**5. Applications**

322 Soybean – Genetics and Novel Techniques for Yield Enhancement

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

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

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

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

In order to evaluate the kinetic constants for the purified protease, the initial velocities of the enzyme reactions were determined at various concentrations of the casein substrate. The kinetic constants, *Km* and *Vmax* values of the protease were calculated from the Lineweaver-Burk plot. The *Vmax*/*Km* value, which is the physiological or catalysis efficiency value, was calculated. The higher *Vmax*/*Km* value means the stronger catalytic activity to hydrolyze the substrate. Therefore the protease produced by *B. subtilis* JM3 exhibited the strongest

> *Vmax*  (U/L)

*Vmax*/*Km*  (U mL / L/mg)

(mg/mL)

*Bacillus subtilis* JM3 1.75 318 181.6 *Bacillus megaterium* KLP-98 2.10 285 135.7 *Aspergillus oryzae* LK-101 1.04 125 119.2 Table 8. Kinetic parameters of the purified protease produced by *Bacillus subtilis* JM3, *Bacillus megaterium* KLP-98, and *Aspergillus oryzae* LK-101 (W.J. Kim and S.M. Kim, 2005; Fu

indicates that AOLK-101 protease is a serine protease (Hwang et al., 2010).

catalytic activity among the three salt –tolerant acid proteases.

Source of protease *Km* 

protease was, therefore, presumed to be a cysteine protease (Fu et al., 2008).

**4.5.7 Effect of inhibitors on enzyme activity** 

cysteine protease (Fu et al., 2008).

**4.5.8 Kinetic parameters** 

et al., 2008; Hwang et al., 2010).

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

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

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

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 soybean paste and soy sauce fermentation.
