**4.5.7 Effect of inhibitors on enzyme activity**

The *B. subtilis* JM-3 protease was strongly inhibited by specific inhibitors for trypsin-like protease, i.e. MTLCK, PMSF, STI and DTT. It was moderately inhibited by inhibitors for chymotrypsin-like proteases, i.e. 2-mercaptoethanol and TPCK. It was not inhibited by inhibitors for cysteine proteases, i.e. NEM and PCMB (Ninojoor, 1985), and inhibitor for metalloproteases, i.e. EDTA. Therefore, the *B. subtilis* JM-3 protease was classified as a trypsin-like serine protease (W.J. Kim and S.M. Kim, 2005).

The *B. megaterium* KLP-98 protease was almost inhibited by NEM, E-64, and egg white cystatin, the specific inhibitors for cysteine protease. Serine protease inhibitors of TLCK and TPCK, aspartic acid protease inhibitor of pepstatin showed no reduction in its activity. Reducing agents such as DTT and 2-mercaptoethanol, and metalloprotease inhibitor, EDTA, even could increase its activity. Based on the results of the substrate specificity and inhibitor studies, *B. megaterium* KLP-98 protease was classified as a cysteine protease (Fu et al., 2008).

EDTA was the strongest inhibitor followed by in order of PMSF, o-phenanthroline, and iodoacetic acid with the inhibition rate of 52, 47, 16 and 8%, respectively. It was moderately inhibited by inhibitors for chymotrypsin-like proteases, i.e. 2,4-dinitrophenol and 2 mercarptoethanol. PMSF is very specific inhibitor for serine protease. Therefore, AOLK-101 protease is classified as a serine protease based on its sensitivity to PMSF. Hence, this indicates that AOLK-101 protease is a serine protease (Hwang et al., 2010).
