Induction Isolation Purification and Characterization of Antimicrobial Peptides from Silkworm larvae Bombyx mori and Philosamia ricini

Abstract

Experimental Biological material silkworm larvae were reared successfully and 5th instar larvae Bombyx mori and Philosamia ricini newline were used for synthesis of Antimicrobial Peptides (AMPs). Mixed bacterial infection of Escherichia coli (NCIM 2137) and Staphylococcus aureus (NCIM 2672) were carried to induce the synthesis of AMPs. Due to infection Mortality (and#8776;30%) was observed in both larvae and hemolymph protein concentration found decreased. Differential protein expression study of healthy larvae s hemolymph and immune challenged larvae s hemolymph were performed using SDS-PAGE, 2D Gel and RP-HPLC. In the immune challenged larvae three extra protein spots were observed as per the 2D Gel comparative study and in the peak graph of chromatography extra peaks were observed. RP-HPLC fractions were screened for Antimicrobial Susceptibility Testing (AST) using two gram negative bacteria Viz. Escherichia coli (NCIM 2137) and Pseudomonas aeruginosa (NCIM 2074), two gram positive bacteria Staphylococcus aureus (NCIM 2672) and Streptococcus faecalis (NCIM 2405), two fungi Aspergillus niger (mold) and Candida albicans (yeast). Standard antibiotic Streptomycin HLS300 and Clotrimazole CC10 were used as reference. Results of HPLC fraction were quit promising as antibacterial and antifungal with respect to standard antibiotics. These potent antimicrobial peptides were further purified by RP-HPLC and authentication of purification performed using SDS-PAGE. Single bands observed from authentication were subjected to MALDI-TOF/TOF analysis and complete amino acid sequences were determined using Peptide Mass Fingerprinting (PMF). After sequencing four AMPs were almost characterized using 15 Bioinformatics tools. Isolated AMPs were found potent hence in future it will be alternative to some antibiotics those were found ineffective. Synthesis of AMPs from silkworm larvae may help to raise the economic status of sericulture farmers. newlineKeywords