Purification and characterization of a thiol amylase over produced by a non-cereal non-leguminous plant, tinospora cordifolia

Abstract

A 43 kDa a-amylase was purified from Tinospora cordifolia by glycogen precipitation, ammonium sulfate precipitation, gel filtration chromatography, and HPGPLC. The enzyme was optimally active in pH 6.0 at 60 C and had specific activity of 546.2 U/mg of protein. Activity was stable in the pH range of 4–7 and at temperatures up to 60 C. PCMB, iodoacetic acid, iodoacetamide, DTNB, and heavy metal ions Hg2+ > Ag+ > Cd2+ inhibited enzyme activity while Ca2+ improved both activity and thermostability. The enzyme was a thiol amylase (3 SH group/mole) and DTNB inhibition of activity was released by cysteine. N-terminal sequence of the enzyme had poor similarity (12–24%) with those of plant and microbial amylases. The enzyme was equally active on soluble starch and amylopectin and released maltose as the major end product.