Rodolfo Quintero

δ-Endotoxins induce cation channels in Spodoptera frugiperda brush border membranes in suspension and in planar lipid bilayers

Membrane potential measurements using a fluorescent dye indicated that two specific toxins active against Spodoptera frugiperda larvae (CryIC and CryID) cause immediate permeability changes in midgut epithelial brush border membrane vesicles (BBMV). The initial response and the sustained permeability change are cationic, notvery K+ selective, and occur at in vivo lethal doses (nM). The toxin response has a different ion selectivity and is more sensitive to Ba2+ than the intrinsic cation permeability of BBMV. Experiments incorporating BBMV into planar lipid bilayers (PLB) demonstrated that these vesicles contain cation channels (31, 47 and 76 pS). A 2–40 fold conductance increase was induced by nM concentrations of toxin in PLB containing BBMV. Cationic single channel transitions of 50, 106, 360 and 752 pS were resolved. Thus, Bacillus thuringiensis δ‐endotoxins induce an increase in cation membrane permeability involving ion channels in BBMV‐containing functional receptors.

Kinetic study of penicillin acylase production by recombinant E. coli in batch cultures

Abstract The pPA102 plasmid, containing the penicillin acylase gene (pac) under the regulation of the lacZ gene promoter was constructed and used to transform E. coli JM101. Batch cultures of the recombinant strain were performed to characterize the kinetics of growth, substrate consumption, and penicillin acylase (PA) production under different induction conditions. A saturation type behaviour of PA activity with respect to the inducer (isopropyl-β-thio-galactopyranoside (IPTG) concentration was observed. No detrimental effect of recombinant protein expression was noted on growth rate, maximum protein and cell concentration, and glucose specific consumption rate. A simplification of an existing mechanistic kinetic model was used to describe the specific PA production rate, which exhibited a maximum at intermediate growth rates. Induction during inoculation resulted in the highest penicillin acylase activity compared to induction during later growth phases. Accumulation of PA precursor protein suggests that postranslational processing and translocation through the cytoplasmic membrane limit PA production.

Exponentially fed-batch cultures as an alternative to chemostats: The case of penicillin acylase production by recombinant E. coli

Exponentially fed-batch cultures (EFBCs), fed with medium containing a highly concentrated carbon source, are commonly employed for attainment of high cell densities. However, large variations in environmental conditions occur, and quasi-steady-state is usually achieved only for the limiting substrate concentration, restricting the use of such cultures in kinetic characterization studies. In this work we report the production of recombinant penicillin acylase (PA) in EFBC of an E. coli JM101 transformed with the pPA102 plasmid, which includes the PA gene under regulation of the lacZ gene promoter and using isopropyl-beta-thio-galactopyranoside (IPTG) as inducer. The culture was fed with nonconcentrated complete medium, resulting in the attainment of quasi-steady-state conditions not only in substrate concentration, but also in cell concentration, and in the specific rates of growth, product production, and substrate consumption. Similar transient behavior was observed between EFBC and chemostat results. At quasi-steady-state, the dilution rate in the EFBC equaled the growth rate. Specific PA production rate during the fed-batch phase remained relatively constant at each dilution rate and followed typical Luedeking-Piret kinetics, with growth-associated and non-growth-associated constants of 142 U gDCW-1 and 7.2 U gDCW-1 h-1, respectively. Specific glucose consumption rate linearly increased from 0.025 to 0.6 g gDCW-1 h-1 as the dilution rate increased from 0.01 to 0.35 h-1. The maximum specific PA activity increased with decreasing dilution rate, reaching its highest value of 2.0 U mg-1 at a dilution rate of 0.01 h-1, the lowest dilution tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of microbial membranes used for the estimation of biochemical oxygen demand with a biosensor

A bacterial mixed culture was immobilized in Millipore filters to construct microbial-membranes for BOD determination using an oxygen electrode. The biosensor response was best when 0.5 mg cells were immobilized per cm2 of membrane, at 30°C, pH 7 and 0.05 M phosphate buffer. Reproducible microbial-membranes can be constructed and they can be stored for up to 20 days without appreciable loss of their response characteristics.

