A quick method for the assessment of activity and inhibition of ®sh amylases I. FERNA Â NDEZ, M. DI Â AZ, T. MARTI Â NEZ & F.J. MOYANO Dpto. Biologõ Âa Aplicada, Escuela PoliteÂcnica Superior, Universidad de Almer  õa, La Can Äada de San Urbano, Spain Abstract A sensitive and quick method was developed to determine the presence of a-amylase in the gut of aquatic organisms, as well as its sensitivity to inhibitors. The assay is based on the utilization of Petri dishes ®lled with starch±agarose gel as a substrate for the enzyme solution, which is placed in small wells punched in the surface. Circular zones produced by the action of amylase remain colourless after staining with lugol. Pure commercial porcine amylase was used to ®t 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 detected 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 dierent sparid ®sh. The method was also used to evaluate the eect produced by a speci®c inhibitor on ®sh amylases, showing a linear response when the ratio inhibitor:enzyme (in units) changed from 20:1 to 2:1. Comparison of the cleared zones produced by amylases of sparid ®sh in the presence or absence of inhibitor, revealed dierences 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 feedstus used in ®sh feeding. KEY WORDS KEY WORDS: a-amylase, ®sh, inhibition, starch±agarose gel Received 4 October 1999, accepted 10 April 2000 Correspondence: F.J. Moyano, Dpto. Biologõ Âa Aplicada, Escuela PoliteÂc- nica Superior, Universidad de Almerõ Âa, La Can Äada de San Urbano, 04120 Almerõ Âa, Spain. E-mail: fjmoyano@ualm.es Introduction After the early works of Vonk (1937), a great number of papers have been focused to the study of amylase activity in fresh water and marine ®sh (Keddis 1957; Fish 1960; Ushiyama et al. 1965; Ugwumba 1993; Hidalgo et al. 1999). The secretion of amylase seems to be greatly determined by the species and the feeding habit. This enzyme is produced in signi®cant amounts in dierent parts of the digestive tract in herbivorous ®sh, being less abundant in carnivorous ®sh, where it is produced only in the pancreas (Nagayama & Saito 1968). A number of chemical methods are routinely used to determine activity of amylase in animal and plant extracts. Some of the more usual are those of Bernfeld (1955) and Somogy±Nelson, described by Robyt & Whelan (1968). Nevertheless, some of those methods are expensive and time consuming. Methods of radial diusion in agar gels are commonly used in immunological applications for the rapid determination of speci®c protein levels in large number of samples. Those methods may oer a useful and time-saving alternative to the spectrophotometric or titrimetric assays. The aim of the present paper was to propose a simple and quick method for both qualitative and quantitative assess- ment of amylase activity in non-puri®ed extracts obtained from aquacultured species. It is based on the utilization of a starch±agarose gel as a substrate for the enzyme, with the activity further revealed using lugol. The method may be useful for a rapid evaluation of the presence of amylase in ®sh digestives and for the evaluation of the eect produced by inhibitors present in plant ingredients. Such an informa- tion may be useful in the preliminary tests of raw materials used in the elaboration of ®sh feeds. Materials and methods Preparation of the starch ^agar plates A melted 5 agar (Panreac, Barcelona, Spain) solution (100 g L )1 in distilled water), was mixed with dierent amounts of soluble starch (Sigma, St. Louis, USA). 6 After mixing, 10 mL were quickly poured into sterile 8.5 cm diameter Petri dishes. Once the gel was solidi®ed, nine wells (4 mm diameter, 3 mm 19 Aquaculture Nutrition 2001 7 ; 19^23 .............................................................................................. .............................................................................................. Ó 2001 Blackwell Science Ltd