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Debayan Ghosh

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EPYGEN IS A BIOTECHNOLOGY RESEARCH AND MANUFACTURING ORGANIZATION
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.

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The company explicitly fosters individual creativity and initiative among employees, encouraging scientists and technologists to keep product pipeline full with products and solutions for tomorrow.

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Dubai Temperature Chart

Jan
Average Maximum Temperature C

Feb

Mar

Apr

May

Jun

Jul

Aug Sep

Oct

Nov

Dec

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 Minimum 14.3 Temperature C

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 5.4 with Rain

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 Mean Sea Temperature C

8.2 20.9

8.5 20.7

8.6 22.3

10.2 25.0

11.3 28.5

11.5 31.2

10.7 32.2

10.5 32.9

10.4 31.9

9.9 29.9

9.3 27.0

8.2 23.4

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 sheets in a protein and uncoils it into a random shape. Something like this..

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Native and denatured states

denatured ensemble unfolded ensemble native state folded state

many different structures fluctuating; not usually very compact; disordered but not a random coil

single structure or ensemble of very similar structures; compact

For some proteins, but not all, this process is readily reversible and occurs without populated intermediate forms--> two-state folding

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Actually what happens when protein feels too hot..

Heat energy disrupts hydrogen bonds and nonpolar hydrophobic interactions as it increases the kinetic energy and causes the molecules to vibrate so rapidly and violently that the bonds are disrupted.
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 hydrogen bonding groups and of hydrophobic groups is to increase the amount of water bound by the protein molecules. The unfolding that occurs increase the hydrodynamic radius of the molecule causing the viscosity of the solution to increase.

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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 folding thermodynamics

Native (folded)

Gu

Denatured (unfolded)

protein becomes less stable at high temp and unfolds when TS exceeds H

Gu = Hu - TSu

Gu Hu TSu T

+
favorable native state interactions broken

unfolded state more disordered

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Amount of unfolded protein as function of T eqn describing Gu as function of T

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Gu = Hu,Tm(1 T/ Tm ) + Cp[T Tm T ln (T/ Tm)] Ku = exp(Gu/RT) = [U]/[F]

Keq for unfolding reaction

fu = Ku / (1 + Ku )
fraction unfolded

concentration unfolded and folded 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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Documents Examples 1.90+ Degrees Centigrade..AMYLASE in Desizing. 2.80+ Degrees Centigrade..Xylanase for Feed Pelletization. 3.100+ Degrees Centigrade.Cellulases for Petroleum Industry Downhole action.

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WHY Most industrial enzymes are produced Recombinantly

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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

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To Render Enzymes more Thermostable

BY PROTEIN ENGINEERING TOOLS.

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Documents Improving Thermostablity by structurally reinforcing or locking -Helix by disulfide links.

What was happening in the Glucoamylase Protein

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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 ( 600 K,..... )6-barrel at simulation temperatures up to

The catalytic base, E400, migrated from its original interior pocket to the surface of the catalytic domain by jumping the hydrophobic barrier provided by -helices 12 and 13 at 800 K.

Ref: Hsuan-Liang Liu, Wen-Chi Wang(2003), Protein Eng., 16, 19-25

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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 to dramatically increase the Thermostability......

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This indicates that the introduced residues with higher hydrophobicity were favorable in the loop between -helices 12 and 13, whereas they partially destroyed the hydrogen bond and salt linkage network in the catalytic centre..... -Helices 12 and 13 can be stabilized by introducing residues with higher hydrophobicity...... This was an example how protein can be stabilized by protein engineering .

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LOOKING INTO NATURE MESOPHILES versus EXTREMOPHILES:

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Mesophiles

35-60oC pH 4-8

Extremophiles organisms that colonize with one or more extreme environmental parameters pH temp salinity pressure

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HYPERTHERMOPHILES;
90oC plus typically anaerobic found at tectonically active sites vents springs geysers

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But often Hyperthermophiles:

Difficult to culture Once has to move to recombinant production of their enzymes Purification easier Just Raise temp..

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Stabilizing Proteins in formulation with Polyols

Polyol-induced water-mediated effects such as an increase in the surface tension of water play a major role in the stabilization of proteins Preferential hydration of proteins observed in Polyol presence is a consequence of the increase in the surface free energy of water.

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How We Measure Thermal Stability in our Dubai Plant

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We believe that thermodynamic measurements coupled with biological activity might be a valuable tool for screening additives in liquid protein formulations for protection against various degradation mechanisms causing protein conformational destabilization associated with loss of (or decline in) biological activity.

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Which Polyol To chose?

No single polyol could provide maximal protection against all protein destabilization routes. Therefore, polyols should be selected on the basis of possible destabilization mechanism for a particular protein likely to be encountered during formulation, process development, handling or storing.

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Effect of Polyols on Midpoint Transition Temperature, Calorimetric Enthalpy, and Specific Enzyme Activ-ity of Lysozyme Subjected to Aggregation/Precipitation by Sodium Chloride

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Polyols

Transition Tem

Calorimetric Enthalpy Cal/mol X 104

Specific Enzyme Activity X 103

Control Mannitol Sucrose Lactose Glycerol Propylene glycol

73.7 0.6 75.9 0.8 75.3 0.4 75.2 0.2 74.9 0.4

5.9 0.5 7.3 0.6 6.8 0.4 6.7 0.3 6.2 0.8

29.0 1.9 36.0 0.8 33.0 0.6 33.0 0.4 30.0 1.2

71.4 0.5

5.4 0.3

27.0 1.2

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 protein Has gone through...

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Circular dichroism

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.

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We are CONCERNED about the Loss of Biological Activity

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, temperature 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.

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EPYGEN CMC-DNS-ASSAY
This assay measures the release of reducing sugars by the action of a cellulase on a cellulosic substrate. One unit of activity liberates 1 micromole of reducing sugar (expressed as glucose equivalents ) in one minute under the conditions described. The sample activity is then related to a standard with a stated CMCase activity.

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Accelerated Stability and Normal stability at Dubai Temperatures after POLYOL stabilization

Normal Stability

Days October to April

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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 should be stored in refrigerated conditions. 3.Immediately after production, move drums in cold room. 4.Prefer cold distribution and reefer containers during July to September. 5.Protein Salting out is first sign to watch for. 6.Check turbidity and consistence 7.Maintain Accelerated and Normal stability data for all new formulations. 8.Compulsory First In First Out inventory. 9.Container to carry temperature rea-ltime logging.

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