Penicillin acylase extraction by osmotic shock

Penicillin acylase was extracted from E. Coli by osmotic shock. The process was optimized by factorial design, scaled up and integrated to a purification process in order to compare it with purification processes reported in the literature. A specific parameter PPEF (Purification Processes Evaluation Factor) was defined for this purpose.

Design of two immobilized cell catalysts by entrapment on gelatin: Internal diffusion aspects

Experimental results obtained during the design of two immobilized cell catalysts by entrapment on gelatin are presented. Strong diffusional limitations are found and explained with the usual parameters and models, introducing an empirical correlation between substrate concentration and effectiveness factor. The effect of particle size, enzyme load, and specific activity in the system is discussed in terms of cooperation between bioengineers and geneticists.

Microbial sensor for penicillins using a recombinant strain of Escherichia coli

E. coli harboring the multicopy plasmid pBR327, which codes for beta-lactamase synthesis, was immobilized on acetylcellulose membranes. These were placed in the vicinity of a flat pH electrode, thus constituting a penicillin biosensor based on the detection of changes in pH using a reference pH electrode. A response time of 8 min was obtained with at least 2 mg of cells cm-2 of membrane. A linear detection range between 5 and 30 mM of penicillin was achieved. Buffering capacity decreased the sensitivity, but to a lower extent as when compared with probes using purified enzyme. The sensor was completely stable for at least 13 days and proved to be useful for penicillin estimation in complex media such as fermentation broths and milk.

Maximizing the expression of recombinant proteins in Escheria coli by manipulation of culture conditions

Abstract The expression of hybrid proteins β-galactosidase-human insulin chain A (constitutive), and β-galactosidase-human insulin chain A (constitutive), and β-galactosidase-human insulin chain B (inducible) was studied. The main aspects covered included plasmid stability and optimization of expression levels. In both systems, the ampicillin used for selective pressure exerted its action for only a few minutes during culture, hardly affecting plasmid segregation trends. Without the antibiotic, segregants were very low and reached a level of 10% only after seven sub-cultures. Expression levels in the A chain system were closely related to the biomass production. Maximum levels were reached developing the inoculum in minimal media, as well as by balancing and statistically optimizing the medium. The B chain system appeared to have higher plasmid stability than the A chain one. By optimizing the medium, similar induction levels to those obtained by using IPTG were attained using lactose as the main carbon source. Hybrid production was inversely related to the cell/glucose yield. In both systems, sub-culturing the bacteria in minimal medium increased substantially the production of hybrid proteins. Sub-culturing in rich medium with small amounts of ampicillin had the opposite effect. It seems that plasmid copy number dynamics could be playing an important role in this phenomenon.

Cell Suspension Culture of Solanum chrysotrichum (Schldl.) - A Plant producing an Antifungal Spirostanol Saponin

The production of an antifungal spirostanol saponin designated SC-1 has been detected in cell suspension cultures of the Mexican species Solanum chrysotrichum. Batch cultures of a cell suspension obtained from hypocotyl derived calluses of this species were grown for 25 days in shake flasks containing Murashige & Skoog (MS) medium. Throughout the growth cycle, fresh and dry weight, SC-1 yield, and uptake of sucrose, glucose and fructose were determined. The effects of inoculum size and sucrose concentration on the biomass accumulation and synthesis of the active metabolite, were studied. The maximum SC-1 production, above 14 mg.g−1 (which was fifty times that of field grown plants), was reached after 20 days using a 2% inoculum and complete MS medium supplemented with 2 mgl−1 2,4-D, 2 mg l−1kinetin, and sucrose between 30 and 45 gl−1..

Design and kinetic characterization of a whole cell penicillin acylase biocatalyst using E. coli

Abstract The design and characterization of a biocatalyst with whole cells of E. coli containing penicillin acylase activity were carried out. The biocatalyst was prepared by gel entrapment using a two-phase dispersion system consisting of a suspension of cells and agar in oil. The catalyst was characterized in terms of its kinetic and diffusional properties as a function of particle size, as well as its storage and operational stability. An economical analysis of the process was taken as the basis for a rational design of a genetic engineering programme to improve the specific activity of the biocatalyst.