α-amylase from mon thong durian (Durio zibethinus murr. cv. mon thong)-nucleotide sequence analysis, cloning and expression

Abstract

Amylase is hypothesized to involve in the initiation of intracellular starch granule digestion in the plastids of ripening durians. A putative α-amylase from Mon Thong durian (Durio zibethinus Murr. cv. Mon Thong; DzAmy3) was successfully isolated and its gene contain a 2,679 base pair open reading frame, which encodes 892 amino acids with a calculated molecular mass of 93.7 kDa and an isoelectric point of 5.77. The DzAmy3 contains starch binding domain with a putative plastid transit peptide and α-amylase like domain. Phylogenetic tree analysis proved it into the family three of plant α-amylases. The predicted structural model proposed a catalytic triad (Asp611, Glu636 and Asp719) for general acid/base hydrolysis. Recombinant DzAmy3 (rDzAmy3) was successfully expressed in Escherichia coli. rDzAmy3 hydrolyzed starch and ethylidene-pNP-G7 which confirms the authenticity of DzAmy3 gene and functional α-amylase. The rDzAmy3 had an optimum activity at pH 8.0 and 30°C. It was stable in the pH range of 7–8 at 37°C, temperature range of 5–20°C and in the presence of 1% (v/v) Tween 20 and Triton X-100. Substrate specificity analysis revealed that rDzAmy3 was active toward β-limit dextrin, starch, amylopectin, amylose and glycogen.