Characterization And Molecular Cloning Of Secreted Alpha-Amylase With Dominant Activity From Mon Thong Durian (Durio Zibethinus Murr. Cv. Mon Thong)

The secreted alpha-amylase with dominant activity was purified from the crude extract of Mon Thong durian by steps of ammonium sulphate precipitation and the affinity column chromatography. The purified alpha-amylase (DzAmy1) had a molecular mass of approximately 44 kDa. Its optimum pH and temperature for activity were 7.0 and 50 degrees C, respectively. The enzyme was stable from pH 6 to 10 and from 30 to 60 degrees C. Many metal ions did not affect amylase activity. The gene cloning of DzAmy1 was carried out and it was confirmed that DzAmy1 gene consisted of 1,254 bp open reading frame, which encoded 23 amino acids of the signal peptide and 395 amino acids of mature protein with a calculated molecular mass of 43.7 kDa. The isoelectric point of the enzyme was 5.78. DzAmy1 was shown to belong to sub-family one of the plant alpha-amylases based on phylogenctic tree analysis. Structural characterization by homology modelling suggested that it consisted of 3 domains with a catalytic triad in domain A. Recombinant DzAmy1 (rDzAmy1) was successfully expressed in Escherichia colt and had hydrolysis activity for starch and ethylidenepNP-G7, which was clearly confirmed the authenticity of DzAmy1 as a functional alpha-amylase.