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Bio-Engineering

Bio-Engineering. Professor Chris G Whiteley. Visiting Professor of Enzymology Department of Biochemistry, Microbiology & Biotechnology Rhodes University, Grahamstown, SOUTH AFRICA. Bio-Engineering. “ Engineering biological processes of living

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Bio-Engineering

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  1. Bio-Engineering Professor Chris G Whiteley Visiting Professor of Enzymology Department of Biochemistry, Microbiology & Biotechnology Rhodes University, Grahamstown, SOUTH AFRICA

  2. Bio-Engineering “Engineering biological processes of living organisms for developing new technologies to improve the living standards of humans” Microbiology Molecular Biology Biotechnology Biochemistry

  3. Neural engineering Nanotechnology Biomimicry Enzymes Bio Engineering Cryobiology Biosensors Biomimicry Synthetic biology Systems biology Artificial intelligence Biomechanics Bioinformatics Tissue engineering Protein design

  4. Studies methods or systems in nature in order to imitate these designs or processes to solve human problems Neural engineering Nanotechnology Biomimicry Enzymes Bio Engineering Cryobiology Biosensors Biomimicry Synthetic biology Systems biology Artificial intelligence Biomechanics Uses 4 billion years of evolution to establish what works and what lasts It is not what we take from the world - - but what we learn. Bioinformatics Tissue engineering Protein design

  5. Ceramics - Abalone Glue - Blue mussell Pigment free colour - Butterfly Biomimicry Water harvester - Beetle Molecular biologists at the Idaho National Engineering and Environmental Laboratory have cloned five mussel proteins for use in a natural, waterproof adhesive. The mussel foot produces a "super-glue“ that remains intact in seawater, is created at low temperatures, and is environmentally safe. Platinum catalysts in fuel cells - Microbes Silicon manufacture – Diatom/sponge Vaccine storage -Anhydrobiosis

  6. Ceramics - Abalone Glue - Blue mussell Pigment free colour - Butterfly On the underside of the Red Abalone (Haliotis Rufescens) shell is a remarkable iridescent ceramic that is twice as tough as our high-tech ceramics. Mother-of-pearl, also called nacre, is composed of alternating layers of calcium carbonate (in a special crystal form called aragonite) and Lustrin-A protein. The combination of hard and elastic layers gives nacre remarkable toughness and strength, allowing the material to slide under compressive force. The “bricks” of calcium carbonate are offset, and this brick-wall architecture stops cracks from propagating. Can toughen windshields, airplanes or anything that needs to be lightweight but fracture-resistant. Biomimicry Water harvester - Beetle Platinum catalysts in fuel cells - Microbes Silicon manufacture – Diatom/sponge Vaccine storage -Anhydrobiosis

  7. Ceramics - Abalone Glue - Blue mussell Pigment free colour - Butterfly Biomimicry Water harvester - Beetle The feathers, scales, and exoskeletons of iridescent birds, butterflies, and beetles have structural features that cause light to diffract and interfere in ways that amplify certain wavelengths. This creates brilliant colors to the viewer through the use of structure rather than the addition of a chemical pigment. Instead of painting a product, simply add surface layers that play with light. Thin-film interference of this sort can create color that is 1) four times brighter than pigment, 2) never needs repainting, 3) avoids the toxic effects associated with pigments. Platinum catalysts in fuel cells - Microbes Silicon manufacture – Diatom/sponge Vaccine storage -Anhydrobiosis

  8. Walking cane - Bat Hearing aid - Cicada Sonar/Radar/ Ulrasound - Bat The UltraCane is a new electronic mobility aid, designed to help people get around more easily and safely. The UltraCane was inspired by the way bats navigate in darkness. The cane uses ultrasonic signals which bounce off objects present in the environment and feed information back to the cane. This covers the areas in front and, uniquely, to the head height of the user. Biomimicry Fan - Molusc Aircraft wings - Whale Solar cells – Leaf Swarm intelligence – Birds Air conditioning – Termite

  9. How do you build a mid-rise building that has no air- conditioning, yet stays cool? Thanks to a termite-inspired ventilation system. Termites maintain the temperature inside their nest to within one degree of 31 °C, day and night, - while the external temperature varies between 3 °C and 42 °C. Digital scans of termite mounds to map the three dimensional architecture to help understand exactly how the tunnels and air conduits manage to exchange gases, maintain temperature, and regulate humidities. The designs may provide a blueprint for self-regulating human buildings. Walking cane - Bat Hearing aid - Cicada Sonar/Radar/ Ulrasound - Bat Biomimicry Fan - Molusc Aircraft wings - Whale Solar cells – Leaf Swarm intelligence – Birds Air conditioning – Termite

