| Abstrakt: |
Marine habitat harbors a wide variety of Bacillusspecies which are dominant producers of biologically active extracellular metabolites and anti-microbial peptides (AMPs). The present study reports a marine bacterial strain MAH84 explored for production of a bioactive compound, its characterization and applications as antibiofilm agent. Cell free supernatant of strain MAH84 (Nutrient broth amended with 1% Glucose), was purified by two resins [Diethylaminoethyl (DEAE) Sepharose and Sephadex G-25]. The peptide mass fingerprinting (PMF) data of column purified cell free supernatant revealed it as zinc metalloproteases that belonged to M30 family of peptidases (hyicolysin) with a zinc motif at N-terminus with sequence AHEYQHMat position 102. Enzyme characterization studies showed that enzyme is thermostable with optimum activity at 60 °C, pH 12, halotolerance, and halo-stability (1–4 M NaCl). The enzyme activity in presence of chloride salts of metal [magnesium chloride (98.75%) and ferric chloride (97.5%)], and solvents [ethyl acetate (83.4%), glycol (98.4%), toluene (92%), methanol (99.6%) and acetone (93%) were found to be more than 80%. In continuation, application wise enzyme exhibited >50% antibiofilm activity against bacterial pathogens (such as E. coliMTCC43, P. aeruginosaMTCC424, K. pneumoniaeMTCC9751, S. aureusMTCC96, and Salmonella entericaSubsp. Entericaserotype Abony- MTCC 6017), and high radical scavenging activity was noted i.e., (2, 2- diphenyl-1-picrylhydrazyl) free radical scavenging (DPPH Assay) 82%, 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS assay) 69%, and Ferric reducing power activity (FRPA assay) 69%. Enzyme was found to be non-cytotoxic towards HeLa cell lines at 10 μg/ml. Additionally, a combinatorial study of ciprofloxacin, H2O2, short-chain fatty acids (acetic acid, propionic acid, and butyric acid) at their respective MIC (as determined) was conducted wherein the enzymes antibiofilm activity increased by 70% when co-administered with column purified cell free supernatant (metalloproteases 10 μg/ml). Further, quantitative Reverse Transcriptase Polymerase Chain Reaction (q-RTPCR) revealed downregulation of two biofilm genes, namely csgD and bcsA in S. Abonywith highest fold downregulation (2.48-fold) with butyric acid (100 μg/ml) in concurrent administration with column purified cell free supernatant (10 μg/ml). From our results, it is inferred that metalloprotease M30 from strain MAH84 can be a compelling quest for biofilm management. |
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