Marine habitat harbors a wide variety of Bacillus species which are dominant producers of biologically active extracellular metabolites and anti-microbial peptides(AMPs).The present study reports a marine bacterial st...Marine habitat harbors a wide variety of Bacillus species 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 AHEYQHM at 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.coli MTCC43,P.aeruginosa MTCC424,K.pneumoniae MTCC9751,S.aureus MTCC96,and Salmonella enterica Subsp.Enterica serotype 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 10μg/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 10μg/ml).Further,quantitative Reverse Transcriptase Polymerase Chain Reaction(q-RTPCR)revealed downregulation of two biofilm genes,namely csgD and bcsA in S.Abony with highest fold downregulation(2.48-fold)with butyric acid(100μg/ml)in concurrent administration with column purified cell free supernatant(10μg/ml).From our results,it is inferred that metalloprotease M30 from strain MAH84 can be a compelling quest for biofilm management.展开更多
基金financial support[EMR/2016/003586(SERB/F/942/2017–2020)]DST FIST,GoI Sanction order No.SR/FST/LSI-613/2014(C)Author PGSM thank UGC-GoI([202223-UGCES-22-GE-TEL-F-SJSGC(Savitribai Jyotirao Phule Single Girl Child)-1823]for fellowship.
文摘Marine habitat harbors a wide variety of Bacillus species 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 AHEYQHM at 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.coli MTCC43,P.aeruginosa MTCC424,K.pneumoniae MTCC9751,S.aureus MTCC96,and Salmonella enterica Subsp.Enterica serotype 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 10μg/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 10μg/ml).Further,quantitative Reverse Transcriptase Polymerase Chain Reaction(q-RTPCR)revealed downregulation of two biofilm genes,namely csgD and bcsA in S.Abony with highest fold downregulation(2.48-fold)with butyric acid(100μg/ml)in concurrent administration with column purified cell free supernatant(10μg/ml).From our results,it is inferred that metalloprotease M30 from strain MAH84 can be a compelling quest for biofilm management.
