To alleviate plasmid instability and to prolong the production phase of subtilisin, integrable plasmid and spore mutants are used. Compared with batch-type shake flask cultures, spore mutants’ ability to produce subtilisin can be well pronounced in fed-batch and continuous cultures. Hence, the two culture methods make it possible to identify the peculiar characteristics of the spore mutants unobtainable in batch culture. Spore mutants can enhance subtilisin productivity and prolong subtilisin production time in fed-batch culture as well as enable us to use very low dilution rates (<0.1 h(-1) without losing productivity in continuous culture, thereby improving the conversion yield of the nitrogen source. At 0.05 h(-1), the spoIIG mutant of Bacillus subtilis DB104 (Delta npr Delta apr) (Em(r)) spoIIG (Blm(r)): : pMK101 (Cm-r) showed a subtilisin yield about ten times higher than that from wild-type DB104 (Delta npr Delta apr)::pMK101 (Cm-r).