Since often denaturation reactions are not strong enough to
break the peptide bonds, the primary structure (sequence of
amino acids) remains the same after a denaturation process.
Denaturation disrupts the normal alpha-helix and beta sheetsin a protein and uncoils it into a random shape.
Something like this..
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Native and denatured states
native statefolded state
denatured ensembleunfolded ensemble
single structure or ensemble
of very similar structures;
compact
many different structures fluctuating;
not usually very compact;
disordered but not a random coil
For some proteins, but not all, this process is readily reversible and occurs
without populated intermediate forms--> two-state folding
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Heat energy disrupts hydrogen bonds and non-polar hydrophobic interactions as it increases thekinetic energy and causes the molecules to vibrateso rapidly and violently that the bonds aredisrupted.
Actually what happens when
protein feels too hot..
As temperature is increased, a number of bonds in the protein molecule are
weakened.
The first affected are the long range interactions that are necessary for the
presence of tertiary structure.
As these bonds are first weakened and are broken, the protein obtains a more
flexible structure and the groups are further exposed.
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Theoretically, even if heating ceases at this stage the
protein should be able to readily refold to the native structure
(renaturation) and you are lucky.....
As heating continues, some of the cooperative hydrogen bonds
that stabilize helical structure will begin to break.....
As these bonds are broken, water can interact with and form
new hydrogen bonds with the amide nitrogen and carbonyl
oxygen of the peptide bonds.
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The presence of water further
weakens nearby hydrogen bonds by
causing an increase in the effective
dielectric constant near them.
As the helical structure is broken,
hydrophobic groups are further
exposed.........
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The effect of exposure of new hydrogenbonding groups and of hydrophobic groups is to
increase the amount of water bound by the proteinmolecules.
The unfolding that occurs increase the hydrodynamic radius of themolecule causing the viscosity of the solution to increase.
The net result will be an attempt by the protein to minimize its free energy by burying
as many hydrophobic groups while exposing as many polar groups as possible to the
solvent.
While this is analogous to what occurred when the protein folded originally, it is
happening at a much higher temperature.
This greatly weakens the short range interaction that initially direct protein folding
and the structures that occur will often be vastly different from the native protein.
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Naive view of fo ld ing
thermodynamics
Native
(folded)
Denatured
(unfolded)
Gu
Gu = Hu - TSu
++
unfolded state
more disordered
favorable native state
interactions broken
Gu
T
Hu
TSu
protein becomes less stable at
high temp and unfolds when TS
exceeds H
0
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Amount of unfo lded
prote in as func t ion of T
Gu = Hu,Tm(1 T/ Tm )
+ Cp[T T
mT ln (T/ T
m)]
Ku = exp(Gu/RT) = [U]/[F]
fu = Ku / (1 + Ku )
fraction unfolded
concentration unfoldedand folded
Keq
for unfolding reaction
eqn describing Guas function of T
set of nested equations
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Drawbacks of Chemical processes:
Many chemical processes inherent drawbacks from
commercial and environmental point
Non-specific reactions result in poor product yields
High process temperatures and pressure required
High energy costs
Often causes devastating environmental implications.
These drawbacks can be eliminated by using
enzymatic conversions.
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Enzyme reactions
Usually require mild conditions;
Highly specific,
Involve very fast reaction rates
Carried out by numerous enzymes playing
different roles in an orchestrated manner.
Robust scope of kinetics
BUT OFTEN ENZYMES ARE
EXPOSED TO HARSH PROCESS
HEAT !!!!!!!!
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Examples
1.90+ Degrees
Centigrade..AMYLASE in
Desizing.
2.80+ Degrees Centigrade..Xylanase
for Feed Pelletization.
3.100+ DegreesCentigrade.Cellulases for Petroleum
Industry Downhole action.
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Why?
A. Higher expression
B. Higher purity (%protein:other junk)C. cheap
D. can engineer protein
E. can express enzymes which are found
in pathogenic organisms
Heterologous Protein Expression
Homologous Protein Expression
WHY Most industrial enzymes are
produced Recombinantly
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BY PROTEIN ENGINEERING
TOOLS.
To Render
Enzymes
more
Thermostable
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After mutating a Glucoamylase Enzyme from Aspergillus species the thermal unfolding of
the catalytic domain , 13
-helices obeyed the random ordered mechanism.....
In which the -helices 8, 1 and 11 unfolded more rapidly than the others.
The catalytic center was well protected by ()6-barrel at simulation temperatures up to
600 K,.....
The catalytic base, E400, migrated from its original interior pocket to the surface of thecatalytic domain by jumping the hydrophobic barrier provided by -helices 12 and 13 at
800 K.
Improving Thermostablity bystructurally reinforcing or locking
-Helix by disulfide links.
