|Introduction (3 hours)
Definition and history of enzyme and enzyme catalysis, prejudices and advantages of enzyme catalysis, chemo-, regio-, and enantioselectivity, biotransformation with isolated enzyme and whole cells, industrial application, enzyme assay; enzyme activity, whole cell activity, space-time-yield.
Enzyme classes and reactions (3 hours)
Enzyme classification (EC numbers), oxidoreductase, co-factor, transferase, hydrolase, lyase, isomerase, examples of enzymatic reactions.
Enzyme selection (3 hours)
Target reaction definition and selection, source of enzymes, enzyme or microorganism screening strategies, microtiter plate, high-throughput screening, detection methods, enantioselectivity assays.
Enzyme purification and characterization (3 hours)
Enzyme purification techniques and general principles: SEC, IEC, HIC, IMAC, affinity matrices, affinity tags, and fusion proteins, examples. Enzyme characterization methods: molecular mass, IEP, multimers, amino terminal sequence, structure determination.
Enzyme biochemistry and enantioselectivity (3 hours)
Michaelis-Menten kinetics, kinetic constants Kcat, Km, inhibition, enzyme selectivity, enantioselectivity, kinetic resolution vs dynamic resolution.
Enzyme engineering (5 hours)
Physicochemical classification of amino acids, replacement strategy, Dayhoff matrix, in vivo and in vitro mutagenesis (random, semi random), site directed mutagenesis, sequence comparisons and predictions, directed evolution, DNA shuffling, examples.
Biotransformation with whole cells (3 hours)
Cell growth, whole cells biotransformation, recombinant microorganisms, Multi-enzyme reactions, secondary metabolites, fermentation.
Biotransformation with enzymes (3 hours)
Cofactor regeneration systems (NADPH, NADH, ATP), coupled substrate approach, coupled enzyme approach, permeabilized cells, enzyme stabilization, immobilization technology, membrane reactors.
Biotransformation in organic solvent (4 hours)
Enzymes and solvents: solubility of enzymes, substrates and products, water activity. organic solvents: ‘Klibanov’ rules, reaction reversal, ‘memory’ effect. Ion-paired enzyme. Enzyme catalysis in ionic liquid.
Product recovery (3 hours)
Product recovery strategies, intracellular and extra-cellular compounds, disruption of cells, separation of cells, primary isolation (solvent extraction, sorption, adsorption, precipitation, membrane filtration, reverse osmosis, pervaporation, perstraction), further purification.
Process economics (3 hours)
Process design, flow sheets, calculations, stringent and relaxed parameters, process evaluation, quantitation, economics of a process, research choices based on process cost considerations.
Biotransformation in industry (3 hours)
Case studies based on operational industrial processes.