C8512 - Advanced Enzymatic Biocatalysis and Sustainable Technologies

Learning outcomes

Upon completion of the course, students will understand the mechanisms of enzymatic catalysis, immobilization techniques, and the stereochemical aspects of biocatalysis. They will be able to apply these skills to solving biotechnological problems and to the design and stereoselective synthesis of new molecules. Specifically, they will be able to: i) select biocatalysts for these syntheses based on criteria of economy, sustainability, and overall process cost-effectiveness; ii) identify potential critical aspects of the enzymatic process and propose solutions; iii) evaluate the feasibility of substituting biocatalytic methods for traditional synthesis technologies based on the principles of industrial process redevelopment. These skills are applicable to a variety of sectors, ranging from macroscale biomass production to energy production, the production of products for the pharmaceutical, food, and textile industries, and even the design of diagnostic tools.

Course contents

Lesson 1 (2h): Organization of lessons and laboratory experiences. Learning assessment methods. Introduction to the program topics. Basics of biocatalysis.

Lessons 2-3 (4h): Different types of selectivity: chemoselectivity, regioselectivity, stereoselectivity. Importance of enantiomeric purity in the pharmaceutical industry. Kinetic resolution of racemic mixtures. Conditions to shift the reaction equilibrium towards the desired product. Dynamic kinetic resolution. Desymmetrization of prochiral symmetric compounds. Generation of enantiopure molecules starting from prochiral systems

Lessons 4-5 (4h): Enzyme classification; Enzymes that require cofactors. Transaminases: mechanism of pyridoxal phosphate, multienzymatic reactions. Techniques to shift the reaction equilibrium and optimize the yield of the process. Industrial example: optimization of a mutated transaminase for sitagliptin synthesis developed by Codexis.

Lessons 6-7 (4h): Enzymes involved in redox reactions: Alcohol dehydrogenase/ketoreductase. Recycling of the cofactor, glucose dehydrogenase. Oxidases, active site and mechanism of catalysis of HRP. Colorimetric diagnostic methods that exploit HRP.

Lesson 8 (2h): Principles of green chemistry and methods for quantifying the eco-compatibility of a reaction. Evaluation of process redevelopment through the introduction of biocatalysts: comparison with classical chemistry protocols. Industrial examples: synthesis of pregabalin and atorvastatin. Biocatalytic retrosynthesis: combination of biocatalysis and classical catalysis.

Lesson 9-10-11 (6h): Immobilization of enzymes, choice of support and technique, immobilization by chelation with metals, by covalent bonding to the support or by cross-linking. Immobilization by entrapment in gels, liposomes or polymers. Biocatalysts immobilized in membrane reactors.

Lessons 12-15 (8 h): Reactions in non-conventional solvents. Cascade enzymatic processes. Coimmobilization of enzymes and cofactors, channeling effect, proximity and cosmotropic effects Coimmobilization of enzymes and cofactors on membranes in continuous flow reactors. Ribozymes, DNAzymes and aptamers. Biocatalysis controlled by self-assembly of oligonucleotides, ABzymes antibodies as catalysts

Lessons 16-20 (10 h): Biocatalysis in industry: biomass recovery, digestion of plant waste material, design of biorefineries for the circular economy; pharmaceutical industry, enzymes for the synthesis of complex molecules, mutation of enzymes for the production of stereodefined molecules; materials sciences, enzyme-induced polymerization, effect of reaction conditions on the chemical-physical characteristics of materials; in textile industry, in the production of detergents and in the food industry

Exercitations in lab (15h): Synthesis of 2-palmitoyl-glycerol: first step, lipase-catalyzed acylation of glycerol

Purification of 1,3-butanoyl-glycerol. Second step, non-catalyzed acylation with palmitic acid of position 2

Purification of 1,3-butanoyl-2-palmitoyl glycerol. Third step, lipase-catalyzed transesterification of the 1,3 positions

Purification of 2-palmitoyl glycerol and characterization

Readings/Bibliography

A. S. Bommarius, B. R. Riebel “Biocatalysis – Fundamentals and applications'” Wiley VCH editor.

K. Faber, “Biotransformations in Organic Chemistry” Springer editor.

R. Patel “Green Biocatalysis”, Wiley VCh Editor

J. A. Tao, R. Kazlauskas, “Biocatalysis For Green Chemistry And Chemical Process Development” John Wiley & Sons, Editor.

K. Drauz, H. Grger, O. May “Enzyme Catalysis in Organic Synthesis”, Wiley VCH editor.

Teaching methods

Lessons in class

Laboratory exercises at the CILDIC building in the month of January. Students will perform biocatalyzed experiments and laboratory attendance will be verified by signature.

In consideration of the types of activities and teaching methods adopted, attendance of this training activity requires all students of modules 1 and 2 to carry out e-learning mode [https://www.unibo.it/it/servizi-e-opportunita/salute-e-sistenza/salute-e-sicurezza/sicurezza-e-salute-nei-luoghi-di-studio-e-tirocinio] and to participate in module 3 of specific training on safety and health in places of study. Information on the dates and methods of attendance of module 3 can be consulted in the specific section of the study course website.

Assessment methods

The exam takes place through a written test lasting 1.5 hours, without the help of notes, books or computer aids. In particular, the student is asked to complete 25 multiple choice questions, each of which is associated with a value of 1 point (no penalty for incorrect or unanswered questions), and an open question which is associated with a value of 8 points. The outcome of the test is calculated as the sum of the score of the multiple-choice questions and the points obtained in the open question, which is evaluated based on the level of understanding, critical analysis and in-depth study of the subject that the student was able to carry out and the ability to describe the mechanisms discussed with diagrams and chemical reactions.

Students with DSA or temporary or permanent disabilities: it is recommended to contact the responsible University office in advance (https://site.unibo.it/studenti-con-disabilita-e-dsa/it): it will be their responsibility to propose any adaptations to the interested students, which must in any case be submitted, 15 days in advance, to the approval of the teacher, who will evaluate the opportunity also in relation to the educational objectives of the course.

Teaching tools

Power point presentations of the lessons are shared via the Virtuale OnLine portal

Office hours

See the website of Alessandra Tolomelli