Learning outcomes of the course unit
This course will provide methodological tools aimed at resolving heterogeneous mixtures of proteins and nucleic acids and qualitatively and quantitatively evaluating specific components of interest in food diagnostics, based on chromatographic and electrophoretic separation techniques, protein and DNA sequencing techniques and a variety of immunological and molecular biology techniques. Also described are the molecular biology techniques used to obtain transgenic organisms and recombinant proteins of interest to the agri-food sector. A particular attention is given to enzymes and their analytical and technological applications in food science and technology.
Course contents summary
Determination of amino acid composition and sequence of proteins. Determination of the amino acid composition of proteins. Fragmentation of proteins into peptides. Chromatographic resolution, sequencing (Edman degradation) and alignment of peptides. Protein identification through sequencing of N-terminus coupled with analysis of protein and DNA sequence databases. Determination and comparison of protein amino acid sequences through analysis of protein and DNA sequence databases.
Protein separation methods. Differentiated protein precipitation through modification of pH, ionic force and dielectric constant of the medium. Protein chromatographic separation techniques: hydrophobic interaction, ion exchange, dimensional exclusion and affinity/immunoaffinity chromatographies. Protein electrophoretic separation techniques: polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE), isoelectric focusing and two-dimensional electrophoresis.
Immunochemical methodologies. Overview of the biochemical basis of immune response and of the structural/functional properties of antibodies. Preparation of polyclonal and monoclonal antibodies. Utilisation of antibodies as analytical reagents: immunoprecipitation, immunodiffusion, immunoelectrophoresis, Western Blotting, immunoenzymatic tests (ELISA).
Molecular biology methodologies. Electrophoresis of nucleic acids. Enzymes used for DNA manipulation: DNA polymerases, nucleases, ligases. DNA sequencing: chemical degradation method and chain termination method; thermal cycle sequencing; use of fluorescent primers for automated sequencing. DNA fusion and renaturation. Qualitative and quantitative PCR (Polymerase chain reactions), RT-PCR and RAPD. Restriction enzymes and RFLP. Cloning of exogenous genes, transformation of host organisms and selection of recombinants. Overview of the methods used in the preparation of transgenic organisms and recombinant proteins for the agri-food sector. Bt corn and RoundUp ready soybean. Analytical methods for the determination of authorised and non-authorised GMOs in food products.
Enzymology and its applications. Enzyme mechanism of action: main factors of enzymatic catalysis. Enzyme kinetics in the stationary phase and determination of kcat, Km and kcat/Km. Enzyme technology. a) Use of enzymes for analytic purposes. b) Technological applications of enzymes in food industry. Production of sugars from starch degradation (amylases and glucose isomerase), dairy product technologies (chymosin, proteases, lipases, lactase, lysozyme), aspartame synthesis (thermolysin), resolution of D- and L-amino acids (aminoacylase). c) Enzyme inhibitors. Reversible and irreversible inhibitors. Pesticides as enzyme inhibitors. d) Enzyme denaturation and stabilisation. Mechanism of action of chemical-physical agents with denaturing activity. Analysis of "folding"/"unfolding" of proteins through the use of spectroscopic techniques. Preparation of stable enzymes: covalent and non-covalent immobilization. Enzymes from thermophilic microrganisms.
Biochemical basis of BSE.
Wilson K., Walker J. Metodologia Biochimica. Raffaello Cortina Editore
Ninfa A.J., Ballou D.P. Metodologie di base per la biochimica e la biotecnologia. Zanichelli Editore.
Classroom lectures with the use of multimedia equipment.
Assessment methods and criteria
Written exam followed by the discussion of written answers.