Protein Processing to Form Extracellular Thermostable α-Amylases from a Gene Fused in a Bacillus subtilis Secretion Vector

SUMMARY: A thermostable α-amylase gene (amyT631) from Bacillus stearothermophilus A631 was cloned into pBR322 and recloned into pUB110: the resulting plasmid was designated pTUB607. To investigate the processing from preproenzyme to mature enzyme, amyT631 from pTUB607, after digestion with BAL31, was introduced into the B. subtilis α-amylase secretion vector pTUB285. Three chimaeric plasmids, pTUB613, pTUB616, and pTUB617, were isolated. The fused α-amylases expressed from the three plasmids seemed to be synthesized as preproenzymes. From analysis of the NH2-terminal amino acid sequences of purified extracellular α-amylases, the precursors of the fused enzymes appeared to be cleaved at first between amino acids 31 and 32 from the translation initiator Met (positions −11 and −10 with respect to the beginning of the mature enzyme), and processed to mature extracellular enzymes in which the NH2-terminal amino acid sequences were the same as that of the parental pTUB607 α-amylase, in spite of the lengths of the prosequences and the amino acid composition near the secondary cleavage sites being different in each enzyme.