High Kanamycin Concentration as Another Stress Factor Additional to Temperature to Increase pDNA Production in E. coli DH5α Batch and Fed-Batch Cultures.

Plasmid DNA (pDNA) vaccines require high supercoiled-pDNA doses (milligrams) to achieve an adequate immune response. Therefore, processes development to obtain high pDNA yields and productivity is crucial. pDNA production is affected by several factors including culture type, medium composition, and growth conditions. We evaluated the effect of kanamycin concentration and temperature on pDNA production, overflow metabolism (organic acids) and metabolic burden (neomycin phosphotransferase II) in batch and fed-batch cultures of Escherichia coli DH5α-pVAX1-NH36. Results indicated that high kanamycin concentration increases the volumetric productivity, volumetric and specific yields of pDNA when batch cultures were carried out at 42 °C, and overflow metabolism reduced but metabolic burden increased. Micrographs taken with a scanning electron microscope (SEM) were analyzed, showing important morphological changes. The high kanamycin concentration (300 mg/L) was evaluated in high cell density culture (50 gDCW/L), which was reached using a fed-batch culture with temperature increase by controlling heating and growth rates. The pDNA volumetric yield and productivity were 759 mg/L and 31.19 mg/L/h, respectively, two-fold greater than the control with a kanamycin concentration of 50 mg/L. A stress-based process simultaneously caused by temperature and high kanamycin concentration can be successfully applied to increase pDNA production.

Developing strategies to increase plasmid DNA production in Escherichia coli DH5α using batch culture.

Plasmid DNA (pDNA) production has recently increased as a result of advances in DNA vaccines. The practical development of pDNA vaccines requires high yield and productivity of supercoiled plasmid DNA (sc-pDNA). The yield and productivity are influenced by the host strain, the plasmid, the production process, and especially by growth conditions, such as the culture type and medium. We evaluated different strategies to increase pDNA production by Escherichia coli DH5α in batch culture. The strategies were driven by the development of a four single-factor experimental design and were based on the change of culture media composition in terms of carbon and nitrogen and the modification of the pH control by using NaOH or NH4OH. The results revealed the carbon (50g/L of glycerol) and nitrogen (8.34g/L of YESP) concentration in the culture medium and starting pH control with NH4OH when most of the organic nitrogen was consumed. Under these conditions, we obtained a volumetric yield of 213mg pDNA/L, a specific yield of 10mg pDNA/g DCW (dry cell weight), 92% of sc-pDNA and a productivity of 17.6mg pDNA/(Lh). The pDNA productivities reached were 42% higher than the productivities reported by other authors applying similar conditions.

Alternative non-chromatographic method for alcohols determination in Clostridium acetobutylicum fermentations.

An economic, simple, quantitative, and non-chromatographic method for the determination of alcohols using microdiffusion principle has been adapted and validated for acetone-butanol-ethanol (ABE) fermentation samples. This method, based on alcohols oxidation using potassium dichromate in acid medium, and detection by spectrophotometry, was evaluated varying, both, temperature (35°C, 45°C, and 55°C) and reaction time (0 to 125min). With a sample analysis time of 90min at 45°C, a limit of detection (LOD), and a limit of quantification (LOQ) of 0.10, and 0.40g/L, respectively. The proposed method has been successfully applied to determine butanol and ethanol concentrations in ABE fermentation samples with the advantage that multiple samples can be analyzed simultaneously. The measurements obtained with the proposed method were in good agreement with those obtained with the Gas Chromatography Method (GCM). This proposed method is useful for routine analysis of alcohols and screening samples in laboratories and industries.

Cloning and expression of a novel, moderately thermostable xylanase-encoding gene (Cflxyn11A) from Cellulomonas flavigena.

The Cfl xyn11A gene, encoding the endo-1,4-beta-xylanase Cfl Xyn11A from Cellulomonas flavigena, was isolated from a genomic DNA library. The open reading frame of the Cfl xyn11A gene was 999 base pairs long and encoded a polypeptide (Cfl Xyn11A) of 332 amino acids with a calculated molecular mass of 35,110Da. The Cfl xyn11A gene was expressed in Escherichia coli and the recombinant enzyme, with an estimated molecular weight of 31kDa was purified and xylanase activity was measured. Cfl Xyn11A showed optimal activity at pH 6.5 and 55 degrees C. The enzyme demonstrated moderate thermal stability as Cfl Xyn11A maintained 50% of its activity when incubated at 55 degrees C for 1h or at 45 degrees C for 6h. This is the first report describing the cloning, expression and functional characterization of an endo-1,4-beta-xylanase-encoding gene from C. flavigena. Cfl Xyn11A may be suitable for industrial applications in the food and feed industries, or in the pre-treatment of lignocellulosic biomass required to improve the yields of fermentable sugars for bioethanol production.

Purification and characterization of two sugarcane bagasse-absorbable thermophilic xylanases from the mesophilic Cellulomonas flavigena.

We report the purification and characterization of two thermophilic xylanases from the mesophilic bacteria Cellulomonas flavigena grown on sugarcane bagasse (SCB) as the only carbon source. Extracellular xylanase activity produced by C. flavigena was found both free in the culture supernatant and associated with residual SCB. To identify some of the molecules responsible for the xylanase activity in the substrate-bound fraction, residual SCB was treated with 3 M guanidine hydrochloride and then with 6 M urea. Further analysis of the eluted material led to the identification of two xylanases Xyl36 (36 kDa) and Xyl53 (53 kDa). The pI for Xyl36 was 5.0, while the pI for Xyl53 was 4.5. Xyl36 had a Km value of 1.95 mg/ml, while Xyl53 had a Km value of 0.78 mg/ml. In addition to SCB, Xyl36 and Xyl53 were also able to bind to insoluble oat spelt xylan and Avicel, as shown by substrate-binding assays. Xyl36 and Xyl53 showed optimal activity at pH 6.5, and at optimal temperature 65 and 55 degrees C, respectively. Xyl36 and Xyl53 retained 24 and 35%, respectively, of their original activity after 8 h of incubation at their optimal temperature. As far as we know, this is the first study on the thermostability properties of purified xylanases from microorganisms belonging to the genus Cellulomonas.