Biomolecular characterization of Antheraea mylitta cocoonase: A secreted protease.

Cocoonase is a protease secreted during the emergence of silk moths. In the present study cocoonase of Antheraea mylitta was collected, purified and secondary structure was determined using circular dichroism (CD) spectroscopy which revealed the presence of α-helix 4.3%, ß-sheet 55%, turn 8% and random coil 32.7%. The thermal stability of cocoonase was studied using CD spectroscopy while the thermal property was observed using Differential Scanning Calorimetry (DSC). Furthermore, MALDI-TOF peptide mass fingerprinting (PMF) was performed for similar protein identification using the MASCOT server. Using casein as the substrate, the kinetic constants Km and Vmax were 13 × 103 mg/ml and 15.09 × 10-2 µg/mg.s1 respectively. The specific activity of cocoonase was observed to be maximum at temperature 40 °C, pH-8.0. The effect of heavy metals Hg2+, Cd2+, Co2+, Pb2+ showed inhibitory activity at higher concentrations, while few metals like Mn2+, Fe3+ enhanced the activity while the effect of Ca2+ was not much on the activity. Soybean trypsin inhibitor and PMSF showed an inhibitory effect on the activity of cocoonase. Additionally, antioxidant scavenging and fibrinolytic properties were also observed. Furthermore, the imperative information generated through the present study will serve to explore cocoonase for its prospective pharmaceutical applications.

Identification of cocoonase and cocoonase like protein using polyclonal antibody of Antheraea mylitta cocoonase.

BACKGROUND: Cocoonase is a proteolytic enzyme released by silk moths during pupal adult emergence. Without damaging the silk fibroin, this enzyme dissolves the shell of the tasar cocoon by exclusively targeting the protein sericin. Prior to this study, there was no available antibody against Antheraea mylitta cocoonase to identify or screen out similar variants or cocoonase like protein. RESULTS: In the present study, naturally secreted A. mylitta cocoonase was purified and used to immunize New Zealand white rabbits. The developed polyclonal antibody of cocoonase was purified and its specific interaction with cocoonase was determined using Indirect ELISA. The confirmation of its specificity and immuno-reactivity was evaluated by western blot using native cocoonase of tasar silkworm A. mylitta. The efficacy and specificity of the polyclonal antibody were further verified and confirmed by western blot which was performed to detect ten different ecotypes of A. mylitta cocoonase. CONCLUSION: The developed antibody successfully detected the cocoonase of different ecotypes. Thus, in future this antibody can serve as one of the molecular detection method for cocoonase and cocoonase-like proteins.

Protein-protein docking and molecular dynamics studies of sericin and cocoonase of silkworm: an insight for cocoon softening.

Cocoonase is known to digest the sericin protein that encapsulates the silkworm cocoon's fibroin protein. Silk fibroin and sericin are two types of proteins that make up silk, and accounts for around 20-30% of the overall cocoon weight. The aim of the study was to see the protein-protein interaction (PPI) and molecular dynamic study of sericin, cocoonase and protein-protein docked complex of silkworm by computational approaches. Here motif analysis, phylogenetic analysis, principal component analysis, root-mean-square deviation (RMSD), root mean square fluctuation, radius of gyration, structural and functional study of cocoonase and sericin as well as molecular docking study were carried out. The 33 amino acid residues of cocoonase shows interaction with 38 aa residues of sericin involving 4 disulphide bonds, 22 hydrogen bonds and 319 non-bonded contacts. The confirmational stability and flexibility of both the proteins as well as protein-protein complex were achieved at 70 ns of MD simulation study. RMSD-based data indicated that cocoonase is more stable than sericin and complex, and complex has a greater fluctuation with more compact (higher Rg) value than cocoonase and sericin, inferring higher conformational stability and flexibility of protein-protein complex than cocoonase and sericin. This study provides a new dimension for PPI study by computational approaches.Communicated by Ramaswamy H. Sarma.

Evaluating the role of trypsin in silk degumming: An in silico approach.

