Enzyme Stability

1. Enzyme Stability

2. Definisi • Stabilitas enzim adalah kondisi di mana enzim dapat mempertahankan konformasi struktural atau aktivitasnya selama proses isolasi, purifikasi, penyimpanan dan perlakuan fisik atau kimia yang lain termasuk enzim proteolitik maupun panas.

3. Enzyme stability Long term stability: • Production, storage, shipment • Enzyme purification • pH, ionic strength, temperature • Frozen, liquid, powder • Presence of additives

4. Operational stability • Medicine • Frequency of administrating a new dose • Reduce costs, and inconvenience for patient. • Laundry • Presence of surface active compounds • High temperatures, alkaline conditions • Resistance of lipases to proteases.

5. Operational stability • Industrial synthetic applications • Process conditions • pH, organic solvents, denaturants etc. • Re-usage of biocatalyst

6. Factors affecting protein folding and activity • Hydrogen bonds • Ionic bonds • Van der Waals forces

7. Non-covalent interactions • Hydrogen bonds C=O …. HN • C=O : Glu, Asp, Gln, Asn • NH : Lys, Arg, Gln, Asn, His • OH : Ser, Thr, Glu, Asp, Tyr • Ionic bonds COO-….+H3N • COO- : Glu, Asp pKa < 5 • NH3 + : Lys, Arg pKa > 10

8. Non-covalent interactions • Van der Waals forces • Electrostatic in nature, short ranges • Dipole-dipole, ion-dipole etc. • Strength of interaction • 0.4 - 400 kJ/mol • Charge, dipole moment • Distance, dielectric constant medium • D = 80 (water) D = 2 - 4 (protein interior)

9. Protein Inactivation • Protease • Temperatur • Ekstrim pH • Oksidasi • Logam berat Radiasi Detergent Unfolding agents Chelating agents Mechanical forces

10. Irreversible inactivation of protein • Proteolysis • Partial unfolding may increase proteolytic susceptibility (surface loops) • Integrity protein can be studied by (limited) proteolysis

11. High temperature • Increase of mobility of protein segments • Exposure of hydrophobic groups • Formation of non-native disulfide bridges • Precipitation, scrambled structures • Aggregation, denaturation.

12. High temperature • Chemical modification • Deamidation of Asn or Gln • Hydrolysis of peptide bonds (Asp) • Destruction of disulfide bonds • Chemical reactions between proteins and other compounds: carbohydrates, polyphenolics

13. Thermostable enzymes • Hyperthermophilic microorganisms • Comparison with mesophilic counterparts • Many different structural reasons for increased thermostability • Compact (multimeric) proteins • Increase of number of salt bridges

14. Low Temparature • Freezing • Concentration of solutes • Changes in pH and ionic strength • Increase in oxygen sensitivity • Storage in liquid nitrogen

15. Extremes of pH • Repulsion of charged amino acid residues • Chemical modification (deamidation) • Hydrolysis of Asp-Pro linkages • High pH: destruction of disulfide bonds

16. Surfactants and detergents • Hydrophilic head, hydrophobic tail • Form micelles above CMC • Monomers interact with proteins • Exposure of buried hydrophobic residues • Anionic detergents SDS • Cationic detergents CTAB • Non-ionic detergents Triton

17. Denaturing agents • Reversible unfolding • Urea, guanidinium hydrochloride • Diminish intramolecular hydrophobic interactions • Chaotropic salts • Polar organic solvents • Chelating agents • Heavy metals and thiol reagents

18. Oxidation and UV Radiation Oxidation • Oxygen, hydrogen peroxide, oxygen radicals • Tyr, Phe, Trp, Cys, Met UV-radiation • Cys, Trp, His

19. Mechanical forces • Stirring and mixing: shear forces • Ultrasound, high pressure, shaking • Deformation and exposure of hydrophobic residues  aggregation • Adsorption to wall of reaction vess

20. General Mechanisms • Charge or hydrophobic interactions • Disturbance of balance of stabilising and destabilising interactions by weakening or strengthening • Covalent modifications • Breaking disulfide bonds

21. Enzyme inactivation Reversible inactivation • Partial unfolding • Chemical alteration Irreversible inactivation • Complete unfolding, aggregation • Chemical modification, proteolysis

22. Monitoring protein stability Thermodynamic approach • Proteins are only marginally stable in the folded active form • Globular proteins: GU = 40 - 80 kJ / mol • Optimum for most proteins between 20 and 40 ºC

23. Monitoring protein stability Biochemical approach • Storage stability as function of pH, temp, salt etc. • Useful information for applications • Useful for insights into enzyme action • Incubation of resting enzyme • Measurement of residual activity with time • Kinetics of enzyme inactivation.

