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.

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 (Results:The enzyme was purified 105-fold with a final specific activity of 59.27 U mgConclusion: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.

Abstract:The 594-amino acid residue sequence of α-amylase, MiAmy, from the Ok-rong mango (Mangifera indica Linn. cv. Ok-rong), in the ripening stage, was determined through Reverse Transcription Polymerase Chain Reaction (RT-PCR). Sequence alignments and evolutionary tree analyses revealed its high similarity to plastid α-amylase from many other plants. The sequence was revealed to have four conserved regions with catalytic amino acid residues for the active site. It was classified as a member of α-amylase family 13 because it has an active Domain A, similar to a (β/α) 8 -barrel structure. Three-dimensional structural predictions revealed that this partial sequence completely covered all of necessary domains for amylase activity.

The secreted α-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 α-amylase (DzAmy1) had a molecular mass of approximately 44 kDa. Its optimum pH and temperature for activity were 7.0 and 50°C, respectively. The enzyme was stable from pH 6 to 10 and from 30 to 60°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 α-amylases based on phylogenetic 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 coli and had hydrolysis activity for starch and ethylidene-pNP-G7, which was clearly confirmed the authenticity of DzAmy1 as a functional α-amylase.