Cloning, Expression And Characterization Of Three Thermophilic Aminotransferases

?-Transaminases are a potential tool for preparing optically pure ?-chiral amines.Since a large number of biologically active compounds or synthetic precursors have?-chiral amine structural moieties,?-transaminases offer a unique opportunity for the asymmetric synthesis of bioactive compounds with chiral amine substructures.In this thesis,three thermophilic aminotransferase genes,separately from Thermotogamaritima,Sphaerobacter thermophilus,and Thermosynechococcus sp.NK55a(TN)were cloned by molecular cloning,the target geneswere thenrespectively ligated with the vector p ET30 a and transformed into E.coli BL21(DE3)cells to construct the engineering strains that couldbe used to prepare heat-resistant transaminases.Afterinducing expression and purification by affinity chromatography,three different thermophilic transaminases,namely recombinant Thermotogamaritima N-acetyl ornithine transaminase(Ac OAT),Streptococcus thermophilus ?-transaminase(?-TAST),and thermophilic amphoteric transaminase(TATN)were obtained.The results of SDS-PAGE electrophoresis indicated that Ac OAT and ?-TAST were expressed as soluble proteins,but TATN was as inclusion bodies.Based on this,the enzymatic properties of the two soluble transaminases Ac OAT and ?-TAST were characterized.The experimental results showed that the optimum acidityfor Ac OAT and ?-TAST were bothat p H 8.0,but the change trend of their activitiesweredifferent in correspondence with the change of acidity;the optimum temperatures for thetwo proteins were 60 °C,but Ac OAT was more heat-resistant and still active at 100 °C while ?-TAST was almost inactivatedat 80 °C;the optimal reaction time for the two enzymes was 1 h.The effect of various metal ions such as Zn2+,Ca2+,Mg2+,Fe2+,Mn2+,and Na+ on the activity of Ac OAT was also measured.It was found that the activity of Ac OAT was inhibited to varying degrees,suggesting an experimental basis for the study of Ac OAT inhibitors.Since the tested substrates of ?-TAST were mostly insoluble in water,cosolvents had to be used to enhance their water solubility,the influence of cosolvents on the ?-TAST activity was determined.It was found that when the methanol concentration was around 10%,only about 10%enzyme activity remained,so methanol was not a suitable cosolvent.When DMSO concentration was as high as about 25%,the ?-TAST was still more than 80% active.So DMSO couldbe used as a good cosolvent for ?-TAST.Furthermore,the Km values of Ac OAT and ?-TAST were also measured in the experiments.The maximum reaction rate Vmax of Ac OAT to N-acetyl-L-ornithine was determined to be 32.71 ?mol/min,and the Km value was 6.7 m M;the maximum response rate Vmax of ?-TAST to(S)-?-MBA was 36.50 ?mol/min and the Km value was 1.25 m M.The relative activities of the two enzymes on different amino donors and amino receptors were investigated as well,the results indicated that the two transaminases prepared fromdifferent sources exhibited significant differences in activity for different amino donors and amino acceptors.