Purification, developmental expression, and in silico characterization of α-amylase inhibitor from Echinochloa frumentacea

Barnyard (Echinochloa frumentacea) and finger (Eleusine coracana) millet growing at northwestern Himalaya were explored for the alpha-amylase inhibitor (alpha-AI). The mature seeds of barnyard millet variety PRJ1 had maximum alpha-AI activity which increases in different developmental stage. alpha-AI was purified up to 22.25-fold from barnyard millet variety PRJ1. Semi-quantitative PCR of different developmental stages of barnyard millet seeds showed increased levels of the transcript from 7 to 28 days. Sequence analysis revealed that it contained 315 bp nucleotide which encodes 104 amino acid sequence with molecular weight 10.72 kDa. The predicted 3D structure of alpha-AI was 86.73% similar to a bifunctional inhibitor of ragi. In silico analysis of 71 alpha-AI protein sequences were carried out for biochemical features, homology search, multiple sequence alignment, phylogenetic tree construction, motif, and superfamily distribution of protein sequences. Analysis of multiple sequence alignment revealed the existence of conserved regions NPLP[S/G] CRWYVV[S/Q][Q/R] TCG[V/I] throughout sequences. Superfam analysis revealed that alpha-AI protein sequences were distributed among seven different superfamilies.