Fed-Batch production of glucose 6-Phosphate dehydrogenase using recombinant saccharomyces cerevisiae

The strain Saccharomyces cerevisiae W303-181, having the plasmid YEpPGK-G6P (built by coupling the vector YEPLAC 181 with the promoter phosphoglycerate kinase 1), was cultured by fed-batch process in order to evaluate its capability in the formation of glucose 6-phosphate dehydrogenase (EC.1.1.1.49). Two liters of culture medium (10.0 g/L glucose, 3.7 g/L yeast nitrogen broth (YNB), 0.02 g/L L-tryptophan, 0.02 g/L L-histidine, 0.02 g/L uracil, and 0.02 g/L adenine) were inoculated with 1.5 g dry cell/L and left fermenting in the batch mode at pH 5.7, aeration of 2.2 vvm, 30 degrees C, and agitation of 400 rpm. After glucose concentration in the medium was lower than 1.0 g/L, the cell culture was fed with a solution of glucose (10.0 g/L) or micronutrients (L-tryptophan, L-histidine, uracil, and adenine each one at a concentration of 0.02 g/L) following the constant, linear, or exponential mode. The volume of the culture medium in the fed-batch process was varied from 2 L up to 3 L during 5 h. The highest glucose 6-phosphate dehydrogenase activity (350 U/L; 1 U=1 micromol of NADP/min) occurred when the glucose solution was fed into the fermenter through the decreasing linear mode.     

Protein binding onto surfactant-based synthetic vesicles

Synthetic vesicles were prepared by mixing anionic and cationic surfactants, aqueous sodium dodecylsulfate with didodecyltrimethylammonium or cetyltrimethylammonium bromide. The overall surfactant content and the (anionic/cationic) mole ratios allow one to obtain negatively charged vesicles. In the phase diagram, the vesicular region is located between a solution phase, a lamellar liquid crystalline dispersion, and a precipitate area. Characterization of the vesicles was performed by electrophoretic mobility, NMR, TEM, and DLS and we determined their uni-lamellar character, size, stability, and charge density. Negatively charged vesicular dispersions, made of sodium dodecylsulfate/didodecyltrimethylammonium bromide or sodium dodecylsulfate/cetyltrimethylammonium bromide, were mixed with lysozyme, to form lipoplexes. Depending on the protein/vesicle charge ratio, binding, surface saturation, and lipoplexes flocculation, or precipitation, occurs. The free protein in excess remains in solution, after binding saturation. The systems were investigated by thermodynamic (surface tension and solution calorimetry), DLS, CD, TEM, 1H NMR, transport properties, electrophoretic mobility, and dielectric relaxation. The latter two methods give information on the vesicle charge neutralization by adsorbed protein. Binding is concomitant to modifications in the double layer thickness of vesicles and in the surface charge density of the resulting lipoplexes. This is also confirmed by developing the electrophoretic mobility results in terms of a Langmuir-like adsorption isotherm. Charges in excess with respect to the amount required to neutralize the vesicle surface promote lipoplexes clustering and/or flocculation. Protein-vesicle interactions were observed by DLS, indicating changes in particle size (and in their distribution functions) upon addition of LYSO. According to CD, the bound protein retains its native conformation, at least in the SDS/CTAB vesicular system. In fact, changes in the alpha-helix and beta-sheet conformations are moderate, if any. Calorimetric methods indicate that the maximum heat effect for LYSO binding occurs at charge neutralization. They also indicate that enthalpic are by far the dominant contributions to the system stability. Accordingly, energy effects associated with charge neutralization and double-layer contributions are much higher than counterion exchange and dehydration terms.     

Adsorption of endotoxins on Ca2+ iminodiacetic acid by metal ion affinity chromatography

Endotoxins (also known as lipopolysaccharides (LPS)) are undesirable by products of recombinant proteins, purified from Escherichia coli. LPS can be considered stable under a wide range of temperature and pH, making their removal one of the most difficult tasks in downstream processes during protein purification. The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration. Immobilized metal affinity chromatography (IMAC) enables the affinity interactions between the metal ions (immobilized on the support through the chelating compound) and the target molecules, thus enabling high efficiency separation of the target molecules from other components present in a mixture. Affinity chromatography is applied with Ca2+ iminodiacetic acid (IDA) to remove most of the LPS contaminants from the end product (more than 90%). In this study, the adsorption of LPS on an IDA-Ca2+was investigated. The adsorption Freundlich isotherm of LPS-IDA-Ca2+provides a theoretical basis for LPS removal. It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ligands on the beads. The factors such as pH (4.0 or 5.5) and ionic strength (1.0 mol/L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin’ concentration values less than 100 EU/mL and 100000 EU/mL. This new protocol represents a substantial advantage in time, effort, and production costs.     

Gevrey solvability near the characteristic set for a class of planar complex vector fields of infinite type

We study the Gevrey solvability of a class of complex vector fields, defined on Ω?=(−?,?)×S¹, given by L=∂/∂t+(a(x)+ib(x))∂/∂x, b?0, near the characteristic set Σ={0}×S¹. We show that the interplay between the order of vanishing of the functions a and b at x=0 plays a role in the Gevrey solvability.     

