B.Subtilis expression plasmids generally require a stringent Shine Dalgarno Sequence(SDS). Site directed mutagenesis was explored to change the Shine Dalgarno Sequence from AAAAATGGGG (mutant type) to AAAAAGGGGG (...B.Subtilis expression plasmids generally require a stringent Shine Dalgarno Sequence(SDS). Site directed mutagenesis was explored to change the Shine Dalgarno Sequence from AAAAATGGGG (mutant type) to AAAAAGGGGG (wild type) in recombinant plasmid PSM604. The single base substitution made the plasmid with wild SDS unstable in structure and segregation. The interaction of SDS with subtilisin leader sequence of PSM604 might be responsible for the instability of plasmid.展开更多
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
文摘B.Subtilis expression plasmids generally require a stringent Shine Dalgarno Sequence(SDS). Site directed mutagenesis was explored to change the Shine Dalgarno Sequence from AAAAATGGGG (mutant type) to AAAAAGGGGG (wild type) in recombinant plasmid PSM604. The single base substitution made the plasmid with wild SDS unstable in structure and segregation. The interaction of SDS with subtilisin leader sequence of PSM604 might be responsible for the instability of plasmid.
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.
