Microbial Biotechnology (408 AMB) PDF

Microbial Biotechnology (408 AMB) Dr. Aya Samir  What is the BIOTECHNOLOGY The United Nations Convention on Biological Diversity defines biotechnology as: "Any technological application that uses biological systems or living organisms to make or modify products or processes for spec...

Microbial Biotechnology (408 AMB) Dr. Aya Samir  What is the BIOTECHNOLOGY The United Nations Convention on Biological Diversity defines biotechnology as: "Any technological application that uses biological systems or living organisms to make or modify products or processes for specific use.“ Biotechnology is any technique that uses living organisms or substances from those organisms: to make or modify a product or processes, to improve plants or animals, to develop microorganisms for specific uses.  What is the BIOTECHNOLOGY Biotechnology is the application of scientific and engineering principles to the possessing of raw materials by biological agents to provide commercial products and services.  Raw Organic materials Materials Inorganic materials Microorganisms Biological Microbial products Agents Animal cells Plant cells  Disciplines of biotechnology Red Green Biotechnology Biotechnology White Blue Biotechnology Biotechnology Disciplines of biotechnology  MICROBIAL BIOTECHNOLOGY Application of scientific and engineering principles to the possessing of raw materials by microorganisms or microbial substances to provide commercial products and services. The microbial products are classified according to the industrial sectors to 6 groups Area of Application application Enzymes, polysaccharides, organic Chemical sector solvents, organic acids, …. etc Antibiotics, therapeutic enzymes, Pharmaceutical vaccines, antibodies, human and health hormones, biosensors, gene sector therapy,……etc. Energy sector Ethanol, methane (biogas) Food sector Baker's yeast, food additives, beverages, single cell protein  Area of Application application Microbial pesticides, microbial fertilizers, tissue culture, transfer of nif-gene for nitrogen fixation from Agricultural legumes to cereals, increasing milk, sector meat and egg production from farm animals, veterinary vaccines, production of animal feed from single cell protein Environmental Removing heavy metals from water, sector biodegradation of oil, plastics, ….. etc.  Antimicrobial Agents Chemical compounds biosynthetically or synthetically produced to Kill or inhibit the growth or metabolism of the microorganisms without serious toxicity to the host. Among the antimicrobial agents are antibacterial drugs, antiviral agents, antifungal agents, and antiparasitic drugs.  Antibiotics Definition: The antibiotics are secondary metabolites produced by some microorganisms to kill or inhibit the growth of other microorganisms even when used with low concentration. Antibiotic - producing microorganisms: Bacteria Actinomycetes Fungi Classification of antibiotics: The antibiotics are classified according to: Antimicrobial Chemical spectrum structure Manner of Producer Mode of biosynthesis strain action  Antimicrobial Spectrum What is the antimicrobial spectrum? The range of microbial species susceptible to specific antimicrobial agent.  Antimicrobial Spectrum According to the antimicrobial spectrum, the antibiotics are classified into 2 groups: A: Broad spectrum B: Narrow spectrum antibiotics antibiotics The antibiotic is The antibiotic is active against a active against a wide range of narrow range of microorganisms microorganisms Antimicrobial Spectrum The spectra of activity may change with acquisition of resistance genes Narrow spectrum Broad spectrum antibiotic antibiotic  Antibiotics Mode of Action The major actions of antibiotics include: 1. Inhibition of cell wall synthesis. Chemical structure of Chemical structure of peptidoglycan in G+ peptidoglycan in G- bacteria bacteria  Antibiotics Mode of Action The major actions of antibiotics include: 1. Inhibition of cell wall synthesis. In order for bacteria to increase their size following binary fission, the following actions must be completed: breaking down links in the peptidoglycan. inserting new peptidoglycan monomers. reformation of the peptide cross links.  Antibiotics Mode of Action The following sequence of steps occur: Step 1. Bacterial enzymes called autolysins are synthesized to: Lysis of the glycoside bonds between the peptidoglycan monomers. Lysis of the peptide cross-bridges. Step 2. The peptidoglycan monomers are synthesized in the cytoplasm and bind to bactoprenol to transport them across the cytoplasmic membrane and interact with transglycosidase enzyme to insert the monomers into existing peptidoglycan.  Antibiotics Mode of Action Step 3. Transglycosylase (transglycosidase) enzymes insert and link new peptidoglycan monomers into the breaks in the peptidoglycan. Step 4. Finally, transpeptidase enzymes reform the peptide cross-links between the glycan chains of peptidoglycan to make the wall strong.  