24. Monitoring protein stability Operational stability • Stability of catalytically active enzyme • Highly relevant for applications • Difficult to measure on a laboratory scale • Influence of substrates • Mimicking of reactor conditions • Product yield with time

25. Monitoring protein stability Optimal stability vs Optimal activity • pH dependence of thermostability • pH dependence of enzyme activity • Temperature dependence of enzyme activity • Absence or presence of substrates or cofactors • Optimum conditions for maximum conversion • Cost aspects (reusage of biocatalyst)

26. Prevention of inactivation • pH, temperature, protein concentration • Addition of stabilisers • Use of thermophilic enzymes • Enzyme immobilisation • Protein engineering • Chemical modification • Apolar organic solvents

27. What is denaturation? • When the unique 3-D structure of proteins is destroyed. • It leads to temporary or permanent loss of activity.

28. Denaturation • Denaturation of proteins happens when there is a loss in the secondary or tertiary or quaternary structure of it.

29. Denaturation • Proteins are maintained in their native state (their natural 3D conformation) by stable secondary and tertiary structures, and by aggregation of subunits into quaternary structures. • Denaturation is caused when the folded native structures break down because of extreme temps. or pH values, which disrupt the stabilizing structures. The structure becomes random and disorganized.

30. Denaturation • Most proteins are biologically active only over a temperature range of 0ºC to 40ºC. • Heat is often used to kill microorganisms and deactivate their toxins. The protein toxin from Clostridium botulinum is inactivated by being heated to 100ºC for a few minutes; heating also deactivates the toxins that cause diphtheria and tetanus.

31. Denaturation • Heat denaturation is used to prepare vaccines against some diseases. The denatured toxin can no longer cause the disease, but it can stimulate the body to produce substances that induce immunity. • Proteins can also be denatured by heavy-metal ions such as Hg2+, Ag+ , and Pb2+ that interact with —SH and carboxylate groups.

32. Effects of denaturation • Decreased solubility • Altered water binding capacity • Loss of biological activity • Destruction of toxins • Improved digestibility • Increased intrinsic viscosity • Inability to crystallize

33. Denaturation

34. Types of protein denaturation • Reversable denaturation: when the effect of denaturation reagent is removed by dialysis, the enzyme will gain its activity again. • Irreverssable denaturation: if the protein is at temperature over 60oC, it will form an insoluble substances called coagulum as in heating the eggs white. It cant be returned to its original state.

35. When and how are proteins denatured? • At Very High or Low pH. • At Very High Temperatures. • By Heavy Metal Ions. • By Small Polar Molecules.

36. Other denaturing agents • Changes in pH, which alters the ability of the acidic and basic side chains to form salt bridges • Organic compounds, which will disrupt the disulfide bonds • Heavy metals that disrupt salt bridges and disulfide bonds • Mechanical agitation, which disrupts hydrogen bonds and London forces

37. Examples of Denaturation • When milk curdles, the acidity increases. • Thermal denaturation by cooking. • Mechanical denaturation when whisking an egg. • Perming hair breaks then reforms the disulphide bonds.

38. Thermal denaturation • Trypsinogen 55°C • Pepsinogen 60°C • Lysozyme 72°C • Myoglobin 79°C • Soy Glycinin 92°C • Oat globulin 108°C Affected by pH, water, solutes

39. Denaturation agents • Heat and ultraviolet light. Disrupt hydrogen bonds and ionic attractions by making molecules vibrate too violently; produce coagulation, as in cooking an egg. • Organic solvents (ethanol and others miscible with water). Disrupt hydrogen bonds in proteins and probably form new ones with the proteins.

40. Denaturation agents • Strong acids or bases. Disrupt hydrogen bonds and ionic attractions; prolonged exposure results in hydrolysis of protein. • Detergents. Disrupt hydrogen bonds, hydrophobic interactions, and ionic attractions. • Heavy-metal ions (Hg2+,Ag+,and Pb2+). Form bonds to thiol groups and precipitate proteins as insoluble heavy-metal salts.