A quick method for the assessment of activity and inhibition of fish amylases

A sensitive and quick method was developed to determine the presence of alp ha -amylase in the gut of aquatic organisms, as well as its sensitivity to inhibitors. The assay is based on the utilization of Petri dishes filled wi th starch-agarose gel as a substrate for the enzyme solution, which is plac ed in small wells punched in the surface. Circular zones produced by the ac tion of amylase remain colourless after staining with lugol. Pure commercia l porcine amylase was used to fit the better conditions for developing the assay (1 g L-1 starch in the gels, 4 h of incubation). The diameter of the cleared zones were related to the activity of enzyme and the method detecte d linearly amylase activity in a range of 2-20 U well(-1) so it was used to reveal the presence of amylase in digestive extracts obtained from differe nt sparid fish. The method was also used to evaluate the effect produced by a specific inhibitor on fish amylases, showing a linear response when the ratio inhibitor:enzyme (in units) changed from 20:1 to 2:1. Comparison of t he cleared zones produced by amylases of sparid fish in the presence or abs ence of inhibitor, revealed differences in their sensitivity to inhibition, which ranged from 15 to 50% of total activity. The assay is proposed for a preliminary evaluation of possible inhibitors contained in feedstuffs used in fish feeding.