The course aims at providing students with advanced knowledge in the field of biocatalysis and new-frontiers application of these abilities to solve biotechnological problems as well as to design and synthesize new molecules.
1. Introduction to the course and general information on biocatalysis: General information on the concepts of bio-resources and eco-sustainability; Main characteristics of enzymes; Cofactors and coenzymes; Nomenclature and classification of enzymes; Isoenzymes, multienzyme systems and complexes.
2. Principles of thermodynamics and kinetics of chemical reactions: Bioenergetics and Thermodynamics; Activation energy and transition state theory; Kinetics of chemical reactions; Rate and order of reactions; Collision theory and the concept of fruitful collisions (Arrhenius equation); Factors that influence the reaction rate.
3. Principles of enzyme catalysis and kinetics: Catalytic power and binding energy; Examples of enzyme catalysis; Determination and meaning of the initial velocity (v0); Michaelis-Menten equation; The meaning of the parameters of the M-M equation: Initial velocity (v0), maximum velocity (Vmax), constant of M-M (Km), number of turnover (kcat) and specificity constant (kcat / Km); Enzyme reactions with multiple substrates.
4. Mechanisms of reaction and regulation of the enzyme activity: Mechanism of reaction of chymotrypsin and application of pre-stationary state kinetics; Effect of pH on enzyme activity; Applications of the pH effect to study reaction mechanisms; Example of reaction mechanisms (aspartyl protease, hexokinase, enolase, lysozyme); Regulatory enzymes (allosteric enzymes), reversible covalent modifications and proteolysis (zymogens)
5. Linearizations of the M-M equation and enzymatic inhibition: Linear representations of the M-M equation: Lineweaver – Burk graphs (of the reciprocal doubles), Eadie – Hofstee and Hanes; Enzyme inhibition (reversible vs irreversible); Chemical agents that modify the enzymes irreversibly; Irreversible inhibitors: directed to the active site (Affinity Labels), suicide substrates (trojan horse) and Transition state analogues (Tight-Binding Inhibitors)
6. Effect of pH and temperature on enzymatic activity: pH dependence of reactions catalyzed by enzymes; Measuring the enzyme activity as a function of pH; Effect of pH on enzymatic activity in the presence of one or two ionizable groups; Dependence of kinetic constants as a function of pH; Van't Hoff and Arrhenius equations; How temperature-dependance activity and thermostability of enzymes are measured; The importance of the thermal stability for industrial applications.
7. Practical aspects of the study of enzymatic kinetics: Study of progression curves; Determination of initial velocity and enzyme units; Limitations on the measurement of the initial speed (dead time and stopped assays); Factors that influence the determination of enzyme activity (solvents, ionic strength, pH and temperature); Enzyme stability and storage methods.
8. Methods to measure enzyme activity (enzyme assays): UV/visible spectroscopy; spectrofluorimetry; Luminescence; Radioactivity; Direct and indirect enzyme assays; Coupled enzyme assays; Enzyme assays for diagnostics; Enzyme immunoassays; Continuous and discontinuous enzymatic assays.
9. Protein engineering and enzyme reactions in unconventional media: Protein engineering (principles and definitions); Chemical modifications; Genetic modifications: Rational design versus Directed evolution; Features of the screening methods;
-Rational design techniques: PCR with MegaPrimer, Whole plasmid PCR, Cassette mutagenesis, the Kunkel method, the QuikChange site-direct mutagenesis method;
- Directed evolution techniques: Error-Prone PCR, MEGAWHOP, Gene Assembly Mutagenesis, Mutator strains, random oligonucleotide-mediated mutagenesis, DNA Shuffling, DNA recombination by random priming, Staggered Extension Process, in vitro recombination methods (RACHITT, ITCHY, SCRATCHY, SHIPREC);
-Semi-rational design techniques: Structure-based combinatorial protein engineering (SCOPE) and SCHEMA structure-guided recombination;
- Enzyme reactions in unconventional mediums (medium engineering); enzymatic reactions in organic solvents; biphasic systems, co-solvents and pure organic solvents; other unconventional media (ionic liquids, supercritical fluids and eutectic mixtures).
10. Homogeneous and heterogeneous enzyme catalysis: Areas of application of homogeneous catalysis; Multienzyme systems (linear, parallel, orthogonal and cyclic enzyme cascades); Practical examples of enzyme cascades (production of perfumed chemical compounds, non-natural amino acids, plastic precursors, di-substituted pyrrolidine, D-phenylalanine derivatives); Heterogeneous catalysis (immobilised enzymes); Advantages and disadvantages of immobilisation; Supports for immobilisation; Immobilisation strategies (covalent, adsorption, entrapment and encapsulation); Carrier-less Immobilisation (CLECs and CLEAs); New frontiers of enzyme immobilisation: viral supports and nanoreactors.
11. Biocatalysis applied to plastics: Physicochemical properties that influence the degradability of plastics; Enzymes that hydrolyse plastic polymers (PET hydrolytic enzymes (PHEs)); Protein engineering to optimise PHEs; Ideonella sakaiensis as a model organism that metabolises PET plastic; Specific enzymes for the hydrolysis of PET (PETase); Lactic acid production from lignocellulosic biomass as a precursor to PLA bioplastics; use of lactic acid bacteria for the production of bioplastic; Pre-adaptation strategy to optimise lactic acid production.
12. The CRISPR-Cas system (principles and applications): CRISPR loci and cas genes; Mechanism of the CRISPR-Cas system; The three main types of CRISPR/Cas systems; Insights on the CRISPR/Cas9 system; Application of the CRISPR/Cas9 system for genome editing and protein engineering; Variants of the Cas9 endonuclease and their applications; the CRISPR/Cas9 system as a library screening tool for mutants generated by directed evolution.
13. Cytochromes P450, a family of promiscuous catalysts: Main features of cytochromes P450; The importance of catalytic promiscuity for the evolution of new enzymes; Types of reactions catalysed by cytochromes P450; Advantages and disadvantages of the use of P450 cytochromes; Main classes of cytochromes P450; The catalytic cycle of cytochromes P450; Uncoupling reactions; Engineering of cytochrome P450BM3 for hydroxylation of alkanes; Development of non-natural reactivity for the cycle-propanation of alkenes; The importance of axial ligand in cytochromes P450 for the development of non-natural reactivities.
During the course the lecturer will provide materials (book chapters, scientific articles and slides), which will be made available through the Moodle platform.
The topics shown are only indicative of the course's contents and may be subject to change by the teacher.
|Hans Bisswanger||Practical Enzymology||Wiley-Blackwell||2011|
The final assessment aims to verify the achievement of the "Learning Outcomes" related to the topics reported in the "Course syllabus".
The final assessment consists of an oral dissertation aimed at ascertaining both that the student has acquired the knowledge defined in the course syllabus as well as the ability to perform the required logical-deductive links. In particular, the completeness of the exposition, the level of integration between the various course topics as well as the scientific appropriateness of the language will be evaluated.
Furthermore, the achievement by the student of a global vision of the topics addressed in class (combined with their critical application), the ability to make connections and the use of an appropriate scientific language will be assessed with a mark of excellence.
The final assessment follows the same guidelines for both attending and non-attending students.