Thermophilic Xylose Isomerase And Its Two Mutant Fluorescence Spectra Gaussian Research

Xylose isomerase(Ⅺ)plays an important role in industrial ethanal fuel production from lignocellulosic biomass,thus the study on its conformation,function and site-directed mutagenesis is of important prospect and wide attention.Termophilic xylose isomerase from Thermus thermophilus HB8 and two mutated types in solution have been investigated theoretically and experimentally by multichannel Fluorolog3-P spectrofluorometor.The fluorescence spectra were processed by the method of second derivative spectroscopy and Gaussian fitting fluorescence spectroscopy. The processed specture can provide information onIn the A3 type,Lys²⁵⁵was modified by site-directed mutagenesis to alanine.In the 3-2 type,Arg⁶⁰and Vla¹⁴⁴were changed to phenylalanine and alanine respectively.At room temperature,the fluorescence specture of three samples under irradiation at 280nm and 300nm were investigated in solution by multichannel Fluorolog3-P spectrofluorometor.The fluorescence spectra of three samples were processed by the method of second derivative spectroscopy and Gaussian fitting spectroscopy,which was used to discriminate the weak signals.The second derivative spectra show a series of minimum points that present fluorescent substance,from which the Gaussian fitting fluorescence spectra were obtained.The Gaussian basis funtion provide the peak position, width and height for further analysis in quantity.Not only the fluorescence spetra of Trp and Tyr,but also the information onⅪconformation and function are included in Gaussian basis function.The fluorescent blue-shift of Trp in A3 reflects the rigid of local protein structure of A3 increased,which leads to activity and thermal statibility increased,while 3-2 is contrary to the situation. The conclution correlate with detection of activity.300nm excitation should be better than 280nm,when fluorescence of Trp is analyzed.The second derivative spectroscopy and Gaussian fitting fluorescence spectroscopy are effective methods to monitor changes in structural characteristics of proteins.These results and research methods of this paper may provide some necessary information for a number of important enzymes on molecular structure and modification research in the fields of industrial microbiology.