Antibiotics Mode of Actions The major actions of antibiotics include: 1. Inhibition of cell wall synthesis. Preventing the transpeptidation reaction Binding the antibiotics to transpeptidase enzymes. Binding the antibiotics directly to the terminal D-alanyl-D-alanine peptide on the peptidoglycan precursors. Binding the antimicrobial agent to Interfering with the transpeptidase enzyme leads to action of transglycosidase Autolysin enzyme Preventing releasing transpeptidation Degrading the existing Preventing the Preventing cross- cell wall insertion of new linkage of the peptidoglycan two glycan linked monomers peptide chains Weak and self- degrading cell wall Mycoplasma is resistant Archaea are resistant to these antibiotics to these antibiotics it does not have cell they have pseudo - wall peptidoglycan in the cell wall Unlike bacteria, archaea lack peptidoglycan in their cell * wall but have pseudopeptidoglycan, which * is different in the chemical structure. It lacks D-amino acids It lacks N-acetylmuramic acid. * N-acetylglucoseamine binds N- acetyltalosaminuronic acid * through β-1,3 glycosidic bond. Cell wall of archaea provides both chemical and physical protection, and prevent macromolecules from contacting the cell membrane. 2. Destruction of the cell membrane Some antibiotics cause formation of pores or channels across the cell membrane. Permeabilization of the cell membrane. The transport into and out of the cell is impaired.  3. Inhibition of protein synthesis Binding to the Binding to the 30s 50s ribosomal ribosomal subunit subunit Blocking the attachment Misreading of Interference of tRNA mRNA with binding of amino acids to the protein 4. Inhibition of nucleic acids synthesis. Some antibiotics can inhibit any of the steps of DNA or RNA replication. 5. Competitive inhibition. Competitive inhibition is a form of enzyme inhibition in which binding the inhibitor to the active site or allosteric site on the enzyme prevents binding the substrate. The antibiotic structure is similar to the enzyme substrate, so the antibiotic can compete with the substrate for the active site of the enzyme and inhibits the synthesis of the end product.  Competitive inhibitors could bind to an allosteric site of the enzyme and prevent substrate binding.  Although there are a number of different antibiotics, they work in one of two ways: Bactericidal antibiotics that kill the bacteria. The action of bactericidal antibiotics are irreversible so once sensitive cells are exposed to a bactericidal antibiotic, they die. A bactericidal antibiotics usually either interferes with the formation of the bacterial cell wall or its cell contents. Bacteriostatic antibiotics that inhibit the bacterial growth and reproduction. Upon exposure to a bacteriostatic antibiotic, cells in a susceptible population stop dividing. However if the agent is removed, the cells once again multiply. Bacteriostatic agent Bactericidal agent Some drugs that are bacteriostatic at lower concentrations can be bactericidal at higher concentrations.  Isolation of Antimicrobials-producing Microorganisms It is used to isolate antimicrobials- Crowded producing microorganisms without plate regarding to what type of the method microorganisms will be sensitive to the antimicrobial compound. The test microorganism is used as Spot-on- an indicator for presence of the lawn antimicrobial activity. method  Crowded Plate Method The aseptic conditions must be considered 1.Preparing serial decimal dilutions. 2.Transferring 1 ml of each selected dilution to 1 plate. 1g 1 ml 1 ml 1 ml 1 ml Soil sample 1 ml 1:10 1:100 1:1000 1:10000 1:100000 3. Melting the nutrient agar medium at 100°c using a water bath. 4. Cooling the melted medium to 50°c. 5. Pouring the medium into the plates. 6. Mixing the medium and inoculum by a circular movement of the plates. 7. Allowing the plates to set. 8. Incubating the plates in the inverted position at 30 °C / 24-48h. After the incubation: The colonies which produce antimicrobial compound are indicated by an area of agar around the colonies that is free of any microbial growth (zone of inhibition). Such colonies are subcultured and purified by streaking method. The purified culture is ready for testing what type of the microorganisms will be sensitive to the antimicrobial compound. Disadvantages of this method: 1. This method is used to isolate an antibiotic – producing microorganism without regarding to what type of the microorganisms will be sensitive to the antibiotic, and it is important to isolate an antibiotic – producing microorganism with activity against specific microorganism not against unknown microorganism. 2. This method does not necessarily select the antibiotic – producing microorganisms because the inhibition zone may be attributed to other causes as rapid utilization of nutrients by some microorganisms causing the growth inhibition of nearby microorganisms.  Spot-on-Lawn Method The aseptic conditions must be considered 1. Melting the nutrient agar medium at 100°c. 2. Cooling the melted medium to 50°c. 3. Pouring the medium into the plates. 4. Allowing the plates to set. 5. Preparing serial decimal dilutions. 6. Spreading 1 ml of the selected dilutions on the surface of nutrient agar medium. 7. Incubating the plates at 30 °C / 24-48 h. 8. Pouring the nutrient agar medium inoculated with the test microorganism into the plates as an overlay. 9. The plates are further incubated at the optimum temperature for the test microorganism / 24-48 h. 10. The antimicrobial activity is indicated by the zone of the inhibited growth of the indicator microorganism around the colony that produce antimicrobial compound.