Breed Of α-Chloropropionic Acid Dehalogenase Production Microorganisms And Isolation Of R-Type Dehalogenase

a-chloropropionic acid (a-CPA) is the basic constitutor of numerous insecticides and herbicides. These agrochemicals have been widely used since 1850's and are indispensable in extractive industry. However, only the agrochemicals which consist (S)-a-CPA work with high physiological and pharmacological activities. The rest, on the contrary, have low activities, and some even work as competitive inhibitors. Therefore, when employing racemic a-CPA to synthesize pesticides, the products have low activities and bring the environmental contamination. So it is valuable to produce optically active (S)-a-CPA. Some dehalogenases possess high stereoselectivities. Thus, we can utilize these specialties to efficiently produce optically active (S)-a-CPA via the resolution of racemic a-CPA.Firstly, a certain a-CPA-degrading strain was isolated from the activated sludge in Hangzhou Pesticide Co. by enrichment. Tracked its dehalogenation process and growth curve. The dehalogenase excreting by the strain was identified a kind of intracellular constituent enzyme.The optimum reaction pH of the dehalogenase was 7.5, and its highest pH stability was at pH 7.5. Considering both the activity and the thermostability, 30癈 was the optimum reaction temperature. Identified the some kinetic parameters of the crude enzyme: the Vmax was 2.23 X lO'Wil'min"1*!/1, and the Km was 1.724X Kr3mol-I/'.Determined the factors affecting the culture of the strain. Employed the orthogonal test to find the suitable amounts of the carbon and nitrogen in the media. When the concentration of glucose was 50g/L, the yeast extraction was 5g/L and the sulfureted ammonium was Ig/L, the yields of strain and dehalogenase were the most. Determined that the optimum culture temperature was 30癈.Purified crude extracts by expanded bed adsorption. The protein concentration of loading sample was 4.6/mL and the elution liquid was sulfureted ammonium at 0.25M.VIAbstractAfter purification, the specific activity increased to 4.722U/mg, which was 28 times to the crude enzyme, and the recovery ratio was 68%. The purification factor was improved greatly comparing with the tradition method, and the total purification process was cut shorter too.Finally, employed the ion exchange chromatography to isolate the (R)-dehalogenase. Utilized sulfureted ammonium ladder elution. At the elution condition of 0.3M sulfureted ammonium, yielded the expectant (R)-dehalogenase. The (R)-dehalogenase was verified to possess high stereoselectivity. Its optimum reaction condition was pH 7.5 and 30癈. And the stereoselectivity did not move according to the changes of pH and temperature.The dissertation firstly developed the method of dehalogenase purification and the production of (S)-a-CPA via the dehalogenase resolution of racemic a-CPA. These will contribute to the fellow researches of the production of optically activated (S)-a-CPA.