Development of an operational synaptobrevin-based fluorescent substrate for tetanus neurotoxin quantification

Tetanus neurotoxin (TTx), produced by Clostridium tetani, is a two-chain polypeptide with a heavy molecular chain (HC; 100 kDa) and a light molecular chain (LC; 50 kDa) linked by a disulphide bridge. the low-molecular-mass chain is classified as a zinc metalloprotease (EC 3.4.24.68) with specific hydrolysis on synaptobrevin. With the known enzymic activity for the LC of TTx, we developed a quantification method using a quenched fluorescence peptide substrate based on the synaptobrevin sequence (fragments 73-82), suitable for direct determination of the whole TTx (HC+LC) even in crude production batches, without the necessity of purification and reduction steps to isolate the LC of TTx. the rate of substrate hydrolysis was 200 nmol/min and it was totally inhibited by EDTA, anti-recombinant fragment C antibody, and the cleavage was in a single bond (Gln-Phe) with purified and crude TTx. Besides, ELISA applied to the anti-TTx serum produced at our Institute showed cross-reaction with every fraction of the crude TTx extract. Another aspect is that TTx activity depends on the storage time, reaching a maximum on day 10. the results obtained suggest that the use of the new fluorescent substrate, Abz-synaptobrevin(73-82)-EDDnp, enables easy and quick determination of TTx. It is a good alternative to some of the existing methods such as flocculation assay, and it can replace, under some conditions, the biological assays (minimal mortal dose).CEPID, Ctr Appl Toxinol, Biochem & Biophys Lab, BR-05503900 São Paulo, BrazilButanan Inst, Immunopathol Lab, BR-05503900 São Paulo, BrazilButanan Inst, Sect Anaerob Vaccine, BR-05503900 São Paulo, BrazilUNIFESP, CEPID, Ctr Appl Toxinol, Dept Biophys, BR-05503900 São Paulo, BrazilUNIFESP, CEPID, Ctr Appl Toxinol, Dept Biophys, BR-05503900 São Paulo, BrazilWeb of Scienc

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 378 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.Butantan Inst, Biochem & Biophys Lab, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilButantan Inst, Dept Bacteriol, Sect Aerob Vaccines, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilWeb of Scienc

Characterization of the Phenol Monooxygenase Gene from Chromobacterium violaceum: Potential Use for Phenol Biodegradation

In this work, the biodegradation mechanism of phenol and sub products (such as catechol and hydroquinone) in Chromobacterium violaceum was investigated by cloning and molecular characterization of a phenol monooxygenase gene in Escherichia coli. This gene (Cvmp) is very similar (74 and 59% of similarity and identity, respectively) to the ortholog from Ralstonia eutropha, bacteria capable of utilizing phenol as the sole carbon source. The phenol biodegradation ability of E. coli recombinant strains was tested by cell-growth in a minimal medium containing phenol as the sole source of carbon and release of intermediary metabolites (catechol and hydroquinone). Interestingly, during the growth of these strains on phenol, catechol, and hydroquinone accumulated transiently in the medium. These metabolites were further analyzed by HPLC. These results indicated that phenol can be initially orto or para hydroxylated to produce cathecol or hydroquinone, respectively, followed by meta-cleavage of aromatic rings. To verify this information, the metabolites obtained from HPLC were submitted to LC/MS to confirm their chemical structure, thereby indicating that the recombinant strains utilize two different routes simultaneously, leading to different ring-fission substrates for the metabolism of phenol. (C) KSBBConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPq (Brasilia, DF, Brazil)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP (Sao Paulo, SP, Brazil