Amplification of the Escherichia coli lacZ gene in Bacillus subtilis and its expression on a by-product growth medium.

A Bacillus subtilis wild type strain and a kinA (spoIIJ) isogenic mutant were compared as hosts for the expression of the Escherichia coli beta-galactosidase gene, lacZ, driven by the B. subtilis aprE promoter in a chromosomal system. The 2 x SG sporulation formula, with some modifications, was used as a basal medium. The specific activity values recorded by the mutant strain at the stationary phase were markedly higher than those achieved by the wild type host. Exposure of the cells to increasing levels of chloramphenicol resulted in significant amplifications of the lacZ region. Gene copy numbers of 19 and 11 were estimated in the amplified wild type and kinA strains, respectively, with high segregational stability records. The magnitude of beta-galactosidase over-expression was dependent on, and roughly proportional to antibiotic resistance levels. Among five examined by-products, a 2.3-times diluted concentration of neutralized cheese whey was successfully used as a sole medium component for over-expression of the recombinant beta-galactosidase gene in B. subtilis.

Identification and molecular characterization of a novel Bacillus strain capable of degrading Tween-80.

A Tween-80-degrading novel marine Bacillus strain, N10, has recently been isolated in Alexandria University, Egypt. The taxonomic position of this endospore forming bacterium was investigated on the basis of fatty acid analysis and 16S rRNA gene sequencing. Comparative computer database analyses revealed that the bacterium is a Bacillus subtilis strain. The gene encoding the small acid-soluble protein gamma-type (SASP-B), sspE, was successfully utilized in this study as a tool for discrimination between the two B. subtilis subspecies W23 and 168. Based on the alignment of 16S rRNA sequences and analysis of SASP-B relatedness, it has been demonstrated that the novel marine B. subtilis strain N10 is more closely related to the B. subtilis reference strain W23 than to 168. The strain, N10, has been deposited in the Bacillus Genetic Stock Center (BGSC) and assigned the accession number 3A17.

Application of the response surface methodology for optimizing the activity of an aprE-driven gene expression system in Bacillus subtilis.

The major targets for improvement of recombinant expression systems in microbial cells are gene dosage, transcriptional control machinery and, to some extent, translation. Here we show that optimization of fermentation conditions by applying statistically designed, multifactorial experiments offers an additional method for potential enhancement of gene expression systems. A chromosomally encoded fusion between the Bacillus subtilis aprE regulatory region and the E. coli lacZ gene carried by the B. subtilis host cells was used. The 2 x SG sporulation medium was used as a basal medium. Among the 11 fermentation factors we examined, the most significant variables influencing beta-galactosidase expression were statistically elucidated for optimization and included peptone, MgSO4.7H2O, and KCl. The optimum concentrations of these variables were predicted by using a second-order polynomial model fitted to the results obtained by applying the Box-Behnken design, a response surface method. Calculated optimum concentrations were predicted to confer a maximum yield of 2,423.5 beta-galactosidase specific activity units. A verification experiment performed under optimal conditions yielded 96% of the predicted specific activity value with an increase by a factor of almost 5 compared with the results obtained under basal conditions.

Cadmium biosorption by a cadmium resistant strain of Bacillus thuringiensis: regulation and optimization of cell surface affinity for metal cations.

A marine bacterial strain putatively identified as Bacillus thuringiensis strain DM55, showed multiple heavy metal resistance and biosorption phenotypes. Electron microscopic studies revealed that DM55 cells are encased in anionic cell wall polymers that can immobilize discrete aggregates of cations. Factors affecting cell surface affinity for metal cations, monitored by means of Cd2+ binding capability, are investigated. The mechanisms of cadmium resistance and Cd2+ biosorption by the bacterium appeared to be inducible and coincident. Medium components affecting metal removal under cadmium-stressed growth conditions were explored based on the application of two sequential multi-factorial statistical designs. Concentrations of potassium phosphates and peptone were the most significant variables. Optimized culture conditions allowed DM55 cells grown in the presence of 0.25 mM CdCl2 to remove about 79% of the metal ions within 24 h with a specific biosorption capacity of 21.57 mg g(-1) of biomass. Both fresh and dry cells of DM55 prepared under cadmium-free optimal nutrient condition were also able to biosorb Cd2+. In addition to the concentration of phosphate in the medium, KinA, a major phosphate provider in the phosphorelay of Bacillus cells, was also demonstrated to regulate the magnitude of cell surface affinity for cadmium ions.

Production of beta-mannanase by B. subtilis from agro-industrial by-products: screening and optimization. Mannanase from wastes.

We have demonstrated that a mixture of wheat bran (35 g l-1), as a main substrate, and palm seed powder (10 g l-1), as a co-substrate, is appropriate for beta-mannanase production by Bacillus subtilis. A 2n factorial experimental design was employed as a primary step for medium optimization. The enzyme activity titters obtained at the optimized growth condition were equivalent to about 319% of the beta-mannanase activity and 114% of the specific activity levels reached by a galactomannan-based culture.

The development of a Bacillus subtilis 168 culture condition for enhanced and accelerated beta-mannanase production.

A shaken flask cultivation condition for enhanced and accelerated beta-mannanase formation by Bacillus subtilis 168 was achieved. Among five examined fermentation media a formula that supported enzyme generation and retarded biomass yield and sporulation was selected. The deficiency of biomass production in this medium was mastered by choosing a seed culture medium that accelerated growth and initiation of beta-mannanase synthesis. With respect to enzyme production, the optimum pH and temperature were 7 and 37 degrees C, respectively. The biosynthesis of the enzyme was extremely influenced by the cell growth state as a modulator, glucose as a catabolite repressor, and galactomannan as an inducer. A galactomannan concentration of 4 g l-1 induced a beta-mannanase activity level of 17.5 U ml-1 after 24 h of incubation at the experimental condition. Higher inducer concentrations supported growth rather than enzyme production. The influence of inoculum size was so remarkable that, at optimum, a crude filtrate with an enzyme activity of 33U ml-1 was yielded within 4 hours. It appears that this is among the highest rates reported for beta-mannanase production. We have also demonstrated that blocking of the sporulation process at stage II do not affect enzyme production significantly. This would allow an extended enzyme production phase especially in a continuous culture.