Ref: Hsuan-Liang Liu, Wen-Chi Wang(2003), Protein Eng., 16, 19-25
What was happening in the GlucoamylaseProtein
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When a disulfide bond is
engineered to lock the -helix 11
on the surface of the catalytic
domain, it was observed todramatically increase the
Thermostability......
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This indicates that the introducedresidues with higher hydrophobicitywere favorable in the loop between-helices 12 and 13, whereas theypartially destroyed the hydrogenbond and salt linkage network in the
catalytic centre.....-Helices 12 and 13 can bestabilized by introducing residueswith higher hydrophobicity......
This was an example how proteincan be stabilized by proteinengineering.
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MESOPHILES versus EXTREMOPHILES:
Mesophiles 35-60oC
pH 4-8
Extremophiles organisms that colonize with
one or more extreme environmental
parameters
pH
temp
salinity
pressure
LOOKING INTO NATURE
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HYPERTHERMOPHILES;
90oC plus
typically anaerobic
found at tectonically active sites
vents
springs
geysers
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Hyperthermophiles:
Difficult to culture
Once has to move to
recombinant production oftheir enzymes
Purification easier
Just Raise temp..
But often
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Polyol-induced water-mediatedeffects such as an increase in thesurface tension of water play a majorrole in the stabilization of proteins
Preferential hydration of proteinsobserved in Polyol presence is aconsequence of the increase in thesurface free energy of water.
Stabilizing Proteins in
formulation with Polyols
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We believe that thermodynamicmeasurements coupled with biologicalactivity might be a valuable tool forscreening additives in liquid proteinformulations for protection against variousdegradation mechanisms causing protein
conformational destabilization associatedwith loss of (or decline in) biologicalactivity.
How We Measure Thermal
Stability in our Dubai Plant
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No single polyol could provide maximalprotection against all protein
destabilization routes.
Therefore, polyols should be selectedon the basis of possible destabilization
mechanism for a particular proteinlikely to be encountered duringformulation, process development,handling or storing.
Which Polyol To
chose?
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Control 73.7 0.6 5.9 0.5 29.0 1.9
Mannitol 75.9 0.8 7.3 0.6 36.0 0.8
Sucrose 75.3 0.4 6.8 0.4 33.0 0.6
Lactose 75.2 0.2 6.7 0.3 33.0 0.4
Glycerol 74.9 0.4 6.2 0.8 30.0 1.2
Propylene
glycol 71.4 0.5 5.4 0.3 27.0 1.2
Polyols Transition Tem Calorimetric Enthalpy Specific Enzyme
Cal/mol X 104 Activity X 103
Effect of Polyols on Midpoint Transition
Temperature, Calorimetric Enthalpy, and SpecificEnzyme Activ-ity of Lysozyme Subjected toAggregation/Precipitation by Sodium Chloride
Source: Somnath Singh, Jagjit Singh, 2003, AAPS PharmSciTech ; 4 (3) Article 42
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Assessment of
Denaturation
Ultraviolet Adsorption Spectroscopy.
This measures the wavelength and amount of ultraviolet radiation absorbed by a
molecule.
In proteins, both the wavelength and extent of absorption depend on the amino acids
present and on their physical environments. Giving a fare idea on changes the proteinHas gone through...
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The interaction of polarized light with protein can be
measured by the techniques of circular dichroism and
optical rotatory dispersion.
These methods yield an indication of the extent of repeating
structures present in protein and are generally utilized to
give estimates of the amount of secondary structure present,
eg. alpha-helix, beat sheet or coil.
Circular dichroism
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For those proteins like our ENZYMES, denaturation can be defined as the loss of
structure to render the enzyme inactive.
Changes in the rate of the reaction, the affinity for substrate, pH optimum, temperatur
optimum, specificity of reaction, etc., may be affected by denaturation of enzyme
molecules.
Loss of enzymatic activity can be a very sensitive measure of denaturation as some
assay procedures are capable of detecting very low levels of product.
We are CONCERNED
about the Loss ofBiological Activity
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This assay measures the release of reducing sugars by the action of acellulase on a cellulosic substrate.
One unit of activity liberates 1 micromole of reducing sugar
(expressed as glucose equivalents ) in one minute under the conditionsdescribed. The sample activity is then related to a standard with astated CMCase activity.
EPYGEN CMC-DNS-ASSAY
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Accelerated
Stability and
Normal
stability at
Dubai
Temperatures
after POLYOL
stabilization
Days October to April
Normal Stability
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Dubai Enzyme Company perspective.
1.Liquid Enzymes are more in danger during summer months.
2.Between Production and formulation, unstable enzyme shouldbe stored in refrigerated conditions.
3.Immediately after production, move drums in cold room.
4.Prefer cold distribution and reefer containers during July toSeptember.
5.Protein Salting out is first sign to watch for.
6.Check turbidity and consistence
7.Maintain Accelerated and Normal stability data for all newformulations.8.Compulsory First In First Out inventory.
9.Container to carry temperature rea-ltime logging.
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