Calcium Chloride → D-Tartaric acid

Participants

Reaction Steps

The cells of Ach. tartarogenes nov. sp. TORAY 1246 (FERM-P 2507), obtained from 3 liters of the culture broth prepared as described in Example 1, were suspended in 10 mM tris-HCl buffer (pH 7.6) to make 200 ml. The suspension contained 5.3 g of the cells and 34200 units of d-tartrate epoxidase activity. One hundred ml of the suspension were treated by a French Press and a sonic oscillator. To the supernatant fluid resulting from centrifugation at 7000 r.p.m. for 15 minutes of the treated cell suspension, 16.2 g (30% saturation) of ammonium sulfate were added. After being kept at 5° C overnight, the precipitates then formed were removed and discarded by centrifugation. To the resulting clear supernatant fluid, 25.9 g (0.70% saturation) of ammonium sulfate were added. After 7 hours, the precipitates formed were collected by centrifugation and dissolved in tris-HCl buffer (10 mM, pH 7.6) to make 46 ml. The solution was dialyzed to remove dialyzable salts to the buffer overnight at 4° C using a Visking cellophane tube. Eighty-one ml of the partially purified enzyme solution thus obtained contained 1.07 g of protein and 13500 units of d-tartrate epoxidase activity. Fifty ml of 20 (w/v)% disodium cis-epoxysuccinate solution buffered by 50 mM tris-HCl buffer to pH 8.0 were mixed with 17 ml of the enzyme solution and water to make a total volume of 100 ml. The mixture was incubated at 40° C for 24 hours without shaking. After the reaction was completed, 14 ml of 4.5 M calcium chloride was added. Precipitates were collected after overnight maturation and 7.77 g of d-tartaric acid was obtained by the same method as in Example 1, yielding 91 - 92%.

Provenance & Evidence