Analysis of secreted protein profile and enzymatic activities from Corynebacterium diphtheriae and Bordetella pertussis on production batch media using peptide quenched fluorescent substrates.
Proteases were identified and characterized from the culture supernatant of the C. diphtheriae and B. pertussis bacteria. The proteases were secreted in the media and detected at the end of the exponential growth phase. Activity was detected in some fluorescent substrates, based on selected protein sequences such as insuline beta-chain, bradykinin, and synaptobrevin. The proteases were purified by means of gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified proteins indicated, for the main secreted proteins, an estimated molecular mass of 30 kDa in C. diphtheriae and 69 kDa in B. pertussis culture media. The proteases were stable and presented enzymatic activity at 37 degrees C. These proteases were not related to the main toxic compounds described in these two bacteria, but could represent good markers for the fermentation process when the enzyme activity was measured with the fluorescent substrates.
Perpetuo
Elen A
EA
Laboratory of Biochemistry and Biophysics, Butantan Institute, Sao Paulo, Brazil.
Lebrun
Ivo
I
Juliano
Luis
L
Juliano
Maria Aparecida
MA
Sakauchi
Maria Aparecida
MA
Prado
Sally M A
SM
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000032:analysis; D001886:Bordetella pertussis / Q000201:enzymology* / Q000254:growth & development; D002021:Buffers; D002850:Chromatography, Gel; D003353:Corynebacterium diphtheriae / Q000201:enzymology* / Q000254:growth & development; D003470:Culture Media / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005374:Filtration; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000032:analysis* / Q000302:isolation & purification; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D037342:Pertussis Toxin / Q000032:analysis; D012965:Sodium Chloride / Q000737:chemistry; D013379:Substrate Specificity; D014325:Tromethamine / Q000737:chemistry
D001427:Bacterial Toxins; D002021:Buffers; D003470:Culture Media; D005456:Fluorescent Dyes; D010455:Peptides; D014325:Tromethamine; D012965:Sodium Chloride; D037342:Pertussis Toxin; D010447:Peptide Hydrolases