Purification Of 2-CPA Dehalogenase

2-CPA(2-chloropropionic acid) is an important material of the synthesis of pesticide, dye, agrochemical or medicine. Because of its optical activity, the chemicals synthesized from (S)-2-CPA can work with high physiological and pharmacological activities, especially for pesticides and herbicides. On the contrary, the rest has low activities and some even work as competitive inhibitors. However, (R)-2-CPA can also be used in the synthesis of some chiral pharmaceutical, such as thiolactomycin.There are three types of 2-CPA dehalogenase, such as (S)-2-haloacid dehalogenase(EC3.8.1.2), (R)-2-haloacid dehalogenase(EC3.8.1.9) and (R,S)-2-haloacid deh-alogenase(EC3.8.1.10 and EC3.8.1.11). So the resolution of 2-CPA by dehalogenase plays an important role in the synthesis of chiral chemical. This dissertation aimed to screen the strains and develop a purification method of dehalogenase. These will contribute to the product of optically activated (S)-2-CPA and (R)-2-CPA.Firstly, certain 2-CPA-degrading strains were isolated from the activated sludge at Hangzhou, Wenzhou, and Fuyang etc. Among these strains, the activity of H9 is very high towards 2-CPA, while W20 and S4 showed certain stereoselectivity towards (R)-2-CPA and (S)-2-CPA respectively.For the widely use of (R)-dehalogenase, the purification of W20 dehalogenase was the main purpose. The purified dehalogenase was obtained after salt out, desalt, ion-exchange chromatography and gel chromatography. The molecular weight estimated by SDS-polyacrylamide gel electrophoresis was 35KDa. The optimum reaction pH of the dehalogenase was pH10, the optimum reaction temperature is 40 ℃.Finally, the cultivate condition of W20 was researched. The optimum combination of carbon resource and nitrogen resource is glucose and urea. And the optimum concentration of glucose and urea were determined to be 20g/L and 1 .Og/L respectively. The optimum cultivate temperature is 30℃, the optimum cultivate pH is 7.0.