RESUMO
BACKGROUND: Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fine industries, particularly in pharmaceuticals, cosmetics, and bioethanol production. METHODS AND RESULTS: In this study, a gene encoding an esterase from Thermobifida fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purified using affinity chromatography. The TfEst kinetic assay revealed catalytic efficiencies of 0.58 s-1 mM-1, 1.09 s-1 mM-1, and 0.062 s-1 mM-1 against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20 min at 70 °C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/ß-hydrolase fold, which is consistent with other esterases. CONCLUSIONS: The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with significant implications for the advancement of biotechnology and biofuels industries.
Assuntos
Acetilesterase , Esterases , Thermobifida , Acetilesterase/metabolismo , Acetilesterase/genética , Acetilesterase/química , Concentração de Íons de Hidrogênio , Cinética , Especificidade por Substrato , Thermobifida/enzimologia , Thermobifida/genética , Esterases/metabolismo , Esterases/genética , Esterases/química , Estabilidade Enzimática , Temperatura , Escherichia coli/genética , Escherichia coli/metabolismo , Clonagem Molecular/métodos , Hidrólise , Xilanos/metabolismo , Butiratos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , NitrofenóisRESUMO
Background Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fne industries, particularly in pharmaceuticals, cosmetics, and bioethanol production. Methods and results In this study, a gene encoding an esterase from Thermobifda fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purifed using afnity chromatography. The TfEst kinetic assay revealed catalytic efciencies of 0.58 s −1 mM−1, 1.09 s−1 mM−1, and 0.062 s−1 mM−1 against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20 min at 70 °C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/β-hydrolase fold, which is consistent with other esterases. Conclusions The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with signifcant implications for the advancement of biotechnology and biofuels industries.
RESUMO
This study investigates the relationship between collective motion and propulsion of bacterial consortia and their biopolymer production efficiency. Rheological tests were conducted for suspensions of two different methanotrophic bacterial consortia obtained after enrichment of sediment samples from mangrove sites in Brazil. We considered the linear viscoelasticity region and analyzed the values of storage and loss moduli as functions of days of cultivation, for different values of the volume fraction. The suspensions' rheological behaviors reflected the bacterial growth stage. We found that the formation of structures over time in some types of consortia can hinder the movement of bacteria in the search for nutrients. The change in complex viscosity of the two consortia followed a different and rich behavior that appears to be closely related to their capacity to capture methane. Our analysis showed a possible correlation between collective motion, viscosity reduction, and biopolymer production. The pieces of evidence from this study suggest that the efficiency of bacterial motion is directly related to biopolymer production, and this could facilitate the process of identifying the best consortium of biopolymer producing bacteria.
Assuntos
Bactérias/crescimento & desenvolvimento , Hidroxibutiratos/metabolismo , Metano/metabolismo , Consórcios Microbianos , Poliésteres/metabolismo , ReologiaRESUMO
Botulinum (BoNT) and tetanus (TeNT) neurotoxins are bacterial zinc metalloproteases that cleave and inactivate cellular proteins essential for neurotransmitter release. There are seven serotypes of BoNT, while TeNT is found in one serotype. In order to characterize their enzymatic activities and to propose serotype-differentiation an enzymatic assay based on their metalloprotease activity was developed. The assays were conducted with FRET peptides derived from SNAP-25, synaptobrevin and syntaxin. The substrates were cleaved by 2 ng/mL of toxin at different rates (K(cat)/K(M) from 0.028 to 75.9 microM.s(-)) at a single bond, as confirmed by Q-TOF mass spectrometry. Inhibition of the hydrolysis was obtained with EDTA or with specific antibodies directed to each neurotoxin. Different substrate selectivities, especially by BoNT- A and E, suggest that these substrates can be used as a putative method for clostridial toxin quantification and serotype differentiation and could be easily adapted to a high-throughput protocols.
Assuntos
Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas/metabolismo , Metaloendopeptidases/metabolismo , Peptídeos/metabolismo , Proteínas R-SNARE/metabolismo , Toxina Tetânica/metabolismo , Sequência de Aminoácidos , Animais , Transferência Ressonante de Energia de Fluorescência , Hidrólise , Cinética , Camundongos , Dados de Sequência Molecular , Peptídeos/química , Proteínas R-SNARE/químicaRESUMO
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.
Assuntos
Bordetella pertussis/enzimologia , Corynebacterium diphtheriae/enzimologia , Meios de Cultura/química , Peptídeo Hidrolases/análise , Peptídeos/metabolismo , Sequência de Aminoácidos , Toxinas Bacterianas/análise , Bordetella pertussis/crescimento & desenvolvimento , Soluções Tampão , Cromatografia em Gel , Corynebacterium diphtheriae/crescimento & desenvolvimento , Eletroforese em Gel de Poliacrilamida , Filtração , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Hidrólise , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Peptídeo Hidrolases/isolamento & purificação , Peptídeos/química , Toxina Pertussis/análise , Cloreto de Sódio/química , Especificidade por Substrato , Trometamina/químicaRESUMO
Peptides that display bradykinin-potentiating activity have been obtained from a number of distinct sources, such as snake venoms, fibrinogen, and casein. This paper describes the characterization of two new peptides generated by tryptic hydrolysis of casein. No homology was found with other known vasoactive or vasopotentiating peptides, especially by the lack of Ile-Pro-Pro motif. The peptides EMPFPK and YPVEPFTE, corresponding to the gamma casein sequence (108-113 and 114-121, respectively), displayed a selective potentiating activity on isolated guinea pig ileum for bradykinin. Besides, the octapeptide YPVEPFTE showed an in vitro competitive inhibitor effect on angiotensin-converting enzyme and thimet oligopeptidase and presented an opiate-like activity, increasing two times the latence time in the hot-plate assay. The results suggest that the isolated bioactive peptides act on conversion and/or inactivation of endogenous peptides by enzymes such as angiotensin-converting enzyme and thimet oligopeptidase by modifying several systemic responses such as blood-pressure regulation and in pain response.
Assuntos
Bradicinina/fisiologia , Caseínas/metabolismo , Oligopeptídeos/farmacologia , Tripsina/metabolismo , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Pressão Sanguínea/fisiologia , Bradicinina/metabolismo , Caseínas/química , Feminino , Cobaias , Hidrólise , Íleo/efeitos dos fármacos , Íleo/metabolismo , Técnicas In Vitro , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/metabolismo , Oligopeptídeos/administração & dosagem , Oligopeptídeos/síntese química , Dor/fisiopatologia , Ratos , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
Peptides that display bradykinin-potentiating activity have been obtained from a number of distinct sources, such as snake venoms, fibrinogen, and casein. This paper describes the characterization of two new peptides generated by tryptic hydrolysis of casein. No homology was found with other known vasoactive or vasopotentiating peptides, especially by the lack of Ile-Pro-Pro motif. The peptides EMPFPK and YPVEPFTE, corresponding to the gamma casein sequence (108-113 and 114-121, respectively), displayed a selective potentiating activity on isolated guinea pig ileum for bradykinin. Besides, the octapeptide YPVEPFTE showed an in vitro competitive inhibitor effect on angiotensin-converting enzyme and thimet oligopeptidase and presented an opiate-like activity, increasing two times the latence time in the hot-plate assay. The results suggest that the isolated bioactive peptides act on conversion and/or inactivation of endogenous peptides by enzymes such as angiotensin-converting enzyme and thimet oligopeptidase by modifying several systemic responses such as blood-pressure regulation and in pain response.
Assuntos
Animais , Cobaias , BradicininaRESUMO
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).