Protein processing to form extracellular thermostable alpha-amylases from a gene fused in a Bacillus subtilis secretion vector.

Abstract

A thermostable alpha-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 alpha-amylase secretion vector pTUB285. Three chimaeric plasmids, pTUB613, pTUB616, and pTUB617, were isolated. The fused alpha-amylases expressed from the three plasmids seemed to be synthesized as preproenzymes. From analysis of the NH2-terminal amino acid sequences of purified extracellular alpha-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 alpha-amylase, in spite of the lengths of the prosequences and the amino acid composition near the secondary cleavage sites being different in each enzyme.