In Bacillus subtilis, extracellular protease-deficient mutants have been used in attempts to increase the productivity of heterologous proteins. We detected protease activity of AprX using protease zymography in the culture medium at the late stationary growth phase. An alpha-amylase-A522-PreS2 hybrid protein, in which the PreS2 antigen of human hepatitis B virus (HBV) is fused with the N-terminal 522-amino-acid polypeptide of B. subtilis alpha-amylase, has been produced in multiple-protease-deficient mutants. The B. subtilis KA8AX strain, which is deficient in eight extracellular proteases and AprX, did not show the proteolysis of alpha-amylase-A522-PreS2 in the late stationary growth phase. Moreover, the production of a-amylase-A522-PreS2 was about 80 mg/l, which was eight times higher than that by the KA8AX strain previously reported. In addition, we showed the degradation of the heterologous protein by AprX that leaked to the culture medium (probably caused by cell lysis) during the late stationary growth phase.