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 composed of: i) a group of 24 questions, to which the Candidate has to provide one among three possible answers; ii) 2 questions to which the Candidate has to reply with a short dissertation. The value of the multiple-choice questions is equal to 24 points, and 6 points can be obtained from the two short dissertations.

Office hours

See the website of Alejandro Hochkoeppler