RESUMEN
This research compared the effects of biosurfactant on the biodegradation of biodiesel and vegetable oils while validating two conceptually diverging methodologies. The two experimental setups were successfully modeled towards the effects of biosurfactants during biodegradation. We established the equivalence of both methodologies from the data output. As expected, the biosurfactants caused an increased oil uptake, thus increasing biodegradation performance. Cooking oils were favored by the microbial consortium as a carbon source when compared with biodiesel fuel, especially after use in food preparation. However, we found that biodiesel substrate standout with the highest biodegradation rates. Our results might indicate that a rapid metabolic change from the original compound initially favored biodiesels during the assimilation of organic carbon for a set specialized microbial inoculum. The data output was successfully combined with mathematical models and statistical tools to describe and predict the actual environmental behavior of biodiesel and vegetable oils. The models confirmed and predicted the biodegradation effectiveness with biosurfactants and estimated the required timeframe to achieve satisfactory contaminant removal.
Asunto(s)
Biodegradación Ambiental , Biocombustibles/análisis , Monitoreo del Ambiente/métodos , Consorcios Microbianos/fisiología , Aceites de Plantas/análisis , Tensoactivos/química , Carbono , Aceites de Plantas/metabolismo , Verduras/metabolismoRESUMEN
A serine proteinase inhibitor was purified from Bauhinia bauhinioides seeds after extraction with 0.15M NaCl by ion-exchange column chromatography on DEAE-Sephadex, gel filtration on Superose 12 column, Mono Q chromatography or alternatively by affinity chromatography on trypsin- Sepharose. The inhibitor is a single polypeptide chain with molecular mass 20 kDa by gel filtration on Superose 12, but was resolved into two peaks by ion - exchange chromatography on Mono Q (FPLC system). The main eluted peak inhibits trypsin (Ki = 0.6 nM), plasma kallikrein (Ki = 0.35 nM), plasmin (Ki = 33.1 nM), and weakly chymotrypsin (Ki = 2,700 nM), being the most effective plasma kallikrein inhibitor isolated from Bauhinia seeds. Therefore, it was denominated Bauhinia bauhinioides kallikrein inhibitor (BbKI). Activity is thermolabile and on trypsin inhibition optimum pH is 8.0. BbKI displays high homology to other plant Kunitz inhibitors, except for the absence of disulfide bridges, and the only cysteine residue is at the C-terminal position (residue 154) characterizes a distinct member of the Kunitz family. The affinity of the inhibitor to trypsin was confirmed by adsorption to trypsin-Sepharose resin and by isolation of the trypsin-inhibitor complex by gel filtration. Peptides with variations around the reactive site of BbKI (GLPVRFESPLRINIIKESY) were synthesized containing a quenched fluorogenic group. Trypsin but not plasma kallikrein substrates, these peptides strongly inhibited plasma kallikrein.
Asunto(s)
Proteínas de Plantas/farmacología , Calicreína Plasmática/antagonistas & inhibidores , Rosales/química , Inhibidores de Serina Proteinasa/aislamiento & purificación , Inhibidores de Tripsina/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Cromatografía de Afinidad , Colorantes Fluorescentes/metabolismo , Humanos , Hidrólisis , Cinética , Datos de Secuencia Molecular , Peso Molecular , Péptidos/síntesis química , Péptidos/química , Péptidos/farmacología , Proteínas de Plantas/aislamiento & purificación , Calicreína Plasmática/metabolismo , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Inhibidores de Serina Proteinasa/genética , Inhibidores de Serina Proteinasa/farmacología , Tripsina/química , Inhibidores de Tripsina/genética , Inhibidores de Tripsina/farmacologíaRESUMEN
Trypsin inhibitors were purified from a saline extract of Bauhinia bauhinioides seeds by ion-exchange column chromatography on DEAE-Sephadex, gel filtration on Superose 12 column, Mono Q ion-exchange chromatography or, alternatively, by affinity chromatography on trypsin-Sepharose. Both B. bauhinioides isolated inhibitors, BbTI-I and BbTI-II, inhibit trypsin being the dissociation constant 0.6 and 0.36 nM, respectively. BbTI-II only inhibits porcine pancreatic kallikrein hydrolysis of H-Pro-Phe-Arg-AMC (Ki 2.0 nM); the bradykinin-containing sequence LGMISLMKRPPGFSPFRSSRI-NH2 and the two kininogen related flanking quenched substrates Abz-MISLMKRP-EDDnp (Ki 2.0 nM) and Abz-FRSSRQ-EDDnp (Ki 2.5 nM).