Teaching code

4S008292

Teacher

Salvatore Fusco

Coordinator

Salvatore Fusco

Credits

6

Language

Italian

Scientific Disciplinary Sector (SSD)

BIO/10 - BIOCHEMISTRY

Period

II semestre dal Mar 1, 2021 al Jun 11, 2021.

Lessons timetable Moodle Seminars 0

Learning outcomes

The course aims to provide the student with the basic and advanced knowledge of biocatalysis applied in the industrial, therapeutic and diagnostic fields. In particular, the main objectives of the course concern the understanding: 1) of the principles of enzyme catalysis; 2) the new frontiers in enzymes applications; 3) molecular methods to optimize the reactivity of enzymes; 4) innovative approaches for the development of artificial enzymes that show non-natural reactivity.

Program

The topics listed below are indicative of the syllabus content and may be subject to changes by the lecturer:

- Introduction to the course and general information on biocatalysis;

- Protein engineering for the optimization of biocatalysts: chemical modifications, site-directed metagenesis (rational design), random mutagenesis (directed evolution), semi-rational mutagenesis approaches, selection and screening methods, case studies (stability improvement: thermal and pH, catalytic efficiency, regio/stereo selectivity, etc.);

- Enzymes in unconventional media (medium engineering); enzyme reactions in organic solvents; biphasic systems, co-solvents and pure organic solvents; other unconventional media (ionic liquids, supercritical fluids and eutectic mixtures);

- Homogeneous enzyme catalysis: Multienzyme systems (linear, parallel, orthogonal and cyclic enzyme cascades); Practical examples of enzymatic cascades (production of non-natural amino acids, plastic precursors, di-substituted pyrrolidine, derivatives of D-phenylalanine, etc.);

- Heterogeneous enzymatic catalysis (immobilization): Advantages and disadvantages of immobilization; Supports for immobilization; Immobilization strategies (covalent, adsorption, entrapment and encapsulation); Immobilization without support (CLEC and CLEA); New frontiers of enzymatic immobilization: viral supports and nanoreactors;

-Biocatalysis applied to plastics: Physicochemical properties that influence the degradability of plastics; Enzymes that hydrolyse plastic polymers (PET hydrolytic enzymes (PHEs)); Protein engineering for optimizing PHEs; Ideonella sakaiensis as a model of bacterium that metabolizes PET plastic; Specific enzymes for the hydrolysis of PET (PETase);

- The CRISPR-Cas system principles and applications: CRISPR loci and cas genes; Operating 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 direct evolution;

- Cytochromes P450, a family of promiscuous catalysts: Characteristics of cytochromes P450; The importance of catalytic promiscuity for the evolution of new enzymes; Types of reactions catalyzed by cytochromes P450; Advantages and disadvantages of using cytochromes P450; Main classes of cytochromes P450; The catalytic cycle of cytochromes P450; Decoupling reactions; Engineering of cytochrome P450BM3 for hydroxylation of alkanes; Development of unnatural reactivity for the cyclo-propanation of alkenes; The importance of the axial ligand in cytochromes P450 for the development of unnatural reactivity.

Teaching material:
-Slides of the course (provided at the end of the lecture)
-Scientific articles (references cited during the lectures)

Examination Methods

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.