@article {48694,
	title = {Domain shuffling and site-saturation mutagenesis for the enhanced inhibitory potential of amaranthaceae α-amylase inhibitors},
	journal = {Protein Journal},
	volume = {42},
	year = {2023},
	month = {OCT},
	pages = {519-532},
	type = {Article},
	abstract = {<p>
	Amaranthaceae a-amylase inhibitors (AAIs) are knottin-type proteins with selective inhibitory potential against coleopteran a-amylases. Their small size and remarkable stability make them exciting molecules for protein engineering to achieve superior selectivity and efficacy. In this report, we have designed a set of AAI pro- and mature peptides chimeras. Based on in silico analysis, stable AAI chimeras having a stronger affinity with target amylases were selected for characterization. In vitro studies validated that chimera of the propeptide from Chenopodium quinoa a-AI and mature peptide from Beta vulgaris a-AI possess 3, 7.6, and 4.26 fold higher inhibition potential than parental counterparts. Importantly, recombinant AAI chimera retained specificity towards target coleopteran a-amylases. In addition, to improve the inhibitory potential of AAI, we performed in silico site-saturation mutagenesis. Computational analysis followed by experimental data showed that substituting Asparagine at the 6th position with Methionine had a remarkable increase in the specific inhibition potential of Amaranthus hypochondriacus a-AI. These results provide structural-functional insights into the vitality of AAI propeptide and a potential hotspot for mutagenesis to enhance the AAI activity. Our investigation will be a toolkit for AAI{\textquoteright}s optimization and functional differentiation for future biotechnological applications.</p>
},
	keywords = {\& alpha, -Amylase, -Amylase inhibitors, Amaranthaceae, Coleoptera, Peptide, Pest control},
	issn = {1572-3887},
	doi = {10.1007/s10930-023-10148-y},
	author = {Rane, Ashwini S. and Nair, Vineetkumar S. and Joshi, Rakesh S. and Giri, Ashok P.}
}
