| Aspartokinase(4-aspartateβ-phosphotransferase, EC 2.7.2.4, AK)is wildly existed in plants, fungi and bacteria, and is a key enzyme of the aspartate pathway. AK catalyzes the phosphorylation of aspartate to produceβ-aspartyl phosphate, which is the first reaction of the pathway, and also is the rate-limited step in the pathway. The activity of AK is under control of the feedback inhibition of the end products amino acids. In plants and microorganisms, the Asp-derived amino acid pathway leads to the synthesis of the essential (regarding animals) amino acids Thr, Lys, Met, and Ile. Therefore, the research on aspartokinase comes into more and more scholars'concern. Through the study of the structure, characterization and metabolic regulationl mechanism of aspartokinase, it makes the enzyme as a excellent target for development of new agrochemical and pharmaceutical compounds controlling weeds, fungi, and bacteria. In the selecting of crops and industrial bacterial, it is also a effective way to raise lysine and threonine production of crops and industrial micro-organisms through creening feedback-insensitive AK with high aspartokinase activity. In addition, lysine, the end product of aspartate pathway, is the precursors of cephamycin C andε-Poly-L-lysine, which are well-used Antibiotics and nature Food preservative respective. Homoserine, the branched pathway product, is the precursors of the biochemical Pesticide-AVG. Through research on AK at the molecular level, it could also be used to engineer thermo-resistant and highly efficient industry bacteria for cephamycin C orε-PL, even to improve the level of AVG in crop in agricultural pest control.Hyperthermophiles grow optimally at temperatures between 80 and 110℃. Thermus thermophilus HB27 is an extremely thermophilic, halotolerant bacterium, years, the complete genome sequence of T. thermophilus HB27 was reported. Extreme thermophilic bacteria has provided us with new protein resources, through the study of characterization and structure of hyperthermophilic Aspartokinase, it does not only help us to understand the heat-resistant protein thermophiles mechanism, the mechanisms for protein thermostability , and understanding for aspartate pathway of Thermus thermophilus HB27. Also the works provides the basic theory for building highly efficient and thermo-resistant bacteria, improving the nutritional content of crops and agricultural pest control techniques. The thermal-unstability of industrial enzyme hampers the application of the product as a"bottleneck", AK from extreme thermophilic bacteria has highly thermal stability, and is expected to be a effective industrial enzyme.To obtain thermostable aspartokinase(AK), the gene ask from an extremely thermophilic bacterium, Thermus thermophilus HB27 was cloned, and its product was overexpressed in Escherichia coli BL21 (DE3). The expression in BL21 (DE3) was more efficient. By the heated treatment of primary purification, the recombinant aspartokinase(tAK)activity keep more efficient when incubated in the buffer of pH 7.5 on 80℃for 3 h. The optimum reactive pH was 7.5, the optimum reactive temperature was 80℃, and tAK was more stable on the temperature of 65-85℃. The half life of tAK under 80℃was 18 h. KmATP was 0.23mmol/L, VmaxATP was 840.34 nmol/(L·min), KmAsp was 0.95 mmol/L, VmaxAsp was 190.76 nmol/(L·min). L-threonine caused obviously inactivation of tAK in concentration dependent manner among three amino acids of L-lysine, L-methionine and L-threonine. Through the expression of tAK in E. coli and the study of the enzymol characterization, it establishes a certain foundation for the next application of the extreme thermophiles, and it also provides reference for the explanation for the metabolism and regulation of aspartate pathway in extreme thermophilic bacteria.
|
PDF Full Text Request