Enzymology and Experimental Biochemistry

The course comprises the theoretical and experimental work on enzymes, proteins and peptides in biological matrices. Identification of new industrial enzymes can be based on search for a specific enzymatic activity. In this course we simulate such a process in the laboratory. Focus is placed on purification of an enzyme from a complex protein mixture followed by characterization of the purified protein.

Topics included are:

• Extraction techniques
• Chromatographic systems
• Solvent and buffer relations
• Enzymatic assay design and development
• Thermodynamics and kinetics in enzymology and biochemical analyses.
• Binding between molecules in equilibrium reactions and thermodynamic stable complexes.
• Receptor and effector sites, activators and inhibitors, and their effects on the active sites.
• Native enzymes: holoenzymes, apoenzymes, co-factors, activation energy and kinetics

The combinations of such knowledge give the students basis for understanding and development of a relevant strategy of methods as used in analytical biochemistry.

Project

The students will perform an activity-based purification of individual enzymes from a complex protein sample originating from an Aspergillus strain. In groups they will design a purification strategy of the enzyme in question. The students will use ÄKTA FPLC-systems for the enzyme purification. 

Upon purification enzymatic properties of the enzyme must be determined.
These includes V_max, K_m and k_cat, and interpretation of such data in relation to ideal catalytic effects. Also, heat stability, pH optimum, analysis of potential inhibitors or competitors can be included in the project.

Digitalization

We will use the graphing program PRISM from GraphPad for the fitting and analysis of data. Bioinformatics tools will be employed to find information on selected enzymes through the CAZy database, the occurrence in other organisms through BLAST searches to qualify strategies for purification and characterization.