**3.6 Enzymes**

Manufacturing processing of fermented soybean product requires to uniform of the quality of the product. However, it is difficult to control the natural fermentation according to the traditional method using microorganisms as starters. Because high salt concentration is needed in fermentation of soybean products, salt-tolerant hydrolytic enzymes, especially

Salt-Tolerant Acid Proteases: Purification, Identification,

Steps Volume

Steps Volume

with 2.9% of yield, respectively.

(mL)

(mL)

microorganism.

**KLP-98** 

Kim, 2005).

2008).

**4.3 Extraction and purification of salt-tolerant acid protease** 

folds with 5.33% of yield and 18.83 folds with 15.3% of yield, respectively.

Total Protein (mg)

Total Protein (mg)

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

Enzyme Characteristics, and Applications for Soybean Paste and Sauce Industry 315

Five kinds of reported salt-tolerant acid proteases were extracellular enzymes. Thus, they were extracted and purified from the culture media of each corresponding

**4.3.1 Purification of salt-tolerant acid protease of** *B. subtilis* **JM-3 and** *B. megaterium* 

As shown in Table 1 and 2, proteases produced by *B. subtilis* JM3 and *B. megaterium* KLP-98 were purified by a similar procedure. Proteases in the culture media were precipitated by ammonium sulfate. The precipitates were dissolved, dialyzed, and applied to a DEAE-Sephadex ion exchange column. Proteins were eluted with an increasing gradient of NaCl. Fractions containing greater than 50% of maximal peak activity were pooled, dialyzed and applied to a Sephadex G-75 gel filtration column, and eluted with sodium acetate buffer (pH 5.5). Proteases produced by *B. subtilis* JM3 and *B. megaterium* KLP-98 were purified by 35.56

> Total activity (U)\*

> Total activity (U)

Crude extract 2,000 1,234 1040 0.84 100.0 1.00 Ammonium sulfate 14 814 1000 1.23 96.2 1.46 DEAE-Sephadex 40 32 172 5.36 16.5 6.37 Sephadex G-75 40 10 159 15.86 15.3 18.83 Table 2. Purification of *Bacillus megaterium* KLP-98 protease from fermented squid (Fu et al.,

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

Crude extract 2,000 600.0 3000.0 5.0 100.0 1.0 Ammonium sulfate 10 13.1 320.5 24.5 10.68 4.9 Ultraltrate 16 8.5 235.2 27.7 7.84 5.5 DEAE-Sephadex 35 2.2 185.2 84.2 6.17 16.8 Sephadex G-75 50 0.9 160.0 177.8 5.33 35.56 Table 1. Purification of *Bacillus subtilis* JM-3 protease from anchovy sauce (W.J. Kim and S.M.

Specific activity (U/mg)

Specific activity (U/mg) Yield (%)

Yield (%)

Purification (fold)

Purification (fold)

protease with high proteolytic activity is used as starter to uniform the quality of the product, and shorten the ripening periods of fermentation. During the fermentation, the pH value decreased down to lower than 5 (Fukushima, 1979), thus the salt-tolerant acid protease is more valuable for the production of fermented soybean products.