  10. Commercial aircraft wings have a straight leading edge. The leading edge of humpback whale flippers are scalloped with prominent knobs called tubercles. The scalloped flipper is a more efficient wing design than the smooth edges used on planes. The scalloped flippers have 32% lower drag, 8 % better lift and withstood stall at a 40 % steeper wind angle. Walking cane - Bat Hearing aid - Cicada Sonar/Radar/ Ulrasound - Bat Biomimicry Fan - Molusc Aircraft wings - Whale Solar cells – Leaf This discovery has the potential to optimize airplane wings, tips of helicopter rotors, propellers, and ship rudders. The improved stall angle adds a margin of safety while making planes more maneuverable, while the drag reduction improves fuel efficiency. Swarm intelligence – Birds Air conditioning – Termite

  11. Boat hulls – Thick skin of dolphins Smart clothing – Pine cones Biomimicry Water repellent paint Lotus flower leaf Velcro – Seed burrs Artificial neurons – Neural networks

  12. Boat hulls – Thick skin of dolphins Smart clothing – Pine cones Glue - Blue mussell Biomimicry Velcro – Seed burrs Pigment free colour - Butterfly Water repellent paint Lotus flower leaf Artificial neurons – Neural networks

  13. Boat hulls – Thick skin of dolphins Smart clothing – Pine cones Glue - Blue mussell Biomimicry Velcro – Seed burrs Pigment free colour - Butterfly Water repellent paint Lotus flower leaf Artificial neurons – Neural networks

  14. Neural engineering Nanotechnology Biomimicry Enzymes Bio Engineering Cryobiology Biosensors Synthetic biology Systems biology Enzymes Artificial intelligence Biomechanics Bioinformatics Tissue engineering Protein design

  15. Biotransformation Inhibition Properties Kinetics Enzymes Reactors Modifications Classification Properties Mechanisms Applications Biocatalysis Immobilisation

  16. Properties of Enzymes Catalytic Power Enzymes can accelerate reactions as much as 1014 over uncatalyzed rates! Urease is a good example: Catalyzed rate: 3x104. sec-1 Uncatalyzed rate: 3x10-10. sec-1 Ratio is 1x1014 ! Energy of activation is changed

  17. Energy of Activation Energy of activation without enzyme [EA] Free energy [G] Energy of activation with enzyme [EA’] Energy of reactants Change in free energy [ΔG] Energy of products Progress of reaction

  18. Enzyme Substrate Complex Enzymesselectivelyrecognize proper substrates over other molecules Enzymes form products invery high yields - often much greater than 95% Specificity is controlled bystructure - the unique fit ofsubstratewith enzyme controls the selectivity for substrate and the product yield Substratebinds to the enzyme at the ‘active site’ Enzyme [E] andsubstrate[S] bind together as anenzyme-substratecomplex [ES]

  19. Enzyme-substrate Active Site Glu Val Ala Ile Asn Asp Trp Trp Asn Substrate Hydrophobic amino acids surround the substrate

  20. Enzyme-substrate Active Site Enzyme [E] [ES] complex Substrate [S] Product [P] Enzyme [E] [EP] complex

  21. Enzyme activity control Substrate availability Covalent modification Zymogens or inactive precursors Allosteric regulation pH Temperature Inhibitors

  22. Covalent modification A new covalent bond is created which acts as a ‘trigger’ and switches off (or on) the enzyme. E E

  23. Zymogen activation: Some enzymes are synthesized in a non-active precursor form, called a zymogen or proenzyme. The zymogen becomes an active protein upon proteolysis at specific sites in the protein. This regulates the activity of a particular enzyme, where you keep it in a nonfunctional state until it is needed. Cutting the protein then switches the protein on. Preproinsulin

  24. Chymotrypsinogen Chymotrypsinogen π-Chymotrypsin Self digestion α-Chymotrypsin 3 Intra disulphide bonds; 2 inter disulphide bonds

  25. Allosteric Regulation Action at "another site" Enzymes situated at key steps in metabolic pathways are modulated by allosteric effectors These effectors are produced elsewhere in the pathway May be feed-forward activators or feedback inhibitors

  26. pH Active site contains ionisable amino acids Ionisation state depends on pH and/or pKa Lysozyme pH optimum = 5.2. • 2 amino acids at active site – Glu-COOH; Asp-COO- • Glutamic acid is not ionised -Aspartic acid is ionised • At low pH: Asp protonated; At high pH: Glu ionised

  27. Temperature Rate of enzyme reaction increase with temperature > 60oC the rate typically declines: Denaturation Some thermophilic enzymes operate at 85oC Enzymatic reactions double in rate every 10oC

  28. Inhibitors Competitive inhibitor is not substrate molecule Binds with an enzyme's active site. No room for the substrate to bind at that site. Prevents the enzyme from any reaction.