Production of nisin by Lactococcus lactis in media with skimmed milk

Nisin is a bacteriocin that inhibits the germination and growth of Gram-positive bacteria. With nisin expression related to growth conditions of Lactococcus lactis subsp. lactis, the effects of growth parameters, media components, and incubation time were studied to optimize expression. L. lactis ATCC 11454 was grown (100 rpm at 30°C for 36 h) in both M17 and MRS standard broth media (pH 6.0–7.0) supplemented with sucrose (1.0–12.5 g/L), potassium phosphate (0.13 g/L), asparagine (0.5 g/L), and sucrose (0.24 g/L), and diluted 1:1 with liquid nonfat milk. Liquid nonfat milk, undiluted, was also used as another medium (9% total solids, pH 6.5). Nisin production was assayed by agar diffusion using Lactobacillus sake ATCC 15521 (30°C for 24 h) as the sensitive test organism. The titers of nisin expressed and released in culture media were quantified and expressed in arbitrary units (AU/L of medium) and converted into known concentrations of “standard nisin” (Nisaplin®, g/L). The detection of nisin activity was <0.01 AU/L in M17 and MRS broths, and 7.5 AU/L in M17 with 0.14% sucrose or 0.13% other supplements, and the activity increased to 142.5 AU/L in M17 diluted with liquid nonfat milk (1:1). The 25% milk added to either 25% M17 or 25% MRS provided the highest levels of nisin assayed.     

Xylanase recovery effect of extraction conditions on the aqueous two-phase system using experimental design

The partitioning of xylanase produced byPenicillium janthinellum in aqueous two-phase systems (ATPS) using poly(ethylene glycol) (PEG) and phosphate (K₂HPO₄/KH₂PO₄) was studied employing a statistical experimental design. The aim was to identify the key factors governing xylanase partitioning. The interactions of five factors (PEG concentration molecular weight, concentration of buffer K₂HPO₄/KH₂PO₄, pH, and NaCl concentration) and their main effects on the partition coefficient (K) were evaluated by means of a 2⁵ full-factorial experimental design with four center points. The %PEG, %NaCl, and pH were the most important factors affecting the response variable (K). Response surface methodology (RSM) was adopted and an empirical second-order polynomial model was constructed on the basis of the results. The optimum partition conditions were pH 7.0, PEG = 8.83% and NaCl = 6.02%. Adequacy of the model for predicting optimum response value was tested under these conditions. The experimental xylanase partition coefficient (K) was 2.21, whereas its value predicted by the model was 2.33. These results indicate that the predicted model was adequate for the process. PEG molecular weight and phosphate concentration did not affect the xylanase partition coefficient.     

The effect of poly(ethylene glycol) on the activity and structure of glucose-6-phosphate dehydrogenase in solution

The effect of poly(ethylene glycol), PEG, on the enzymatic activity of glucose-6-phosphate dehydrogenase (G-6-PDH) in the oxidation of glucose-6-phosphate (G-6-P), using NADP+ as co-enzyme was investigated. The enzymatic activity was determined by means of spectrophotometry in three different media: pure Tris–HCl buffer, solution of PEG400 (20 wt.%) and of PEG4000 (20 wt.%), both in buffer. Comparing the enzymatic activity values measured in pure buffer with those in the polymer solutions, an increase in the enzymatic activity of 20% was observed in the presence of PEG400 as well as in PEG4000. Calorimetric studies indicated the absence of preferential interactions between G-6-PDH and PEG400 or PEG4000. Nevertheless, the interaction enthalpy, ΔH_int, between NADP+ and PEG400 and PEG4000 amounted to −9.3 and −26.7 kJ/mol, respectively. Small angle X-ray scattering (SAXS) measurements were performed in a higher concentration range. Data analysis performed from SAXS curves by means of the intra-particle distance distribution function p(r) and Guinier plots yielded for G-6-PDH in pure buffer and PEG400 solutions radius of gyration, R_g, of about 70 Å and in PEG4000 solutions, R_g of about 40 Å. The latter has the same dimension as that found in the dimeric crystallographic structure of G-6-PDH, evidencing that G-6-PDH preserves its dimeric configuration in PEG4000 solution. On the contrary, different aggregates of G-6-PDH are formed in the presence of either buffer or PEG400. These findings show that the presence of PEG in solution can exert an effect on the enzyme structure and activity.     

Dynamics of DNA adsorption on and release from SDS-DDAB cat-anionic vesicles: a multitechnique study

DNA adsorption and release from cat-anionic vesicles made of sodium dodecylsulfate-dodecyldimethylammonium bromide (SDS-DDAB) in nonstoichiometric amounts was investigated by different electrochemical, spectroscopic, and biomolecular strategies. The characterization of the vesicular system was performed by dynamic light scattering, which allowed estimating both its size and distribution function(s). The interaction dynamics was followed by dielectric spectroscopy and zeta-potential, as well as by agarose gel electrophoresis, AGE. Also, circular dichroism, CD, measurements were carried out, to ascertain possible structural rearrangements of DNA, consequent to the interactions with the cat-anionic vesicles. CD demonstrates that vesicle-bound DNA retains its native conformation. The results obtained by the aforementioned techniques are consistent and indicate that binding saturation is obtained at a [DNA/vesicles] charge ratio close to 0.8, considering only the excess surface charges on the vesicles. This result is apparently in contradiction with a purely electrostatic approach and is tentatively ascribed to the distance between charges on the biopolymer and the vesicle surface, respectively. A possible interpretation is discussed. The nucleic acid can be completely retrieved from the vesicles upon addition of adequate amounts of SDS, which is the defective surfactant in the vesicular system. Precipitation of the poorly soluble SD-DDA salt results in an almost complete release of DNA.