**4.4 Determination of protease activity**

316 Soybean – Genetics and Novel Techniques for Yield Enhancement

Crude enzyme 385000 25300 15.2 100 1.0 Ammonium sulfate 225000 3150 71.4 58.4 4.7 First CMC column 26880 20.5 1311 7 86.25

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

A B

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

> Total protein (mg)

Crude extract 97,270 490.7 198.2 100.0 1.00 Ammonium sulfate 21,229 19.4 1,094.3 21.8 5.52 Ultrafiltration 13,351 10.8 1,236.2 13.7 6.23 DEAE-Sephadex 8,913 5.24 1,700.9 9.2 8.57 Sephadex G-100 6,581 2.86 2,301.0 6.8 11.59 Table 4. Purification of *A. oryzae* LK-101 from the traditional Korean soybean paste (Hwang

Specific activity (U/mg)

Yield (%)

Purification (Fold)

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

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

Total activity (Unit)

Protease I 6050 2.4 2521 1.6 165.5 Protease II 11000 4.1 2683 2.9 176.5

Specific activity (U/mg)

Yield (%) Purification

(fold)

Total protein (mg)

Total activity (Unit)

Purification Steps

protease activity (Chung, 1984).

purified by 11.6 folds with 6.8% of yield.

Purification steps

et al., 2010).

Second CMC column 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.
