Enzyme technology - part 2

Biotechnology & Genetic Engineering Lecture (5-2) Assi. Prof. Rajaa Al –Anbagi


Enzyme technology (production, purification and immobilization of enzymes)



Enzymes extraction and purification


In order to test the activity of the enzyme we must first of all have an assay:-
a quantitative method for measuring the conversion of substrate into product by two methods direct and indirect (Fig. 10). In some cases conversion of substrate to product can be monitored directly by ultraviolet (UV) spectroscopy,*if the substrate or product has a distinctive UV absorbance. Failing this, *a chromatographic method can be used to separate substrate from product and hence monitor conversion. In order to quantify a chromatographic assay a radioactive label ( radiochemical ) is usually required in the substrate, so that after separation from substrate the amount of product can be quantitated by scintillation counting. Such an assay is highly specific and highly sensitive, but unfortunately is rather tedious for kinetic work. A more convenient assay for kinetic purposes is to monitor consumption of a stoichiometric cofactor or cosubstrate, for example the cofactor nicotinamide adenine dinucleotide (NADH) by UV absorption at 340 nm, or consumption of oxygen by an oxygenase enzyme using an oxygen electrode. In other cases a coupled assay is used, in which the product of the reaction is immediately consumed by a second enzyme (or set of enzymes) which can be conveniently monitored.