Teruaki Shiroza

Length and structural effect of signal peptides derived from Bacillus subtilis alpha-amylase on secretion of Escherichia coli beta-lactamase in B. subtilis cells.

The precursor of Bacillus subtilis alpha-amylase contains an NH2-terminal extension of 41 amino acid residues as the signal sequence. The E. coli beta-lactamase structural gene was fused with the DNA for the promoter and signal sequence regions. Activity of beta-lactamase was expressed and more than 95% of the activity was secreted into the culture medium. DNA fragments coding for short signal sequences 28, 31, and 33 amino acids from the initiator Met were prepared and fused with the beta-lactamase structural gene. The sequences of 31 and 33 amino acid residues with Ala COOH-terminal amino acid were able to secrete active beta-lactamase from B. subtilis cells. However beta-lactamase was not secreted into the culture medium by the shorter signal sequence of 28 amino acid residues, which was not cleaved. Molecular weight analysis of the extracellular and cell-bound beta-lactamase suggested that the signal peptide of B. subtilis alpha-amylase was the first 31 amino acids from the initiator Met. The significance of these results was discussed in relation to the predicted secondary structure of the signal sequences.

Stable hyper-production of Escherichia coli β-lactamase by Bacillussubtilis grown on a 0.5 M succinate-medium using a B. subtilisα-amylase secretion vector

Abstract Extracellular production of Escherichia coli β-lactamase by Bacillus subtilis , using a B . subtilis secretion vector constructed from its own α-amylase gene, was promoted when the cells were grown on LG-medium containing 0.5 M succinate under poor aeration conditions. The amount of the enzyme secreted was 50 to 60 times as large as that obtained by cultivation of the cells on LG-medium under good aeration conditions. The effect of protease-deficient mutant of B . subtilis on the enzyme production by B . subtilis was significant while that of protease inhibitors was negligible or inexistent.