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Biocatalysis

Biocatalysis. Discussion. This Week Agenda Today: Biocatalysis Wednesday: Biodiversity Final Paper Discussions Exam 4 – Monday, October 27. What are limitations of enzyme catalysis? What could be done to overcome these limitations?. Methods of enzyme immobilization.

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Biocatalysis

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  1. Biocatalysis

  2. Discussion This Week Agenda Today: Biocatalysis Wednesday: Biodiversity Final Paper Discussions Exam 4 – Monday, October 27 What are limitations of enzyme catalysis? What could be done to overcome these limitations?

  3. Methods of enzyme immobilization Adsorption: Suitable adsorbents: ion-exchange matrices, porous carbon, clays, hydrous metal oxides, glasses and polymeric aromatic resins Covalent binding: cyanogen bromide on sepharose, ethyl chloroformate on cellulose, carbodiimodeon various COO- matrices, glutaroaldehyde, 3-aminopropyltrioxysilane Entrapment: fibers and gels Membrane confinement: calcium alginate gel http://www1.lsbu.ac.uk/water/enztech/immethod.html

  4. Generalized comparison of different enzyme immobilization techniques

  5. Covalent Binding

  6. Immobilization of enzymes: Cross inking with glutaraldehyde • 1) Improves various aspects of stability: • - Heat • pH • Exogenous proteases • Organic solvents • 2) Elongates enzyme activity • 3) Reuse of an enzyme • 4) Increases final productivity

  7. ENCAPSULATION OF ENZYMES

  8. Use of Enzymes in organic solvents • Advantages: • Water-sensitive/ water-insoluble compounds could be used • Thermal stability is improved • Water-dependent side reactions are suppressed (including hydrolysis) • Problems: • Proteins are usually insoluble in organic solvents (or denaturated) • Hydrogen bonding and 3D structure of enzyme could be altered • Solvent could be active site inhibitor • Rates drop dramatically • Solvents used: • Tetrahydrofuran, DMSO, sc CO2, acetonitrile, hexane, toluene, methanol, ionic liquids • Monomolecular layer of water should be present on enzyme surface!

  9. Extremophiles and extremozymes Extremophiles - microorganisms, typically Archarea, populating extreme habitats Extreme conditions → Extreme enzymes High-temperature stable enzymes Food preparation: separation step is not required for sterilization Cold-temperature stable enzyme Food preparation: tenderization of meat Detergents used in cold water

  10. Structural features of hyperthermophilic enzymes • Lower surface area/volume ratio • High number of salt bridges • Tungstopterin cofactor Tungstopterin Aldehydeferredoxinoxidoreductase

  11. Catalytic antibodies (ABZYMES) A hapten is a small molecule that can elicit an immune response only when attached to a large carrier such as a protein; the carrier http://nptel.ac.in/courses/104103018/module3/lec8/images/3.png

  12. The range of biocatalysis • Oxidation and Reduction • Preparation and Hydrolysis of Esters • Preparation of Acids, Amides, and Nitriles • Compounds with Two or More Hydroxyl Groups • Preparation of Aromatic Compounds Are there any products traditionally synthesized from petroleum?

  13. Essentials Steps in DNA Cloning Cutting target DNA at precise locations. Sequence-specific endonucleases (restriction endonucleases) provide the necessary molecular scissors Selecting a small carrier molecule of DNA capable of self-replication. These DNAs are called cloning vectors (typically plasmids or viral DNAs). Joining two DNA fragments covalently. DNA ligase links the cloning vector and the DNA to be cloned. Resulting DNA – recombinant DNA. Moving recombinant DNA from the test tube to a host cell that will provide the enzymatic machinery for DNA replication. Selecting or identifying host cells that contain recombinant DNA. Recombinant DNA technology or genetic engineering E.coli is a hero of genetic engineering

  14. Cleavage of DNA by Restriction Endonucleases

  15. Cloning Vectors Allow Amplification of Inserted DNA Segments Plasmids (E.coli) 2. Bacterial artificial chromosomes (E.coli) 3. Vector from yeasts Plasmids-circular DNA, 5 kbp to 400 kbp long, replicates separately from host chromosome, usually symbionts Classic E. coli plasmid pBR322 constructed in 1977

  16. Cloned Genes Can Be Expressed to Amplify Protein Production • Alteration of the sequence around a cloned gene to trick protein overexpression • Transcription and translation sequences must be inserted in the vector DNA (expression vectors)

  17. Alteration of Cloned Genes Produces Altered Proteins Site-direct mutagenesis – replacement of individual amino acid Oligonucleotide-directed mutagenesis creates a change in specific DNA sequence Fusion protein – the product of a fused gene

  18. Probably the most controversial company http://rinf.com/alt-news/wp-content/uploads/2014/05/monsanto-620x330.jpg

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