The trypsin being universal enzyme forming family of proteases catalyzes the hydrolysis of proteins into amino acids and regenerates the serine hydroxyl an active site. The trypsin enzyme from D. saccharalis, uses sericin as its preferred substrate. Presence of catalytic triad (serine, aspartic acid and histidine) at the substrate binding site of this enzyme is very important for the catalytic activity. In the current study, the interacting mechanism between the substrate sericin protein and enzyme trypsin protein were explored by integrating various computational approaches including physico-chemical properties, biophysical properties, dynamics, gene ontology, molecular docking, protein - protein interactions, binding free energy calculation and structural motifs were studied. The evolutionary study performed by MEGA X showed that trypsin protein sequence (ALE15212.1) is closely related to cocoonase protein sequence (ADG26770.1) from Antheraea pernyi. 3-D models of trypsin and sericin proteins were predicted using I-TASSER and further validated by PROCHECK, and ProSAweb softwares. The predicted trypsin structure model was assigned E.C. no. 3.4.21.4 which refers hydrolytic mechanism. Gene Ontology predicted by QuickGO showed that trypsin has serine hydrolase activity (GO: 00017171), and part of proteolysis (GO: 0006508) as well as protein metabolic process (GO:0019538) actvity. Molecular docking studies between trypsin and sericin proteins were conducted by the HADDOCK 2.4 having best docked protein complex with Z-score - 1.9. 2D and 3D protein-protein interaction was performed with LIGPLOT+ and HAWKDOCK, PDBsum, respectively. The amino acid residues interacting across proteins interface are sericin_chain A representing "Ser133, Tyr214, Thr188, Thr243, Ser225, Ser151, Ser156, His294, Arg293, Gly296″ and trypsin_chain B "Lys120, Tyr246, Asn119, Glu239, Ser62, Tyr194, Ile197, Ser171, Tyr169, Gly170″. Based on our results trypsin shows similarity with cocoonase and presumably trypsin can be used as an alternative source in cocoon degumming.

Study on cocoonase, sericin, and degumming of silk cocoon: computational and experimental.

BACKGROUND: Cocoonase is a proteolytic enzyme that helps in dissolving the silk cocoon shell and exit of silk moth. Chemicals like anhydrous Na2CO3, Marseille soap, soda, ethylene diamine and tartaric acid-based degumming of silk cocoon shell have been in practice. During this process, solubility of sericin protein increased resulting in the release of sericin from the fibroin protein of the silk. However, this process diminishes natural color and softness of the silk. Cocoonase enzyme digests the sericin protein of silk at the anterior portion of the cocoon without disturbing the silk fibroin. However, no thorough characterization of cocoonase and sericin protein as well as imaging analysis of chemical- and enzyme-treated silk sheets has been carried out so far. Therefore, present study aimed for detailed characterization of cocoonase and sericin proteins, phylogenetic analysis, secondary and tertiary structure prediction, and computational validation as well as their interaction with other proteins. Further, identification of tasar silkworm (Antheraea mylitta) pupa stage for cocoonase collection, its purification and effect on silk sheet degumming, scanning electron microscope (SEM)-based comparison of chemical- and enzyme-treated cocoon sheets, and its optical coherence tomography (OCT)-based imaging analysis have been investigated. Various computational tools like Molecular Evolutionary Genetics Analysis (MEGA) X and Figtree, Iterative Threading Assembly Refinement (I-TASSER), self-optimized predicted method with alignment (SOPMA), PROCHECK, University of California, San Francisco (UCSF) Chimera, and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) were used for characterization of cocoonase and sericin proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein purification using Sephadex G 25-column, degumming of cocoon sheet using cocoonase enzyme and chemical Na2CO3, and SEM and OCT analysis of degummed cocoon sheet were performed. RESULTS: Predicted normalized B-factors of cocoonase and sericin with respect to α and ß regions showed that these regions are structurally more stable in cocoonase while less stable in sericin. Conserved domain analysis revealed that B. mori cocoonase contains a trypsin-like serine protease with active site range 45 to 180 query sequences while substrate binding site from 175 to 200 query sequences. SDS-PAGE analysis of cocoonase indicated its molecular weight of 25-26 kDa. Na2CO3 treatment showed more degumming effect (i.e., cocoon sheet weight loss) as compared to degumming with cocoonase. However, cocoonase-treated silk cocoon sheet holds the natural color of tasar silk, smoothness, and luster compared with the cocoon sheet treated with Na2CO3. SEM-based analysis showed the noticeable variation on the surface of silk fiber treated with cocoonase and Na2CO3. OCT analysis also exemplified the variations in the cross-sectional view of the cocoonase and Na2CO3-treated silk sheets. CONCLUSIONS: Present study enlightens on the detailed characteristics of cocoonase and sericin proteins, comparative degumming activity, and image analysis of cocoonase enzyme and Na2CO3 chemical-treated silk sheets. Obtained findings illustrated about use of cocoonase enzyme in the degumming of silk cocoon at larger scale that will be a boon to the silk industry.

