Isolation And Purification Of Acetyl-Coenzyme A Carboxylase

Since the usage of 2,4-D in 1946,chemical herbicides have been used for more than 60 years,and a large number of selective herbicides have been developed successfully.Chemical herbicides have provided strong support to the modernization of agriculture.With the development of computer science and synthesis technology,drug discovery and drug design entered into a new stage,a series of highly effective herbicides targeted as enzyme inhibitors have been developed,such as acetolactate synthase inhibitors,acetyl coenzyme A carboxylase inhibitors,etc.Acetyl coenzyme A carboxylase(Accase) is one member of the biotin-dependent enzyme family.It catalyzes the reaction of carboxyl transfer from Acetyl-CoA to Malonyl-CoA and plays a key role in fatty acid synthesis.Accase is the first dedicated enzyme in the de novo fatty acid biosynthetic pathways.During the fatty acid synthesis pathway,the reaction catalyzed by Accase is not only the key step in the first reaction, but also the rate-limiting step,so Accase is considered as a new efficient herbicide targets. CHDs(cyclohexanediones) and AOPPs(Aryloxyphenoxypropionates) are two classes of herbicides that used to inhibit Accase activity,although these herbicides have been used for nearly 30 years as dominant herbicides,their relative high toxicity,and most of all, weed resistance to these compounds found recently restrict their further application in agriculture.Therefore it is important to optimize the existing inhibitors structure or develop new effective inhibitors.For the purpose of optimization of the existing inhibitors structure and developing new inhibitors,Accase with biological activity should be extracted and evaluated from related biological tissues to establish screening method for its inhibitor.In this thesis, studies mainly covers two aspects:(1) Isolation and purification of Accase from etiolated maize seedlings;(2) Determination of specific activity and Michaelis constant(K_m) of Accase.(1).Isolation and purification of Accase from etiolated maize seedlings Crude Accase was firstly extracted from fresh etiolated maize seedlings by grinding and homogenizing with buffer solution,then partially purified by fractionally salting out with 30%～50%saturation of ammonium sulfate.The precipitate was further purified by Sephacry S-300 gel filtration chromatography and Blue Sepharose CL-6B dye affinity chromatography,respectively,resulting in a relative better purity;(2).Determination of specific activity and Michaelis constant K_m of Accase ATP was chosen as the hydrolytic substrate for the analysis of Accase catalytic activity.The hydrolytic products were separated and assayed by Mono Q anion exchange chromatography.The specific activities of Accase in each step of the purification processes were obtained with the highest specific activity of 1.09μmol·mg^-1·min^-1. Dynamic data demonstrated that Michaelis constant(K_m) for ATP is 37.05μmol·L^-1.