Lecture 4. Applications Biotechnology and Genetic Engineering PDF

Walaa A. Eraqi, PhD Department of Microbiology and Immunology [email protected] Biotechnology and Genetic Engineering Categories of products in biotechnology 1. Enzyme technology 2. Acids 3. Biopolymers 4. Biomass 5. Single Cell Proteins 6. Vitamins 7. Amino acids 8. Biotransfor...

Walaa A. Eraqi, PhD Department of Microbiology and Immunology [email protected] Biotechnology and Genetic Engineering Categories of products in biotechnology 1. Enzyme technology 2. Acids 3. Biopolymers 4. Biomass 5. Single Cell Proteins 6. Vitamins 7. Amino acids 8. Biotransformation 9. Antibiotics 10. Immunologic products 11. Gene Therapy/ Stem Cell technology 12. Gene expression 13. Bioremediation 14. Biotechnology and Mining 15. Bioterrorism Secondary metabolites Antibiotics  Antibiotics Antibiotics: natural product with small molecular weight that inhibit or kill microorganisms, selectively at low concentrations. Penicillin was first discovered by Alexander Fleming in 1929 as a product from Penicillium notatum (contaminating a bacterial culture and producing inhibition zones around the fungal growth). Antibiotics could be: Antibiotic Producing microorganism ❑Natural ❑Synthetic Bacitracin Bacillus lichenformis ❑Semi-synthetic Polymyxin Bacillus polymyxa Spectinomycin Streptomyes spectabilis Kanamycins A, B & C Streptomyces kanamyceticus  Penicillin Penicillin is an acylated 6-aminopenicillanic acid (6-APA). Biosynthesis of penicillin depends on the condensation of valine and cysteine amino acids to yield 6APA and L--aminoadipate as side chain, which is then replaced by various side chains during the fermentation. If the fermentation of penicillin is conducted without the addition of side chain precursors, a mixture of natural penicillins are produced. 6 APA Penicillin Only benzyl penicillin (penicillin G) of this mixture is therapeutically effective and all the other produced penicillins are by-products that create problems during the down-stream processing. However, if phenyl-alanine or phenyl acetic acid are added as a side-chain precursor, so that only the desired penicillin G is produced. Penicillin G Penicillin Penicillin was originally produced by a strain of Penicillium notatum, which produced pigments (Surface culture). The yield of penicillin increased by the use of the non pigmented Penicillium chrysogenum mutants (Submerged fermentation). Culture medium: Glucose (or molasses), Lactose (slowly utilizable sugar), Corn steep liquor (contain phenyl alanine), Phenyl acetic acid, CaCO3 and K2HPO4 buffers (Continuous feed). Penicillium notatum Penicillium chrysogenum  Phenyl alanine phenyl acetic acid (add here) Growth Glucose utilization pH change Lactose utilization Penicillin production Time  Phases of penicillin fermentation Phase I- 36 h (Growth phase) Glucose is utilized with acid production → pH 4.0 Then increase in pH 7.5 due to utilization of proteins in corn steep liquor producing NH3. At the end of this phase, phenylacetic acid should be added. Phase II- 36-75h (Production phase) Lactose is slowly utilized by the fungus so that pH remains constant at 7.5. During this phase, the fungal growth stopped, and penicillin is produced in large amount. Penicillin is recovered at the end of this phase. Phase III After 75 h, the fungal cells are autolysed with the production of NH3 which rise pH, destruction of penicillin. Recovery of penicillin Remove mycelia by filtration (rotating vacuum filter). The fungal biomass, dried and used as animal feed supplement. The filtrate is cooled to 2-3°C, then adjusted to pH 2 to liberate the free acid. Penicillin is extracted by organic solvent from the filtrate and then extracted back into the aqueous solution after adjusting the pH. Potassium ions are added to obtain crystalline potassium salt of penicillin G. Crystalline potassium salt is removed by filtration (or centrifugation) pure. Modification of penicillin Modification by: removing their natural acyl group, leaving 6-amino penicillanic acid (6- APA) to which other acyl group can be added to confer new properties. These semisynthetic penicillins such as methicillin, carbenicillin, oxacillin and ampicillin. Semisynthetic exhibit various improvement including: ❑Resistance to acids (Oral administration) ❑Degree of resistance to ß-lactamase ❑Broad spectrum of antibacterial activity. Biosensors Biosensors A sensor that integrates a biological element with a physiochemical transducer to produce an electronic signal proportional to a single analyte which is then conveyed to a detector. Biosensors main components A. Analyte Example of Biosensor: Glucose measuring device The electrodes contain glucose oxidase immobilized over a platinum oxygen electrode. Glucose oxidase oxidizes glucose producing hydrogen peroxide detected by the electrode B. Biological element (Bio-element) The component used to bind the target molecule (analyte), it must be: ❑Highly specific ❑Stable under storage conditions ❑Immobilized How do they specifically recognize the analyte? Recognition (Detection)  C. Transducer (Signal Detector) Acts as an interface, measuring the physical change that occurs with the reaction at the bioreceptor then transforming that energy into measurable electric output that conveyed to the detector.  