Molecular, Structural and Functional Characterization of Antheraea mylitta Expressed Cocoonase: An Integrative Approach

Abstract

Cocoonase, a vital naturally secreted protease, enables moth emergence by specifically degumming sericin from cocoons, leaving fibroin intact. This unique characteristic makes it highly promising for protein-based silk degumming. Present study comprehensively characterizes molecular, structural, and functional attributes of Antheraea mylitta cocoonase. Here 786 bp full-length cocoonase gene from A. mylitta pupae head RNA has been successfully cloned and expressed in E. coli and sequence obtained deposited in NCBI (Accession ID: ON261685). Extensive in-silico analyses including 3D structure prediction, motif analysis, phylogenetic relationships, TMHMM, InterProScan, STRING-based protein interactions, and flexibility predictions provided deeper structural insights. Gene ontology classified cocoonase as a serine-type endopeptidase. A. mylitta cocoonase protein of 261-amino acid exhibited a theoretical molecular weight of 27,663.35 Da and an isoelectric point of 9.10 while molecular weight of ~ 28 kDa by SDS-PAGE. Its presumed extracellular localization was supported by the absence of signal peptides and transmembrane helices. STRING analysis revealed functional interacting networks, while ProtScale elucidated protein flexibility. Optical Coherence Tomography based study demonstrated distinct morphological changes in cocoon sheets treated with recombinant cocoonase. This study represents the first successful cloning, expression, and comprehensive in-silico characterization of the A. mylitta cocoonase gene.