**4.5.6 Substrate specificity**

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

Salt-Tolerant Acid Proteases: Purification, Identification,

these proteases will be salt-tolerant enough during the fermentation.

decreased from one year of the commercial production to 2 months and 2 days.

soybean paste and soy sauce fermentation.

paste and soy sauce fermentation.

**6. Conclusions** 

The principle of fish sauce fermentation is somewhat similar to the soybean product fermentation. The successful application of the *B.* megaterium KLP-98 protease to anchovy sauce fermentation indicated the potential application of salt-tolerant acid protease to

From the collective information on salt-tolerant acid proteases, we conclude that proteases are important enzymes naturally purified from variety of halotolerant microorganisms. A few salt-tolerant acid proteases, i.e. proteases produced by *Bacillus subtilis* JM3, *Bacillus megaterium* KLP&98, and *Aspergillus oryzae* LK&101, have a remarkable production and possess excellent properties such as high specific activity, excellent temperature and pH stability and salt-tolerant ability, which enlighten their potential applications in sauce bean

**5. Applications** 

Enzyme Characteristics, and Applications for Soybean Paste and Sauce Industry 323

The applications of the purified proteases to soybean paste and soy sauce fermentation haven't been investigated yet. However, the characteristics of the proteases indicated their potential application. The specific activities were high, and the kinetic parameters were excellent. These proteases were stable below temperature 30 and 40 °C, respectively. For the fermentation of soybean paste and soy sauce, the temperature was around 30 to 35 °C, thus these proteases should be stable during the fermentation. The optimal pH for the activity of the proteases was 5.5 and 6.5, respectively, and they were also stable near this pH value. During the fermentation of soybean paste and soy sauce, the pH decreased to around 5. Therefore, these proteases will exhibit high activities and remain stable during the fermentation. These proteases showed high activities at 10% NaCl concentration and still remain moderate activities at 20% NaCl concentration. Different categories of the fermented soybean paste and soy sauce have different salt concentrations ranging from 10-21%, thus

The application of the salt-tolerant acid protease produced by *B. megaterium* KLP-98 to the anchovy sauce processing was investigated (Fu et al., 2008). Anchovy sauce was made by mixing anchovy with 20% NaCl at 30 °C for 2 months. The purified *B. megaterium* KLP-98 protease was lyophilized (15.8 U/mg) and thoroughly mixed with 2 month-ripened anchovy sauce in a ratio of 1 mg/100 g. Samples were stored in screwed boxes in dark at 40 °C for two days and various quality characteristics of the liquid fraction were determined every 12 hours. The degree of hydrolysis (DH), which is defined as the percentage of the free amino group cleaved from protein, was calculated. The yield was determined as the volume of liquid fraction obtained per 100 g fish. The final production was obtained with satisfactory color, flavors and taste (data not shown). The improvement of various parameters related to fish protein hydrolysis was discussed. All parameters of the control samples slightly changed during two days fermentation. However, all the parameters of the fish sauce samples with *B. megaterium* KLP-98 protease were greatly changed. The values of pH, TN, DH of the commercial fish sauces are: Vietnam (5.75, 2590 mg/100 mL, 61.6%), China (6.15, 1490 mg/100 mL, 57.8%) and Korea (5.49, 1270 mg/100 mL, 68.2%) (Park et al., 2001). These values of the anchovy sauces in this study are 5.17, 1252 mg/100 mL, 57.4%, which are slightly lower than the commercial products; however, the fermentation period

Substrates with higher specificity for serine and cysteine proteases were also used. *B. megaterium* KLP-98 protease had high affinity to Z-Phe-Arg-NMec (95.56%), which is ideal substrate for cysteine proteases, but also some trypsin-like serine proteases can hydrolyze it. However, *B. megaterium* KLP-98 did not hydrolyze TAME and BTEE, which were model substrates for trypsin and serine protease. Based on the above results, *B. megaterium* KLP-98 protease was, therefore, presumed to be a cysteine protease (Fu et al., 2008).