Basic characteristics of Biosensors 1. Linearity: Linearity of the sensor should be high for the detection of high substrate concentration. 2. Sensitivity: Value of the electrode response per substrate concentration. 3. Selectivity: Chemicals Interference must be minimized for obtaining the correct result. 4. Response Time: Response time refers to the duration required for the biosensor to achieve 95% of its maximum response. A shorter response time is desirable for real-time monitoring and rapid detection of analytes. Applications of biosensors: Glucose Biosensor Analyte (substrate): Glucose in patient blood Enzyme: Glucose oxidase enzyme Transducer: Platinum oxygen electrode Signal: Electric current Detector: Electric current detector Applications of biosensors: Wearable biosensors (Ex. Ring Sensor) They are wearable They rely on wireless The data recorded monitoring devices sensors enclosed in using these systems that allow continuous items that can be are then processed to monitoring of worn, such as ring or detect the patients' physiological signals. watch. clinical situations. Wearable biosensors: Ring Sensor It is a pulse oximetry, based on the concept of photoconductor. Monitor heart rate and oxygen saturation. Principle: 1. Light source: Light is emitted by LED (that emits both red and infrared light) and transmitted through the artery. 2. Light absorption: The emitted light is passed through a body part, usually a fingertip, or toe. Blood vessels under the skin contain oxygenated and deoxygenated blood, which absorb light differently. ❑Oxygenated Blood: It absorbs less red light and more infrared light. ❑Deoxygenated Blood: It absorbs more red light and less infrared light. Wearable biosensors: Ring Sensor By measuring the amount of each type of light that passes through the body part, the pulse oximeter can determine the oxygen saturation level in the blood. 3. Photoconductor (transducer) & Photodetector : Measures the amount of light that passes through the tissue. It converts the variations in light intensity caused by the pulsatile flow of blood (i.e., the pulse) into an electrical signal Biofuel Cells  Biofuel cell Bioelectrochemical systems or devices that generate electric power by exploiting naturally occurring catalytic or metabolic processes of enzymes, nano enzymes, or even whole cells. Convert biochemical energy to electrical energy. Biofuel: simple high-energy substrates such as glucose, fructose, and saccharose to complex organic molecules. Uses: Generate electric power from pure substrates Help treat wastewater by simultaneously producing power and reducing organic waste.  Biofuel cell ❑Enzymatic biofuel cells: use enzymes for energy conversion from substrate stored to electric power (selectivity towards the substrate). ❑Nonenzymatic biofuel cells: where nanomaterials with catalytic properties which mimic natural enzymes are used to produce energy (high stability, and extended lifetime). ❑Microbial biofuel cells: are driven by microorganisms. The whole metabolic process to be used for energy generation. Organic toxin para-aminophenol is an excellent fuel for the Scedosporium dehoogii. Used to reduce this toxicity in wastewater (Bioremediation). Shewanella loihica reduces metals. Used to decrease chromium pollution by the production of chromium nanoparticles (Bioremediation). Production of vaccines  Production of vaccines Highly controlled operating conditions and strict GMP. It usually involves the growth of bacterial cultures in sophisticated high-grade fermenter. Bioreactor internal pressure should not exceed the atmospheric pressure to reduce risk of leakage. Exhaust gases from fermenters must pass through sterilizing filters, incinerator or both. Types of vaccines: ❑Whole cells (live or inactivated) ❑Surface antigens ❑Toxoids  Production of vaccines Inactivated whole cell vaccines: cell inactivation by heat treatment or by addition of formaldehyde. The microbial cells, inactivated or live, are then separated by centrifugation, then downstream purification (Aim to maximize cell growth). Excreted toxins and loosely bounded surface antigens are purified from the culture broth and the harvested cells must be safely discarded (Aim to maximize toxins and antigens). Toxoids: are inactivated toxins either by heat treatment or by the addition of formaldehyde to produce, which possess antigenic activity without toxicity. THANK YOU