Use of a triple protease-deficient mutant of Bacillus subtilis as a host for secretion of a B. subtilis cellulase and TEM beta-lactamase.

The mature portion of the TEM beta-lactamase (BLA) gene (bla) derived from pBR322 was fused with the promoter and signal region of Bacillus subtilis cellulase (BSC) gene (bsc), and the productivity was compared with that of the cloned native bsc gene, using a wild-type B. subtilis strain and a strain deficient in three proteases (i.e., extracellular serine protease, extracellular neutral protease, and the major intracellular serine protease) as hosts. The effects of the sen, sacQ, and prtR genes, carried on the same plasmids, were tested as for the productivities of BSC and BLA. The production of BSC was increased 9-fold by using the combination of the triple-protease deficient strain and the sacQ gene, compared with that by using the wild-type strain as a host, and no degradation of BSC was observed in the triple-protease deficient strain. On the other hand, though the production of the BSC-BLA fusion protein increased 2.5-fold in the triple-protease deficient strain, the BLA activity was decreased after the cell reached the stationary phase of growth, possibly due to some proteolysis. These observations show different sensitivity of secretary proteins to cellular proteases, and suggest that BLA is decomposed by remaining minor proteases.