A loop-controlled rrnB P1 promoter for high-level expression of heterologous proteins in Escherichia coli.

An effective protein expression system was constructed in Escherichia coli utilizing the rRNA rrnB P1 promoter and the regulatory element of the lac operon (lacO). To regulate the transcriptional activity of the rrnB P1 promoter, we designed two lacO sites with an intervening loop structure; expression was verified by measuring the levels of the ß-1,4-glucanase gene, cel5G. Basal expression from the looped promoter construct was reduced by 92% when compared to expression from the T7 promoter. We also found that the host cell type had a significant effect on the regulation of the rrnB P1 promoter: E. coli DH5α and DH10B had high expression levels, whereas the expression in BL21(DE3) was more stringent.

Characterization of a novel GH36 α-galactosidase from Bacillus megaterium and its application in degradation of raffinose family oligosaccharides.

A novel α-galactosidase gene (agaB) from Bacillus megaterium 3-7 was cloned and expressed in Escherichia coli. The gene coded for a protein with 741 amino acids and a calculated molecular mass of 85.4kDa. The native structure of the recombined AgaB was determined to be a homotrimer. AgaB showed the highest identity of 57% with the characterized glycosyl hydrolase family 36 α-galactosidase from Clostridium stercorarium F-9. The enzyme exhibited a specific activity of 362.6U/mg at 37°C and pH 6.8. The enzyme showed strong resistance to proteases and great tolerance to galactose (Ki=12.5mM). AgaB displayed wide substrate specificity toward pNPGal, melibiose, raffinose and stachyose, with a Km of 0.42, 12.1, 17.0 and 25.4mM, respectively. Furthermore, AgaB completely hydrolyzed raffinose and stachyose present in soybean milk at 37°C within 4h when combined with trypsin. These favorable properties make AgaB a potential candidate for applications in the food and feed industries.