|Budget Amount *help
¥5,800,000 (Direct Cost : ¥5,800,000)
Fiscal Year 1990 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 1989 : ¥4,500,000 (Direct Cost : ¥4,500,000)
A large number of proteins and hormones are synthesized as precursors and are converted into the mature forms by selective enzymatic cleavage of peptide bonds. As extracellular alpha-amylase, neutral protease and alkaline protease in Bacillus subtilis are synthesized as prepro enzymes, their signal peptides for secretion are removed at first, and then the prosequences in the secreted proenzymes are processed to form the mature enzymes. In order to analyze the processing system of the prosequences in. subtilis, we constructed artificial hybrid genes, amyE^1 - ^1pBR322^1 - ^<>amyT.
In these hybrid genes, small peptides encoded in the fragments of pBR322 DNA were inserted between the signal peptide of B. subtilis alpha-amylase (amyE^1) and the mature thermostable alpha-amylase (^1amyT) of. stearothermophilus A631 using dlll linker DNA. Among the constructed hybrid genes, the gene in the plasmid pTUBE638 induced a 1.7-fold higher production of extracellular thermostable alpha-amylase in. Subtilis than by the parental amyE^1 - ^1amyT gene. In B. Coli (pTUBE638), an N-terminally extended thermostable alpha-amylase accumilates in the periplasm of the cells, where it retains the 21-amino-acid-extra-peptide. In contrast, another N-terminally extended thermostable alpha-amylase, in which an 18-amino-acid-extra-peptide, is secreted from B. Subtilis (pTUBE638). We purified the N-terminally extended thermostable alpha-amylase from. Coli (pTUBE638). Then we developed an in vitro processing system for the analysis of the protein processing in B. Subtilis. Then we demonstrated that the extracellular alkaline protease acts as the major processing enzyme for the selective cleavage of the ide.