&#x3b1;-Amylase from Mon Thong durian (<i>Durio zibethinus</i> 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.

Purification and characterization of thermostable α-amylase from germinating Sword bean (<i>Canavalia gladiata</i> (Jacq.) DC.) seeds

“…The samples were prepared under non-reducing conditions and separated on 13% polyacrylamide gels containing 0.1% w/v soluble starch (Posoongnoen et al 2015). The gel was washed with 1% v/v triton X-100 to remove SDS and then incubated with 0.2% w/v starch in 50 mM sodium acetate buffer (pH 5.5) for 6 h at 37°C.…”

Amino Acid Sequence of Amylase Type Alpha, MiAmy, from Ok-Rong Mango (<i>Mangifera indica</i> Linn. cv. Ok-Rong)

“…However, the difference between VD and IE in this region does not affect the mechanistic properties. In this study, nearly all critical domains and region of MiAmy are found, which are as follows: (1) the two consecutive tryptophan residues for starch-granule binding, which correspond to W469 and W470 (Tangphatsornruang et al, 2005), (2) the conserved residues thought to be the Ca 2+ binding site, (3) the residues involved in hydrogen bonding with the α-amylase inhibitor acarbose, (4) Tyr 570 in domain C, corresponding to Tyr807 of DzAmyF3, which was suggested to be a sugar tongs surface binding site (Posoongnoen et al, 2015). Thus, the MiAmy α-amylase gene is predicted to be valuable for further studies, including its modification.…”

Transcriptome analysis during fruit developmental stages in durian (Durio zibethinus Murr.) var. D24

“…Transcriptomic studies are essential in understanding multiple pathways, gene expression patterns, and secondary metabolites biosynthesis in developing fruits or tissues of interest. Several studies on gene expression patterns and gene expression profiles at the molecular level in Durian varieties have been reported ( Nyffeler and Baum, 2000 ; Ruwaida, 2009 ; Vanijajiva, 2011 , 2012 ; Hariyati et al, 2013 ; Santoso and Saleh, 2013 ; Posoongnoen et al, 2015 ; Teh et al, 2017 ; Husin et al, 2018 ). In transcriptomics of Durian, Musang King var., ripening-related gene sets included genes regulated by the MADS-BOX transcription factor family 29, SEPALLATA transcription factor family, and ethylene-related genes such as ACS (aminocyclopropane-1-carboxylic acid synthase), a key ethylene-production enzyme, are involved in ripening ( Teh et al, 2017 ).…”