Abstract
BackgroundSilkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens.Methodology/Principal FindingsSilkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/− bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5′- and 3′-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense.Conclusions/SignificanceThe study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba.
Highlights
Sericulture is one of the oldest agro-based industries in the world
The present study reports the characterization of novel germin-like proteins (Glp) from white mulberry, M. alba by the method of purification of its mature form from silkworm fecal matter
The N-terminal amino acid sequence of the purified protein extracted from silkworm fecal matter was found exactly similar to the deduced amino acid sequence of the full length cDNA from the food plant M. alba
Summary
Sericulture is one of the oldest agro-based industries in the world. From time to time, sericulture practices have undergone changes to improve productivity. Researchers have characterized antiviral proteins from midgut and haemolymph of silkworm, Bombyx mori.The inhibitory and antiviral properties of digestive juice of Bombyx mori larvae have been investigated and an unknown substance of high molecular weight in the gut juice has been reported that could inactivate nuclear polyhedrosis virus in vitro [4,5]. The role of Bmlipase-1 (Bombyx mori Lipase-1) and BmSP-2 (Bombyx mori Serine Protease-2) from digestive juice having strong antiviral activity against BmNPV has been demonstrated [1,11]. Such substances with some pattern recognition proteins in haemolymph have presented unity and diversity in antiviral substance mechanisms. Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens
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