Hisato Yamazaki

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.

Nucleotide sequence of the promotor and NH2-terminal signal peptide region of Bacillussubtilis α-amylase gene cloned in pUB110

Abstract The nucleotide sequence of the promotor and NH2-terminal signal peptide region of the α-amylase gene derived from the α-amylase hyperproducing strain B . subtilis NA64 was determined. DNA sequences of the NH2-terminal region of the mature α-amylase, 41 amino acid residues of the signal peptide, a Shine-Dalgarno sequence (AGGAG), a potential RNA polymerase recognition site (TTGAAA), and a potential Pribnow box (AAGTAA) were identified. The DNA sequence was quite different from that of the α-amylase gene of B . amyloliquefaciens .