  29. Biotransformation Classification Inhibition Properties Kinetics Enzymes Reactors Modifications Classification Mechanisms Applications Biocatalysis Immobilisation

  30. Classification • Enzyme commission • Six classes • 1st Main class 1.Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases • 2nd Sub class • 3rd Sub subclass • 4th molecule Lyase C - C C-COOH Eg: Histidine carboxylyase: EC. 4.1.1.22 Histidine

  31. Classification Oxidoreductases • Enzyme commission • Six classes • 1st Main class 1.Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases • 2nd Sub class • 3rd Sub subclass • 4th molecule Lyase C - C C-COOH Eg: Histidine carboxylyase: EC. 4.1.1.22 Histidine

  32. Classification Transferases • Enzyme commission • Six classes • 1st Main class 1.Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases • 2nd Sub class • 3rd Sub subclass • 4th molecule Lyase C - C C-COOH Eg: Histidine carboxylyase: EC. 4.1.1.22 Histidine

  33. Classification Hydrolases • Enzyme commission • Six classes • 1st Main class 1.Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases • 2nd Sub class • 3rd Sub subclass • 4th molecule Lyase C - C C-COOH Eg: Histidine carboxylyase: EC. 4.1.1.22 Histidine

  34. Classification Lyases • Enzyme commission • Six classes • 1st Main class 1.Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases • 2nd Sub class • 3rd Sub subclass • 4th molecule Lyase C - C C-COOH Eg: Histidine carboxylyase: EC. 4.1.1.22 Histidine

  35. Classification Isomerases • Enzyme commission • Six classes • 1st Main class 1.Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases • 2nd Sub class • 3rd Sub subclass • 4th molecule Lyase C - C C-COOH Eg: Histidine carboxylyase: EC. 4.1.1.22 Histidine

  36. Classification Ligases • Enzyme commission • Six classes • 1st Main class 1.Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases • 2nd Sub class • 3rd Sub subclass • 4th molecule Lyase C - C C-COOH Eg: Histidine carboxylyase: EC. 4.1.1.22 Histidine

  37. Biotransformation Inhibition Properties Kinetics Enzymes Reactors Modifications Classification Mechanisms Kinetics Applications Biocatalysis Immobilisation

  38. Enzyme Kinetics • What is the rate of an enzymatic reaction? • What factors influence this rate? • Enzyme concentration • Substrate and product concentration • Inhibitor/activator • pH • Temperature • Ionic strength

  39. Enzyme concentration Rate 4x v v 2x 1x Time [E] Enzyme concentration

  40. Vmax 0.5 Vmax Km Michaelis Menten and Lineweaver Burk kinetics

  41. v = [Vmax(S)] Lineweaver Burk equation Michaelis-Menten equation Km + (S)] 1/v = [Km/Vmax](1/S) + 1/Vmax Vmax is aconstant. It is themaximal rate of the reaction. Km is a measure of the affinity of enzyme for the substrate Small Km means tight binding High Km means weak binding

  42. The turnover number, kcat A measure of catalytic activity Number of substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate. kcat = Vmax/Et [Et = enzyme concentration] Values of kcat range from less than 1/sec to many millions per sec The catalytic efficiency, kcat/Km An estimate of "how perfect" is an the enzyme.

  43. Bio-Engineering Professor Chris G Whiteley Visiting Professor of Enzymology Department of Biochemistry, Microbiology & Biotechnology Rhodes University, Grahamstown, SOUTH AFRICA

  44. Biotransformation Inhibition Properties Kinetics Enzymes Immobilisation Reactors Modifications Classification Mechanisms Applications Biocatalysis Immobilisation

  45. Enzyme Immobilisation • covalently bound • loosely associated • encapsulated behind a semi-permeable membrane • ‘trapped’ within extrapolymeric substances [EPS] • Advantages: • increased stability to temperature and pH • not effected by products or other pollutants • not ‘flushed away’ • Disadvantages • Distorting enzyme within constrained conformation • Partitioning of the enzyme within the support • If substrates or products are insoluble.

  46. E Enzyme non-covalently adsorbed to an insoluble particle Ion exchangers; porous carbon; clays; hydrous metal oxides; polymeric aromatic resins E E Insoluble particle E Enzyme covalently attached to an insoluble particle Sepharose (CNBr); chloro- Formates; carbodiimides; glutaraldehydee E E Martin Chaplin; London University: South Bank

  47. E Enzyme non-covalently adsorbed to an insoluble particle Ion exchangers; porous carbon; clays; hydrous metal oxides; Polymeric aromatic resins E E Insoluble particle E Enzyme covalently attached to an insoluble particle Sepharose (CNBr); chloro- formates; carbodiimides; glutaraldehydee E E

  48. Enzyme entrapped within an insoluble particle by a porous cross-linked polymer E E Gels; fibres E Cross-linked polymer Enzyme confined within a semipermeable membrane. E Allow substrate in and product Out but impermeable to enzyme. E E Semipermeable membrane

  49. Enzyme entrapped within an insoluble particle by a Porous cross-linked polymer E E Gels; fibres E Cross-linked polymer Enzyme confined within a semipermeable membrane. E Allow substrate in and product out but impermeable to enzyme. E E Semipermeable membrane

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