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

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

Fig. 3. Effects of pH on activity (A) and stability (B) of purified *Bacillus subtilis* JM3 protease (W.J. Kim and S.M. Kim, 2005).

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

Salt-Tolerant Acid Proteases: Purification, Identification,

0

trypsin-like serine protease (W.J. Kim and S.M. Kim, 2005).

20

40

Relative enzyme activity (%)

*oryzae* LK-101protease (C).

**4.5.6 Substrate specificity** 


tolerance (●)

60

80

100

120

sauce productions.

Enzyme Characteristics, and Applications for Soybean Paste and Sauce Industry 321

101 still remained 65, 75 and 50% activity at 10% NaCl concentration and 21, 35 and 22% at 20% NaCl concentration, respectively (W.J. Kim and S.M. Kim, 2005; Fu et al., 2008). Thus, these proteases were salt-tolerant proteases which can be used in soybean paste and soy

A B

0 5 10 15 20 25 30 NaCl concentration (%)

C Fig. 6. Effects of NaCl concentration on the activity of purified *Bacillus subtilis* JM3 protease (A), *Bacillus megaterium* KLP-98 protease (B), and stability and activity of purified *Aspergillus* 

The relative activities of the *B. subtilis* JM-3 protease on specific substrates for trypsin-like serine proteases, i.e. BSA, casein, azocasein and L-BApNA, were 120.81, 98.07, 100.00 and 119.24%, respectively. But it did not hydrolyze BTEE, which was the specific substrate for chymotrypsin-like protease. The *B. subtilis* JM-3 protease was therefore presumed to be a

Relative activity (%)

5 10 15 20 25 30 NaCl concentration (%)

0

20

40

Relative enzyme activity (%)


60

80

100

120

pH 4.0-5.5 of weak acidic conditions. The pH stability range of *B. megaterium* KLP-98 protease and *B. subtilis* JM-3 protease was narrow.

Fig. 4. Effects of pH on activity (A) and stability (B) of purified *Bacillus megaterium* KLP-98 protease (Fu et al., 2008).

Fig. 5. Effects of pH on activity (A) and stability (B) of purified *Aspergillus oryzae* LK-101protease (Hwang et al., 2010).

The optimum pH for *A. oryzae* AOLK-101 protease activity was determined to be 6.5. It had higher proteolytic activity at weak acidic conditions than at the alkaline region. Thus, it was an acid protease. It remained more than 80% of activity in the region of pH 4.5-8.5 (Fig. 5). Thus the stability of this protease was better than those of *B. subtilis* JM-3 and *B. megaterium*  KLP-98 proteases.

### **4.5.5 Effects of NaCl concentration on enzyme activity**

The relative activities of purified proteases decreased as NaCl concentration increased. However, the protease produced by *B. subtilis* JM3, *B. megaterium* KLP-98 and *A. oryzae* LK-

pH 4.0-5.5 of weak acidic conditions. The pH stability range of *B. megaterium* KLP-98

Relative activity (℃)

Fig. 4. Effects of pH on activity (A) and stability (B) of purified *Bacillus megaterium* KLP-98

3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5 pH

The optimum pH for *A. oryzae* AOLK-101 protease activity was determined to be 6.5. It had higher proteolytic activity at weak acidic conditions than at the alkaline region. Thus, it was an acid protease. It remained more than 80% of activity in the region of pH 4.5-8.5 (Fig. 5). Thus the stability of this protease was better than those of *B. subtilis* JM-3 and *B. megaterium* 

The relative activities of purified proteases decreased as NaCl concentration increased. However, the protease produced by *B. subtilis* JM3, *B. megaterium* KLP-98 and *A. oryzae* LK-

optimum pH stability

Fig. 5. Effects of pH on activity (A) and stability (B) of purified *Aspergillus oryzae* LK-

> 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 pH

> > 0

20

40

Relative activity(%)


stability(○)

60

80

100

120

protease and *B. subtilis* JM-3 protease was narrow.

3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 pH

0

**4.5.5 Effects of NaCl concentration on enzyme activity** 

20

40

Relative enzyme activity (%)

101protease (Hwang et al., 2010).

KLP-98 proteases.


60

80

100

120

A B

protease (Fu et al., 2008).

Relative activity (℃)

101 still remained 65, 75 and 50% activity at 10% NaCl concentration and 21, 35 and 22% at 20% NaCl concentration, respectively (W.J. Kim and S.M. Kim, 2005; Fu et al., 2008). Thus, these proteases were salt-tolerant proteases which can be used in soybean paste and soy sauce productions.

Fig. 6. Effects of NaCl concentration on the activity of purified *Bacillus subtilis* JM3 protease (A), *Bacillus megaterium* KLP-98 protease (B), and stability and activity of purified *Aspergillus oryzae* LK-101protease (C).
