Partial Purification and Characterization of Amylases from the Digestive Tract of the Indian Medium Carp Labeo fimbriatus (Bloch, 1797)

Abstract

Partial purification of amylases from the digestive tract of the Indian medium carp Labeo fimbriatus through acetone fractionation and Sephadex G-75 gel filtration resulted in 5-fold purification with 29% recovery. Characterization of amylase activity revealed two pH optima at 4.5 and 6.5-7.0. Activity was stable over a wide pH range of 4.0-10.0. Optimum incubation temperature was 25°C. The enzyme lost 80% activity at 50°C within 30 min and was inhibited by 1 mM p-chloromercuri benzoate, ethylene diamine tetra-acetate, and phenyl methyl sulphonyl fluoride. The heavy metal ions Hg++, Cd++, Cu++, Zn++, Fe++, Pb++, Bi++, and Ag+ strongly inhibited enzyme activity whereas Ca++ activated it. Native polyacrylamide gel electrophoresis of the purified amylase fractions revealed four bands, with corresponding molecular weights of 72, 68, 66, and 65 kDa. Amylase activity from L. fimbriatus exhibited linear hydrolysis of starch up to 7% concentration.