46675 - Protein Engineering

Learning outcomes

The aim of the Course is to get the Student acquainted with Enzymology and Protein Engineering. In particular, the lectures will provide to the Student: i) the general theory of enzyme kinetics and the molecular mechanisms of a selected group of enzymes; ii) principles and practice of Protein Engineering. Finally, the Student will be able to evaluate, both technically and economically, the engineering of enzymes for industrial applications.

Course contents

Kinetics of enzyme-catalyzed reactions. The Michaelis-Menten and the Briggs-Haldane model. Reaction order and its determination. Enzyme inhibitors: competitive, non-competitive, uncompetitive. Activity assays. Definitions of enzyme unit and the marketing of enzymes. Molecular mechanisms of selected enzymes: α-chymotrypsin, DNA polymerases, and lactate dehydrogenase. Rational design of artificial proteins. Principles and practice of rational protein engineering. Construction of enzyme variants featuring improved thermostability or altered catalytic performances. Entropy of unfolding and thermostability of enzymes. Introduction of artificial disulfide bonds and thermostability. Grafting of the seryl-proteases catalytic triad into target proteins and construction of artificial proteases. Multifunctional enzymes. Directed evolution of enzymes. Principles and practice of directed evolution. Generation and recombination of mutant libraries by means of mutagenic PCR, DNA shuffling, STEP (staggered extension process). Screening and selection strategies. Construction of thermostable enzymes by means of directed evolution. Bacterial mutator strains as a tool for the construction of random mutant libraries.

Readings/Bibliography

Daniel Purich, Enzyme kinetics: catalysis and control, 2010, Elsevier

Sheldon J. Park & Jennifer R. Cochran, Protein Engineering and design, 2009, CRC Press

Teaching methods

PowerPoint presentations.

Assessment methods

The Test is an oral colloquium. To the Student, 3 different questions are proposed, each one providing a maximum score equal to 10/30. The 3 different questions are related to, respectively, Enzymology, Protein Engineering, and to one topic selected by the Student. The scoring outcome is assessed by verifying the knowledge and critical analysis skills of the Student. In particular, the aim of the Discussion of a subject chosen by the Student is to ascertain the capability of autonomous analysis.

Office hours

See the website of Alejandro Hochkoeppler