Purification and characterization of two isoforms of native α amylase from Ok-Rong mango (Mangifera indica Linn. cv. Ok-Rong)

Abstract

Abstract Introduction The tropical plant amylases involved in the fruit ripening stage is outstanding for their high activities in converting starch to sugars within a short period at high temperatures over 40°C. Methods The α amylase iso-enzymes from Ok-Rong mango (Mangifera indica Linn. cv. Ok-Rong) were purified in 2 steps, using 70% ammonium sulfate precipitation and affinity chromatography on a β-cyclodextrin sepharose 6B column, and characterized for biochemical properties. Results The enzyme was purified 105-fold with a final specific activity of 59.27 U mg−1. SDS-PAGE revealed two bands of 60 and 64 kDa. pI were supposed to be 4.6 and 5.0. Those were resolved into isoforms I and II by a zymographic method. They were matched with α amylase Amy1 from Vigna mungo and α amylase-like isoform I from Theobroma cacao after LC-MS/MS analysis. Isoforms I and II exhibited maximum activity at pH 4, retained more than 50% of their activity after 1 h of incubation at pH 5–9. Two isoforms showed high activity over a wide range of temperatures at 30°–90°C, with the highest activity at 70°C. They retained more than 50% of their activity at 30°C–40°C after 1 h of incubation. The enzymes were confirmed to be metalloenzymes by the effect of EDTA. In addition, limit-dextrin, amylopectin and soluble starch were suggested to be good substrates. Conclusion Two α amylase iso-enzymes were classified as members of the low-pI group of amylases with identical structure, properties and functions. They are mesophilic with high possibilities for application for many purposes.