Purification and characterization of two isoforms of native α amylase from Ok-Rong mango (Mangifera indica Linn. cv. Ok-Rong)
TURKISH JOURNAL OF BIOCHEMISTRY-TURK BIYOKIMYA DERGISI(2017)
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 degrees C. Methods: The alpha 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 beta-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 alpha amylase Amy1 from Vigna mungo and alpha 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 degrees-90 degrees C, with the highest activity at 70 degrees C. They retained more than 50% of their activity at 30 degrees C-40 degrees 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 alpha 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.
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Key words
alpha Amylase,Ok Rong mango,Purification,Mesophilic,Mangifera indica
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