Calcium Chloride → D-Tartaric acid

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%.

1. customTORAY 1246 (FERM-P 2507), obtained from 3 liters of the culture broth
2. customprepared
3. customTo the supernatant fluid resulting from centrifugation at 7000 r.p.m
4. customthe precipitates
5. customthen formed
6. customwere removed
7. additionTo the resulting clear supernatant fluid, 25.9 g (0.70% saturation) of ammonium sulfate were added
8. waitAfter 7 hours
9. customthe precipitates formed
10. customwere collected by centrifugation
11. dissolutiondissolved in tris-HCl buffer (10 mM, pH 7.6)
12. customThe solution was dialyzed
13. customto remove dialyzable salts to the buffer overnight at 4° C
14. customEighty-one ml of the partially purified enzyme solution thus obtained
15. waitThe mixture was incubated at 40° C for 24 hours
16. customPrecipitates
17. customwere collected after overnight maturation

ORD record

ord-8eccc929646e45fdb4c1098dbd37f653

Source dataset

uspto-grants-1976_05

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US03957579