Leksmono, CS;
Manzoni, C;
Tomkins, JE;
Lucchesi, W;
Cottrell, G;
Lewis, PA;
(2018)
Measuring Lactase Enzymatic Activity in the Teaching Lab.
JOVE-Journal Of Visualized Experiments
(138)
, Article e54377. 10.3791/54377.
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Abstract
Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 °C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.
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