62 Indian J. Fish., 67(2): 62-68, 2020 DOI: 10.21077/ijf.2019.67.2.59187-09 Partial purifcation and characterisation of α-amylases from the digestive tract of the Indian major carp Labeo rohita (Hamilton, 1822) UMALATHA, N. SRIDHAR, JAIRAM PRASAD KUSHWAHA AND VADLAPUDI KUMAR * Regional Research Centre, ICAR-Central Institute of Freshwater Aquaculture, Hessaraghatta Lake Post Bangalore-560 089, Karnataka, India * Department of Biochemistry, Davangere, P. G. Centre, Kuvempu University, Shimoga, India e-mail:sridharcifa@yahoo.co.uk ABSTRACT Partial purifcation of α-amylases from the digestive tract of the Indian major carp Labeo rohita (Hamilton, 1822) through acetone fractionation and ion exchange chromatography (DEAE-SephadexA-50) resulted in 8-fold purifcation with 86% recovery. Characterisation of amylase activity revealed two pH optima at 4.5 and 6.5. Activity was stable over wide pH ranges of 3.5 to 4.5 and 7 to 12. Optimum incubation temperature was 35°C. The enzyme lost 91% activity at 60°C within 15 min and was inhibited by Amylase inhibitor Type-1 (wheat); 1, 10 Phenanthroline; Ethylene diamine tetra-acetate (EDTA) and Phenyl methyl sulphonyl fuoride (PMSF). Heavy metal ions Hg ++ and Cu ++ strongly inhibited the enzyme activity, while Zn ++ and Bi ++ inhibited to a lesser extent. Native polyacrylamide gel electrophoresis of the purifed α-amylase fractions revealed four bands, with corresponding molecular weights of 43.59; 52.36; 55.42 and 54.01 kDa. α-Amylase activity from L. rohita exhibited linear hydrolysis of starch upto 7% concentration in 60 min. Keywords: α-Amylase, Characterisation, Digestive tract, Labeo rohita, Purifcation Introduction The production of freshwater fsh in India was 5.29 million t during 2017-18 and about 33% of this production comprised the Indian major carp (IMC) Labeo rohita (Hamilton, 1822) which contributed 1.75 million t next to Catla catla (GOI, 2018). The rohu Labeo rohita (Family Cyprinidae) is the most popular species among the IMCs. In aquaculture operations, feed accounts for 50% of the cost of production and the protein sources for the feed are more expensive than the carbohydrate sources. The protein sparing action of carbohydrate is well known in fshes (Wilson, 1994; Stone, 2003; Krogdohl et al., 2005). Starch, the predominant carbohydrate in fsh feed is made available to the fsh by the action of α-amylases and therefore understanding the nature of amylases in fsh species will pave ways for selection of appropriate carbohydrate source. The information pertaining to the purifcation of amylases in L. rohita and their characterisation is limited (Moreau et al., 2001; Roychan and Chaudhari, 2001; Kushwaha, et al. 2012). This paper reports on the partial purifcation and characterisation of α-amylases in L. rohita. Materials and methods Enzyme extracts Specimens of L. rohita (average length 45 cm; 995 g) were obtained from the culture ponds of the Regional Research Centre of ICAR-Central Institute of Freshwater Aquaculture (ICAR-CIFA) at Bangalore, Karnataka, India. The digestive tract (DT) and liver (L) of the specimens were dissected out under ice cold conditions and washed repeatedly with ice-cold distilled water. The tissues were homogenised individually with distilled water (4 ml g -1 ; 15 g tissue in 60 ml) and centrifuged at 16,000 rpm for 20 min at 4°C. The supernatants (crude enzyme extract) were frozen and stored at -20°C in 20 ml aliquots for use in purifcation studies. Enzyme estimation α-Amylase activity was estimated using 1% starch solution in Tris-HCl buffer (0.1 M, pH 7.0) as the substrate. The assay mixture contained 0.05 ml crude enzyme extract plus 1.0 ml substrate and was incubated at 25°C for 1 h. The resulting reducing sugars were determined by the method of Nelson (1944) and Somogyi (1952) using glucose as the standard. Enzyme activity was expressed as μg glucose liberated per mg protein per hour. Protein in the crude enzyme extract and other enzyme fractions was estimated by the method of Lowry et al. (1951) using bovine serum albumin as the standard. All assays were carried out in triplicate. - Acetone fractionation (AF) The crude enzyme extract obtained was subjected to solvent fractionation by the addition of chilled acetone