Gum ghatti based hydrogel: Microwave synthesis, characterization, 5-Fluorouracil encapsulation and 'in vitro' drug release evaluation.

The release attributes of anticancer therapeutic embedded in programmable gum ghatti hydrogel matrices has been evaluated. Hydrogel has been programmed simply by adjusting the product composition (monomer-acrylamide and crosslinker- N, N Methylenebisacrylamide (MBAm)) to control mesh size during copolymerization of gum through microwave based technique. The synthesized system has been characterized for physicochemical traits and 'in-vitro' drug release in various physiological pH conditions by USP standard method. The degree of crosslinking analysed through equilibrium swelling ratio (ESR) has been correlated with release kinetics. The release profiles yielded from the measurements of cumulative drug delivery showed that the system with lower ESR included slower release rate than that of the system with higher ESR, consequently generated higher t50 values. Additionally, the release rate rises with rise in pH milieu.

Grafted sesbania gum: A novel derivative for sugarcane juice clarification.

This work describes the significance of graft copolymer induced flocculation process in sugarcane juice clarification. The naturally occurring cinnamic acid has been successfully incorporated into the structure of sesbania gum through microwave assisted technique (hybrid of conventional and microwave based method) to develop novel, non-toxic, eco-friendly product with highly extended structure. The customized structure of gum derivative has been characterized by a number of modern techniques like 13C NMR, FTIR, SEM, XRD, TGA, DSC, elemental and viscometric analysis. The separation of suspended colloidal impurities from cane juice has been achieved through the application of synthesized gum derivative as a flocculant. The clarification performance of the graft copolymer has been evaluated in terms of turbidity, absorbance, and degree brix (°bx). The flocculant exhibited maximum efficiency at 1.0ppm (optimized dose) without altering the nutritional parameters (% sugar content). The clarified juice thus obtained may be used as a beverage for direct consumption or for the manufacture of white sugar.

Sesbania gum based hydrogel as platform for sustained drug delivery: An 'in vitro' study of 5-Fu release.

The purpose of this study is to fabricate 5-Fluorouracil sustained release matrix based on a novel, nontoxic, eco-friendly modified biopolymer. The sesbania gum based hydrogel has been prepared by microwave assisted method using acrylamide as a monomer and N,N Methylenebisacrylamide as a crosslinker. The crosslink copolymerization has been confirmed by several modern techniques such as FTIR, SEM, XRD, TGA, DSC, elemental analysis etc. The bioactive 5-Fluorouracil has been encapsulated via solvent swelling method and its release rate has been investigated in various pH dissolution medium through USP standard protocol. The synthesized hydrogel with higher degree of crosslinking exhibited slower release rate than that of hydrogel having lower degree of crosslinking. Thus, resulting higher t25 value, the release rate increases with increase in pH of the medium. Release kinetics suggests the non-Fickian release behaviour of the hydrogel.

Generation of cytotoxic molecules and oxidative stress in haemolymph of pebrinised tasar silkworm Antheraea mylitta Drury.

The present study was carried out to investigate the effects of microsporidial infection on redox regulation mechanism and oxidative stress in tasar silkworm Antheraea mylitta. High level of superoxide radical (p < 0.05), nitric oxide (p < 0.001) and lipid peroxidation (p < 0.001) was observed in haemolymph of pebrinised larvae, which indicated the resultant generation of cytotoxic molecules and oxidative damage. Increased phenol oxidase (PO) activity in haemolymph of pebrinised larvae indicated the activation of immune defences during pathological conditions. In addition, higher level of glutathione-S-tranferase (GST) activity and reduced glutathione (GSH) level observed in pebrinised larvae indicated adaptive behaviour of tissue against toxic oxyradicals (p < 0.05). Conversely, low level of ascorbic acid (ASA) content suggested that the larvae might have used these compounds to counteract stress in tissues or low uptake under microspridial infection (p < 0.05). Present findings provide new insights into the cellular and biochemical bases of host-pathogen interactions in tasar silkworm A. mylitta.

Immobilization of organophosphate hydrolase on biocompatible gelatin pads and its use in removal of organophosphate compounds and nerve agents.

