Expression, Purification And Preliminary Characterization Of A Thermostable Adenylate Kinase From Thermus Thermophilus HB27

Adenylate kinase(EC 2.7.4.3, AK)is wildly existed in bacteria, archaebacteria and mammal, especially in the cells where high level of ATP is synthesized and used. Adenylate kinase can catalyze the following reversible reaction: ADP + Mg²⁺ADP?AMP + Mg²⁺ATP. The main function of AK is to keep the balance of nucleotide and regulate the energy metabolism. ATP is the major resource of energy in all creatures. ATP is wildly existed in cytoblast, cytoplasm, mitochondria and so on in cells and takes part in movement of creatures, transhipment of nutrient and metabolite, transition of physiological signal and all kinds of biosynthesis activities. ATP is being used in clinic as biodrug and acting more and more important role. Other more ATP is one important biochemistry reagents with an application in scientific research, manufacture and food industry as chelator.Hyperthermophies grow optimally at temperatures between 80℃and 110℃. Thermus thermophilus HB27 is an extremely thermophilic, halotolerant bacterium and can grow at temperature 85℃optimum temperature 75℃. At the year 2004, the complete genome sequence of Thermus thermophilus HB27 was reported and extreme thermophilic bacteria has provided us with a new protein resources. The study of characterization and structure of hyperthermophilic Adenylate kinase, not only help us understand the heat-resistant protein thermophiles mechanism, the mechanism for protein thermostability, also help development of new enzyme used in industry. 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 an effective industrial enzyme.To obtain thermostable adenylate kinase, the gene from Thermus thermophilus HB27 was cloned, and its product was overexpressed in Escherichia coli BL21 (DE3) successfully with a yield of 0.0765 mg per mL fermentation liquor. By the heated-treatment of purification and His6-tag affinity chromatography, high purified AK was obtained. The enzymatic characterization at the direction of ATP synthesized was studied. The optimum reactive pH was 8.5, the optimum temperature was 90℃and AK was more stable at the temperature rank of 60-80℃. Km for ADP and Vmax were 1.02 mmol/L and 0.16 mmol/(L.min), respectively, with a specific activity of 440 U/mg under the condition of 80℃. We also reported that AP5A, a specific inhibitor for AK can inhibit the activity of AK by 70% at concentration of 2.0 mmol/L, with Ki value 46.39μmol/L for ADP. Through the expression of AK in E.coli and the study of the enzymatic characterization, it establishes a certain foundation for the next application of the extreme thermophilic enzyme.