**4.3.2 Purification of salt-tolerant acid proteases of** *Rhizopus japonicus*

As shown in Table 3, two acid proteases were produced by *R. japonicus*. They were precipitated by ammonium sulfate, and purified by twice applications of CMC column (Fig. 1). Proteases I and II were purified by 165.5 folds with 61.6% of yield and 176.5 folds with 2.9% of yield, respectively.

Salt-Tolerant Acid Proteases: Purification, Identification,

**4.5 Characterization of salt-tolerant acid protease** 

**4.5.1 Enzyme activity** 

**4.5.2 Molecular mass** 

the *B. megaterium* KLP-98 was the lowest.

three kinds of proteases were compared and discussed in this study.

**4.4 Determination of protease activity** 

Enzyme Characteristics, and Applications for Soybean Paste and Sauce Industry 317

Protease activity was determined according to the modified method of Anson (1939). One mL of enzyme solution was added to 5 mL of 0.6% casein solution in 1/15 M phosphate buffer, pH 6.5 and reacted at 37 °C for 10 min. The reaction was stopped by adding 5 mL of 0.44 M trichloroacetic acid (TCA) and then stood for 30 min. The solution was then filtered with Whatman No. 2. Two mL of the filtrate was mixed with 5 mL of 0.55 M Na2CO3 solution and 1 mL of 1 N Folin reagent, and then stood for at room temperature for 30 min. The absorbance was measured at 660 nm with spectrophotometer, and then converted to the amount of tyrosine equivalent based on a standard curve. One unit (U) of protease activity was defined as the amount of enzyme releasing 1 mol of tyrosine equivalent per 10 min. Protein concentration was determined according to the method of Lowry (Lowry et al., 1951) with egg ovalbumin as the standard. During column chromatography, protein

concentration in the fractions was estimated by measuring the absorbance at 280 nm.

Characteristics of proteases purified from the culture medium of *B. subtilis* JM3, *B. megaterium* KLP-98 and *A. oryzae* LK-101 were well studied, while those of the protease from *Rhizopus japonicus* except specific activity were not reported. Thus, the characteristics of the

As shown in Table 5, the protease from different origin varied a lot in specific activity (W.J. Kim and S.M. Kim, 2005; Fu et al., 2008; Chung, 1984; Hwang et al., 2010). The specific activity of the *R. japonicus* protease II was the highest among these five proteases, and that of

Source of protease Specific activity

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).

Source of protease Mr (kDa) *Bacillus subtilis* JM3 17 *Bacillus megaterium* KLP-98 64 *Aspergillus oryzae* LK-101 25 Table 6. Comparison of molecular masses of purified salt-tolerant acid proteases.

Protease II 2683

*Bacillus subtilis* JM3 177.8 *Bacillus megaterium* KLP-98 15.86 *Rhizopus japonicus* Protease I 2521

*Aspergillus oryzae* LK-101 2301 Table 5. Comparison of specific activities of purified salt-tolerant acid proteases.

(U/mg)


Table 3. Purification of acid salt-tolerant proteases from *Rhizopus japonicus* (Chung, 1984).

Fig. 1. First (A) and second (B) CMC column chromatography. - - - - Abs 280nm, — protease activity (Chung, 1984).

### **4.3.3 Purification of salt-tolerant acid proteases of** *A. oryzae LK-101*

As shown in Table 4, purification of protease produced by *A. oryzae* LK-101 was carried out as follows. Culture medium was centrifuged. Ammonium sulfate was added to precipitate the protein. The precipitate was dissolved, dialyzed, and concentrated by ultrafiltration. The resulting concentrate was purified by DEAE-Sephadex ion exchange column and Sephadex G-100 gel filtration column consequently. Protease produced by *A. oryzae* LK-101 was purified by 11.6 folds with 6.8% of yield.


Table 4. Purification of *A. oryzae* LK-101 from the traditional Korean soybean paste (Hwang et al., 2010).