Bacterial organophosphate hydrolases (OPH) have been shown to hydrolyze structurally diverse group of organophosphate (OP) compounds and nerve agents. Due to broad substrate range and unusual catalytic properties, the OPH has successfully been used to develop eco-friendly strategies for detection and decontamination of OP compounds. However, their usage has failed to gain necessary acceptance, due to short half-life of the enzyme and loss of activity during process development. In the present study, we report a simple procedure for immobilization of OPH on biocompatible gelatin pads. The covalent coupling of OPH using glutaraldehyde spacer has been found to dramatically improve the enzyme stability. There is no apparent loss of OPH activity in OPH-gelatin pads stored at room temperature for more than six months. As revealed by a number of kinetic parameters, the catalytic properties of immobilized enzyme are found to be comparable to the free enzyme. Further, the OPH-gelatin pads effectively eliminate OP insecticide methyl parathion and nerve agent sarin.

Indigenous organophosphate-degrading (opd) plasmid pCMS1 of Brevundimonasdiminuta is self-transmissible and plays a key role in horizontal mobility of the opd gene.

A fosmid library of the 66kb indigenous organophosphate-degrading (opd) plasmid pCMS1 of Brevundimonas diminuta was tagged with mini-transposon EZTn5 , to determine its sequence using transposon-specific primers. The sequence revealed the presence of a number of tra genes suggesting their role in conjugal transfer of pCMS1. Consistent with the presence of the tra genes, the B. diminuta plasmid, pCMS1::tet, generated by replacing the opd gene with opd::tet, served as a donor for transferring pCMS1::tet into recipient strain Pseudomonas putida. The self-transmissibility of the opd-containing plasmid pCMS1 and the existence of identical opd genes on otherwise dissimilar plasmids suggests a probable role of indigenous opd plasmids like pCMS1 in transferring the opd gene among soil bacteria.

Organophosphate hydrolase in Brevundimonas diminuta is targeted to the periplasmic face of the inner membrane by the twin arginine translocation pathway.

A twin arginine translocation (Tat) motif, involved in transport of folded proteins across the inner membrane, was identified in the signal peptide of the membrane-associated organophosphate hydrolase (OPH) of Brevundimonas diminuta. Expression of the precursor form of OPH carrying a C-terminal His tag in an opd-negative background and subsequent immunoblotting with anti-His antibodies showed that only the mature form of OPH associated with the membrane and that the precursor form of OPH was entirely found in the cytoplasm. When OPH was expressed without the signal peptide, most of it remained in the cytoplasm, where it was apparently correctly folded and showed activity comparable to that of the membrane-associated OPH encoded by the wild-type opd gene. Amino acid substitutions in the invariant arginine residues of the Tat signal peptide affected both the processing and localization of OPH, confirming a critical role for the Tat system in membrane targeting of OPH in B. diminuta. The localization of OPH to the periplasmic face of the inner membrane in B. diminuta was demonstrated by proteinase K treatment of spheroplasts and also by fluorescence-activated cell sorting analysis of cells expressing OPH-green fluorescent protein fusions with and without an SsrA tag that targets cytoplasmic proteins to the ClpXP protease.

mRNA secondary structure modulates the translation of organophosphate hydrolase (OPH) in E. coli.

Organophosphate hydrolases (OPHs), involved in hydrolytic cleavage of structurally diverse organophosphates are coded by a plasmid borne, highly conserved organophosphate degrading (opd) gene. An inverted repeat sequence found in the signal coding region of the opd gene was found to be responsible for inducing a stable stem loop structure with a DeltaG of -23.1 kcal/mol. This stem loop structure has shown significant influence on the expression levels of organophosphate hydrolase (OPH) in E. coli. When the signal coding region comprising the inverted repeat sequence was deleted a approximately 3.28 fold increase in the expression levels of OPH was noticed in E. coli BL21 cells. Mutations in the inverted repeat region, especially at the third position of the codon, to a non-complementary base destabilized the secondary structure of opd mRNA. When such opd variant, opd' was expressed, the expression levels were found to be similar to expression levels coded by the construct generated by deleting the signal peptide coding region. Deletion of signal peptide did not influence the folding and activity of OPH. Though high level induction has resulted in accumulation of OPH as inclusion bodies, modulation of expression levels by reducing the copy number of the expression plasmid, inducer concentration and growth temperature has produced majority of the protein in soluble and active form.