**4.5.2 Molecular mass**

The molecular masses and purities of the protease were determined by SDS-PAGE electrophoresis (Fig. 2) (W.J. Kim and S.M. Kim, 2005; Fu et al., 2008; Hwang et al., 2010).


Table 6. Comparison of molecular masses of purified salt-tolerant acid proteases.

Salt-Tolerant Acid Proteases: Purification, Identification,

al., 2010).

proteases.

Source of protease

(W.J. Kim and S.M. Kim, 2005).

**4.5.4 Effects of pH on enzyme activity and stability** 

Enzyme Characteristics, and Applications for Soybean Paste and Sauce Industry 319

Each purified protease was homogeneous on SDS-PAGE. As shown in Table 6, different

As shown in Table 7, the optimal temperature for the activity of protease produced by *A. oryzae* LK-101 was lower than those of the other two proteases, while the stability of proteases produced by *A.* LK-101 and *B. megaterium* KLP-98 was better than that of the protease produced by *B. subtilis* JM3 (W.J. Kim and S.M. Kim, 2005; Fu et al., 2008; Hwang et

> Optimal temperature for enzyme activity (°C)

*Bacillus subtilis* JM3 60 ≤ 30 *Bacillus megaterium* KLP-98 60 ≤ 40 *Aspergillus oryzae* LK-101 50 ≤ 40 Table 7. Effects of temperature on activity and stability of purified salt-tolerant acid

Relative activity increased up to pH 5.5 and decreased rapidly at higher pH (Fig. 3A). The optimal pH for the hydrolysis of azocasein was 5.5. Therefore, the *B. subtilis* JM-3 protease was classified as an acid protease. *B. subtilis* JM-3 protease showed the optimal stability at

Fig. 3. Effects of pH on activity (A) and stability (B) of purified *Bacillus subtilis* JM3 protease

Relative activity increased up to pH 5.5 and then decreased significantly (Fig. 4A). The optimal pH for the hydrolysis of azocasein was 5.5, and it had high proteolytic activity at weak acidic conditions (pH 5.0-6.0). The optimal pH of *B. megaterium* KLP-98 protease was similar to those of *B. subtilis* JM-3 protease. *B. megaterium* KLP-98 protease was stable around

pH 5.5 (Fig. 3B) of weak acidic condition, but was unstable above pH 6.0.

Temperature range for enzyme stability (°C)

protease had different molecular masses ranging from 17 kDa to 64 kDa.

**4.5.3 Effects of temperature on enzyme activity and stability** 

Fig. 2. SDS-PAGE electrophoresis of proteases produced by *B. subtilis* JM3, *B. megaterium* KLP-98 and *A. oryzae* LK-101.

Each purified protease was homogeneous on SDS-PAGE. As shown in Table 6, different protease had different molecular masses ranging from 17 kDa to 64 kDa.
