Prilimary Study On The Kinetics Of The Enzymatic Activity And The Structural Of Halohydrin Dehalogenase Infrared Spectroscopic

Haloalcohol dehalogenase HheC from the soil bacterium Agrobacterium radiobacter AD1 is able to cleave carbon-halogen bonds and can be used in produced optical purity epoxide with mostly R configuration.It plays an important role in the degradation of organic pollutants and the production of chiral drug intermediates.So there is a major work to establish a rapid and sensitive screening method for improving the performance of HheC because of the disadvantages of the wild type.At present,the measure method of dehalogenation is mature but the method of ring opening reaction has not been reported.Infrared spectroscopy technology which can detect the small structure changes caused by the combination of enzyme and substrate has a great advantage in the research of the structural dynamics of enzyme catalyzed reaction.Haloalcohol dehalogenase HheC catalyzed epoxide ring opening reaction with various nucleophiles such as N3-(N=N=N),CN-(C=N),OCN-(O=C=N),SCN-(S=C=N)has a characteristic infrared absorption peak which is different from the amide I band.Therefore,infrared spectroscopy can be used to detect the activity of epoxide catalyzed by Haloalcohol dehalogenase.In this paper,a new infrared spectroscopy method for measuring activity of HheC based on the CHBE epoxide ring opening reaction was proposed.First,we find the position of the stretching vibration peaks of the free N3-and the open ring product of CHBE,which are in the 2048 cm-1 and 2115 cm-1 wave numbers.In the enzymatic reaction,the curve of the absorbance was measured in 2115 cm-1 with time,and then the curve of the concentration of 4-azide-3-ethyl ester with time was obtained.Finally,we calculated the specific enzyme activity of HheC was 0.19 U/mg which is similar to the results measured by gas chromatography(GC).The above results show that it is feasible to use FTIR method to detect the activity of halogenated alcohol,and our detection method has lots of advantages compared with the traditional GC method.Although the infrared spectroscopy technology has a great advantage in the field of the research on the structure dynamics of enzyme catalyzed reaction,the biggest obstacle of this method is that there is no suitable infrared probe.At present,the introduction of unnatural amino acids into proteins as infrared probe has widespread concern because of its no site limitation.Through the codon expansion technology,we put an tRNA and the matched aminoacyl-tRNA synthetase with orthogonal transformation into E.coli MC1061,so we can obtain the HheC with infrared probe of unnatural amino acids.The results showed the expression system which can put unnatural amino acids into proteins as infrared probe has been successfully constructed and it laid the foundation for the further study on the structural dynamics of HheC.