RESUMEN
BACKGROUND: Nonribosomal peptide synthases (NRPS) can synthesize functionally diverse bioactive peptides by incorporating nonproteinogenic amino acids, offering a rich source of new drug leads. The bacterium Escherichia coli is a well-characterized production host and a promising candidate for the synthesis of nonribosomal peptides, but only limited bioprocess engineering has been reported for such molecules. We therefore developed a medium and optimized process parameters using the design of experiments (DoE) approach. RESULTS: We found that glycerol is not suitable as a carbon source for rhabdopeptide production, at least for the NRPS used for this study. Alternative carbon sources from the tricarboxylic acid cycle achieved much higher yields. DoE was used to optimize the pH and temperature in a stirred-tank reactor, revealing that optimal growth and optimal production required substantially different conditions. CONCLUSIONS: We developed a chemically defined adapted M9 medium matching the performance of complex medium (lysogeny broth) in terms of product concentration. The maximum yield in the reactor under optimized conditions was 126 mg L-1, representing a 31-fold increase compared to the first shaking-flask experiments with M9 medium and glycerol as the carbon source. Conditions that promoted cell growth tended to inhibit NRPS productivity. The challenge was therefore to find a compromise between these factors as the basis for further process development.
Asunto(s)
Péptido Sintasas/metabolismo , Reactores Biológicos/microbiología , Escherichia coli , Temperatura , Biotecnología , Carbono/metabolismo , Modelos Estadísticos , Electroforesis en Gel de Poliacrilamida , Bioingeniería , Concentración de Iones de HidrógenoRESUMEN
Plants endue a key role against illnesses caused by oxidative stress. These attributes are frequently associated with polyphenolic compounds. However, presence and concentration of secondary metabolites are affected by abiotic factors. The in vitro culture techniques can solve these drawbacks. Peppers can be a suitable alternative to obtain polyphenols. Aiming to optimise the callus culture stage from Capsicum baccatum to produce polyphenols, this work evaluated systemically the effects of the explant's origin (root, hypocotyl and cotyledon), growth hormone type (2,4-dichlorophenoxyacetic acid (2,4-D), benzylaminopurine (BAP) and a combination of 2,4-D/BAP at five-to-one ratio) and concentration (0.023-10.000 mg L-1) on callus culture efficiency parameters using a multilevel factorial design. The root explant in combination with BAP at 1.138 mg L-1 ensured the optimal values of the assessed responses; âcallus mass (225.03 mg), antioxidant activity (35.95%), total phenols (11.48 mg of GAE/g DE) and flavonoids (15.92 mg of RU/g DE) production.
Asunto(s)
Antioxidantes/farmacología , Capsicum/citología , Modelos Estadísticos , Técnicas de Cultivo de Tejidos/métodos , Ácido 2,4-Diclorofenoxiacético/farmacología , Antioxidantes/química , Compuestos de Bencilo/farmacología , Capsicum/efectos de los fármacos , Capsicum/metabolismo , Medios de Cultivo/química , Medios de Cultivo/farmacología , Flavonoides/metabolismo , Hipocótilo/citología , Fenoles/metabolismo , Extractos Vegetales/química , Extractos Vegetales/farmacología , Raíces de Plantas/citología , Polifenoles/farmacología , Purinas/farmacología , Metabolismo Secundario , Técnicas de Cultivo de Tejidos/estadística & datos numéricosRESUMEN
The mutual interactions among the consortium constructed by four indigenous bacteria and five inter-kingdom fusants and the effects of nitrogen and carbon supplementations on lignin degradation and laccase activity were investigated. Analyzed by Plackett-Burman and central composite design, the microbial consortium were optimized, Bacillus sp. (B) and PE-9 and Pseudomonas putida (Pp) and PE-9 had significant interactions on lignin degradation based on a 5% level of significance. The nitrogen and carbon supplementations played an important role in lignin degradation and laccase production. The ultimate lignin degradation efficiency of 96.0% and laccase activity of 268U/L were obtained with 0.5g/L of ammonium chloride and 2g/L of sucrose. Results suggested that a stable and effective microbial consortium in alkalescent conditions was successfully achieved through the introduction of fusants, which was significant for its industrial application.
Asunto(s)
Bacterias/metabolismo , Biodegradación Ambiental/efectos de los fármacos , Carbono/farmacología , Microbiología Industrial/métodos , Lignina/metabolismo , Consorcios Microbianos/fisiología , Nitrógeno/farmacología , Lacasa/metabolismoRESUMEN
New ß-1,3;1,4-glucanase was purified from Aspergillus niger US368. The pure glucanase has a molecular mass of about 32 kDa. The N-terminal sequence of the purified enzyme (A-G-T-N-P-P-I-G-V) was determined. The optimum pH and temperature recorded for enzyme activity were 5 and 60 °C, respectively. It also displayed marked thermostability with a half-life of 30 min at 70 °C. At 37 °C, the enzyme showed 100% stability from pH 3 to 10. The Km and Vmax values exhibited by the enzyme on barley ß-glucan were 0.62 mg ml(-1) and 34.46 U ml(-1), respectively. The enzyme is a retaining-one and was only active toward glucan containing ß-1,3;1,4-linkages. The production of ß-glucanase with barley flour as the sole carbon source was optimized. This is the first report on the purification and characterization of a ß-1,3;1,4-glucanase from A. niger. This lichenase could be considered as a candidate for future application particularly in the animal feed industry.
Asunto(s)
Aspergillus niger/enzimología , Glicósido Hidrolasas/aislamiento & purificación , Glicósido Hidrolasas/metabolismo , Temperatura , Secuencia de Aminoácidos , Alimentación Animal , Fenómenos Químicos , Estabilidad de Enzimas , Glicósido Hidrolasas/química , Concentración de Iones de Hidrógeno , Hidrólisis , Industrias , Cinética , Metales/farmacología , Especificidad por SustratoRESUMEN
We have developed a strategy for the optimization of plant cell suspension culture media using a combination of fractional factorial designs (FFDs) and response surface methodology (RSM). This sequential approach was applied to transformed tobacco BY-2 cells secreting a human antibody (M12) into the culture medium, in an effort to maximize yields. We found that the nutrients KNO3, NH4NO3 and CaCl2 and the hormones 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP) had the most significant impact on antibody accumulation. The factorial screening revealed strong interactions within the nutrients group (KNO3, NH4NO3 and CaCl2) and also individually between 2,4-D and three other components (KNO3, NH4NO3 and BAP). The RSM design resulted in a fivefold increase in the antibody concentration after 5 days and a twofold reduction in the packed cell volume (PCV). Longer cultivation in the optimized medium led to the further accumulation of antibody M12 in the culture medium (up to 107 µg/mL, day 10). Because the packed cell volume was reduced in the optimized medium, this enhanced the overall yield by 20-fold (day 7) and 31-fold (day 10) compared to the conventional MS medium.