Increasing the stability of sacB transcript improves levansucrase production in Bacillus subtilis.

Aims: To develop a strategy to increase the stability of transcripts of structural genes expressed under the control of sacR, the leader region of Bacillus subtilis levansucrase gene. Methods and Results: Insertion of Shine Dalgarno like sequences in the 5'-untranslated sacR region controlling the expression of sacB. Depending on the number of stabilizing sequences inserted and the position of these sequences with respect to the translation start codon, it was observed that the mRNA stability and the final protein production could be increased or decreased. Conclusions: This mRNA stabilization can be used to increase exocellular protein production in the degU32 (Hy) mutant. Significance and Impact of the Study: This approach can be applied to the expression of heterologous genes of biotechnological interest

In vitro analysis of aggregation-disaggregation of the folding intermediate of Bacillus subtilis α-amylase.

The refolding intermediate of Bacillus subtilis α-amylase is prone to aggregate at 37°C and pH 7 when the protein concentration is relatively high (≥ 1µM). Low concentrations of 2,2,2 trifluoroethanol greatly increased the rate of aggregation. Aggregation made the folding intermediate resistant to proteases and there was kinetic competition between aggregation and proteolytic degradation. Analysis by Fourier transform infrared spectroscopy indicated that the secondary structure of the refolding intermediate is sightly different under soluble or aggregated states. Aggregates were readily solubilized by guanidium chloride (D1/2 = 1.25 M), but disaggregation was slow when aggregates were resuspended in solutions of various foreign native proteins under physiological conditions of pH and temperature. This destabilizing effect resulting from protein-protein interactions may make the aggregation process reversible in vivo