Purification of Thermolysin

The purification procedure of thermolysin is essentially that described by Endo (1). The culture broth (sample I; 10 L) of Bacillus thermoproteolyticus is filtered with the aid of Celite, and the enzyme in the filtrate (sample II; 9.7 L) is precipitated by 45% saturation with ammonium sulfate, pH 7.0, at 4°C. After centrifuga-tion, the precipitate is dissolved in 20 mM Ca-acetate buffer (1.5 L), pH 7.0, and the solution (sample III) is brought to 30% saturation with ammonium sulfate. The precipitate is removed by centrifugation, and the supernatant is brought to 45% saturation with ammonium sulfate. After centrifugation, the precipitate is dissolved in 20 mM Ca-acetate buffer (1.0 L), pH 7.0. The solution (sample IV) is dialyzed overnight against 10 mM Ca-acetate buffer, pH 7.0, at 5°C. To the dialyzed solution (sample V; 1.1 L), chilled acetone (0.88 L, 0°C) is added and the precipitate formed is removed. Chilled acetone (1.88 L, at 0°C) is added slowly, and the precipitate is collected. The precipitate is dissolved in 20 mM Ca-acetate buffer (0.2 L), pH 7.0; the enzyme activity in the solution should be 100 x 103 U/mL. The solution is kept at 4°C. Crystals of thermolysin appear within 30 min. The solution is held overnight. Crystals (sample VI; 1.55 g) are collected by centrifugation. A lyophilized preparation of these crystals shows an activity of 6.5 x 103 U/g. In the same manner, crystallization is repeated three times. The activities of the two-times- and three-times-crystallized preparations (samples VII and VIII) are 8.5 x 103 and 10.0 x 103 U/g, respectively. Crude preparations (lyophilized acetone precipitate of culture broth) and three-times-crystallized preparations of thermolysin are available from Daiwa Kasei (Osaka, Japan). The purification process is summarized in Table 1.

Table 1 Purification of Thermolysin

Sample No.

Volume or weight


Total activity (x106 units)

Yield (%)

0 0

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