Abstract
The current study deals with a digestive α-amylase in the larvae of Pieris brassicae L. through purification, enzymatic characterization, gene expression, and in vivo effect of a specific inhibitor, Acarbose. Although α-amylase activity was the highest in the whole gut homogenate of larvae but compartmentalization of amylolytic activity showed an equal activity in posterior midgut (PM) and anterior midgut (AM). A three step purification using ammonium sulfate, Sepharyl G-100 and DEAE-Cellulose Fast flow revealed an enzyme with a specific activity of 5.18 U/mg, recovery of 13.20, purification fold of 19.25 and molecular weight of 88 kDa. The purified α-amylase had the highest activity at optimal pH and temperature of 8 and 35°C. Also, the enzyme had Vmax values of 4.64 and 3.02 U/mg protein and Km values of 1.37 and 1.74% using starch and glycogen as substrates, respectively. Different concentrations of acarbose, ethylenediamine tetraacetic acid, and ethylene glycol-bis (β-aminoethylether) N, N, N′, N′-tetraacetic acid significantly decreased activity of the purified α-amylase. The 4th instar larvae of P. brassicae were fed on the treated leaves of Raphanus sativus L. with 0.22 mM of Acarbose to find in vivo effects on nutritional indices, α-amylase activity, and gene expression. The significant differences were only found in conversion efficiency of digested food, relative growth rate, and metabolic cost of control and fed larvae on Acarbose. Also, amylolytic activity significantly decreased in the treated larvae by both biochemical and native-PAGE experiments. Results of RT-PCR revealed a gene with 621 bp length responsible for α-amylase expression that had 75% identity with Papilio xuthus and P. polytes. Finally, qRT-PCR revealed higher expression of α-amylase in control larvae compared to acarbose-fed ones.
Highlights
Carbohydrates gained from plant tissues must be broken down being absorbable via epithelial cells of insect midgut
Amyloytic activity was found in all midgut preparations of P. brassicae larvae, the soluble fraction of the total, posterior, and anterior midgut preparations showed the highest amylolytic activity compared to other preparations (Figure 1A)
Herbivorous insects utilize plant tissues full of carbohydrates (Mainly starch and glycosides) which are digested by the activities of different carbohydrases to provide monomers like glucose to be absorbed via epithelial cells (Nation, 2008). α-Amylases are one of the important classes of digestive enzymes that break down starch within plant tissues to oligosaccharides prior to be further hydrolyzed to glucose by glucosidases (Kaur et al, 2014)
Summary
Carbohydrates gained from plant tissues must be broken down being absorbable via epithelial cells of insect midgut. It must be noted that providing such a transgenic plants requires precise and sufficient knowledge on the biochemical properties of insect α-amylases and interactions with host plants or specific inhibitors (Kaur et al, 2014). Several experiments were carried out determining α-amylase importance in digestive process of the 4th instar larvae of P. brassicae as (i) compartmentalization of α-amylase activity in different preparations of larval midgut, (ii) purification of the enzyme by a three step purification, (iii) characterization of the purified α-amylase such as optimal pH and temperature as well as in vitro responses to specific inhibitors, (iv) in vivo effects of acarbose on nutritional indices and amylolytic activity, and (v) determination of the gene responsible for α-amylase secretion and its expression in response to larval feeding on acarbose
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
