Optimization by factorial analysis of caprylic acid precipitation of non-immunoglobulins from hyperimmune equine plasma for antivenom preparation.

Optimization of caprylic acid precipitation of equine plasma non-immunoglobulin proteins for antivenom preparation was achieved by regression analysis of the responses of three highly significant factors assayed by factorial design. The factors studied were caprylic acid concentration, plasma pH and temperature, and their response was assessed in terms of filtration speed, residual albumin, total protein content and turbidity. The results evidenced that the three variables are involved in the precipitation process. Moreover, the factors displayed significant interactions, indicating that their levels distinctly affect the optimization procedure. The best combination was 3% caprylic acid, 37 °C and plasma pH 4.9; under these conditions, all immunoglobulins and only 0.1% albumin remained in the supernatant, in a very fast and simple procedure. After formulation, the antivenom obtained by this procedure presented full lethality neutralizing activity and absence of protein aggregates.

Rapid affinity purification processes for cyclodextrin glycosyltransferase from Bacillus circulans.

Two rapid and easy-to-scale-up methods for the purification of cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans were developed: affinity precipitation with starch and aqueous two-phase partition. The first method, optimised by a factorial design, gave an 80% CGTase adsorption at 11% starch and 1.6% ammonium sulphate, and a 65% recovery after elution with 10 mM alpha-cyclodextrin. The purification factor was 17. Aqueous two-phase partition yielded a 72% CGTase recovery in a two-step procedure; CGTase was obtained in the bottom phase with a purification factor of 37.

Detection and characterization of quorum sensing signal molecules in Acinetobacter strains.

Quorum sensing is a widespread regulatory mechanism among Gram-negative bacteria. In this study, Acinetobacter strains were assayed for the presence of quorum sensing signal molecules capable of activating N-acylhomoserine lactone biosensors. By using an Agrobacterium tumefaciens reporter strain it was shown that all the cultures produced two to four detectable signal molecules with different chromatographic patterns. In A. calcoaceticus BD413 supernatants four compounds were detected in a time-dependent manner, and maximal activity was reached at stationary phase. The number of signal molecules was dependent on medium composition; typically, cultures in minimal medium displayed one or two more signals, as compared to complex medium. None of the Acinetobacter supematants showed autoinduction activity with an Chromobacterium violaceum reporter strain, neither in direct or competition assays.

Purification of a lipase from Acinetobacter calcoaceticus AAC323-1 by hydrophobic-interaction methods.

The performance of hydrophobic-interaction chromatography (HIC) for the purification of Acinetobacter calcoaceticus AAC323-1 lipase was compared with that of various aqueous two-phase systems. While a 42% lipase yield with a purification factor of 140 could be recovered by HIC, higher yields were achieved by using aqueous two-phase systems, either those formed by poly(ethylene glycol) and dextran or those based upon the use of a detergent. Triton X-114-based aqueous two-phase partition showed the best performance, with a yield of 81% and a purification factor of 68. Further detergent removal was easily achieved with an adsorbent, with no significant decrease in yields. Owing to its simplicity, the method should be easy to scale-up.

Stability of Escherichia coli strains harboring recombinant plasmids for L-threonine production.

Escherichia coli recombinant strains bearing the thr operon have been previously selected for threonine production and phenotypically classified according to antibiotic resistance properties (Nudel et al. 1987). Further analysis of those strains permitted the isolation and restriction mapping of two different plasmids of 13 kb and 18.6 kb. The smaller one, which expressed tetracycline resistance gave better results on threonine accumulation but it was rather unstable when grown without antibiotic pressure. Therefore, other hosts were transformed with those plasmids to improve stability. A threonine-auxotrophic strain was a better host for plasmid maintenance and expression of thr operon. Host influence in plasmid-mediated threonine production was studied in terms of specific yields (the ratios of threonine accumulated to biomass values) and of plasmid maintenance (percent of AprTcr clones after cultivation in non selective media). We also determined that semisynthetic media of defined composition were better than rich media for threonine expression, due to feed-back controls exerted by undesired catabolites accumulated in complex media.

[Selection of bacterial strains for the production of threonine]. / Selección de cepas bacterianas para la producción de treonina.

The production of L-threonine in submerged culture was studied in the following bacterial strains; Brevibacterium flavum ATCC 21269, Corynebacterium acetoacidophilum ATCC 21270, Escherichia coli ATCC 21149, E. coli NRRL 12098, E. coli NRRL 12099 and E. coli NRRL 12100. Erlenmeyer flasks with different volumetric relations liquid/recipient, were used to study the influence of the volumetric oxygen transfer rate. B. flavum reached levels of threonine of 0.72 g/l at 96 hours of culture with a volumetric relation liquid/recipient of 1:5. With a relation 1:25 the maximal level was reached at 48 hours (0.60 g/l of threonine) (Table 1). In addition to threonine this strain accumulated in the culture media glutamic acid (+/- 2 g/l), alanine or glycine and proline. With E. coli ATCC 21149 the aeration favored the production of threonine reaching levels of 0.38 g/l in six day cultures with valine and alanine at levels approximate to 2 g/l. Excepting C. acetoacidophilum, all the strains produced threonine at levels of 0.30 to 3.55 g/l (Table 5). E. coli NRRL 12098 and E. coli NRRL 12100 produced only threonine, the culture medium being free from other aminoacids. With E. coli NRRL 12098 levels of 2 g/l were attained but the production was restricted to the presence of yeast extract in the media (Table 2). E. coli NRRL 12100 was the best strain and the inoculum media influenced the production (Table 3) and the best threonine levels were reached in the best aeration conditions assayed (Table 4) with 3.55 g/l for volumetric relations 1:10 and 1.25 g/l for 1:5.

Selección de cepas bacterianas para la producción de treonina / [Selection of bacterial strains for the production of threonine]

The production of L-threonine in submerged culture was studied in the following bacterial strains; Brevibacterium flavum ATCC 21269, Corynebacterium acetoacidophilum ATCC 21270, Escherichia coli ATCC 21149, E. coli NRRL 12098, E. coli NRRL 12099 and E. coli NRRL 12100. Erlenmeyer flasks with different volumetric relations liquid/recipient, were used to study the influence of the volumetric oxygen transfer rate. B. flavum reached levels of threonine of 0.72 g/l at 96 hours of culture with a volumetric relation liquid/recipient of 1:5. With a relation 1:25 the maximal level was reached at 48 hours (0.60 g/l of threonine) (Table 1). In addition to threonine this strain accumulated in the culture media glutamic acid (+/- 2 g/l), alanine or glycine and proline. With E. coli ATCC 21149 the aeration favored the production of threonine reaching levels of 0.38 g/l in six day cultures with valine and alanine at levels approximate to 2 g/l. Excepting C. acetoacidophilum, all the strains produced threonine at levels of 0.30 to 3.55 g/l (Table 5). E. coli NRRL 12098 and E. coli NRRL 12100 produced only threonine, the culture medium being free from other aminoacids. With E. coli NRRL 12098 levels of 2 g/l were attained but the production was restricted to the presence of yeast extract in the media (Table 2). E. coli NRRL 12100 was the best strain and the inoculum media influenced the production (Table 3) and the best threonine levels were reached in the best aeration conditions assayed (Table 4) with 3.55 g/l for volumetric relations 1:10 and 1.25 g/l for 1:5.