Studying Of Cloning, Expression And Bioactivity Analysis Of Xylose Isomerase And Xylulokinase

Energy is the basic of the development of human being society, with the decrease of petroleum fuels and other non-renewable energy resources, more and more people keep eyes on those new, clean, especially renewable energies, such as bioethanol. Converting the cellulose and hemicellulose to ethanol is one of the hot topics in this field. Xylose is the main component of hemicellulose. Saccharomyces cerevisiae can’t metabolize xylose to ethanol because it can’t product xylose isomerase to transfer xylose to xylulose which can be utilized by saccharomyces cerevisiae. Saccharomyces cerevisiae metabolize xylulose-5-P to ethanol is following the pentose phosphate pathway and the xylulokinase is the key enzyme in this passway. Though saccharomyces cerevisiae can product xylulokinase but it’s activity is not enough to hydrolyze xylulose to xylulose-5-P. Here we Studyied of cloning xylose isomerase and xylulokinase from E. coli and expressing them in yeast and bioactivity analysis of the expressed products.The genes of xylose isomerase and xylulokinase were amplified and the His tag was amalgamated to the each 3’end by PCR technic and approved by DNA sequence determine. Using DNA operations, the xylose isomerase and xylulokinase were cloned into Pichia pastoris’s inducible expression vector of pPIC9K or constitutive expression vector of pGAP9K to generate pPIC9K-xi, pPIC9K-xk, pGAP9K-xi and pGAP9K-xk.Then the linearized expressive vectors were transformed into P. pastoris GS115 with electroporation technique and using the 700μg/mL of G418 containing plate to select high gene copies recombinants:GS115(pPIC9K-xi)、GS 115(pPIC9K-xk)、GS115(pGAP9K-xi)、GS115(pGAP9K-xk). While fermentation those engineering strains in the sharking flash, the methanol was selected as the only carbon source to induce the GS115(pPIC9K-xi) and GS115(pPIC9K-xk) to express foreign protein and the glucose was the only carbon for GS115(pGAP9K-xi) and GS115(pGAP9K-xk). The results showed on the SDS-PAGE indicated that the xylose isomerase and xylulokinase can successful expression in P.pastoris and the expressed D-xylose isomerase behave enzyme activity according to the test of glycolysis.And also the more copies engineering strains of S. cerevisiae JL1(pGAP9K-xi)、JL1(pGAP9K-xk), were obtained by transforming the pGAP9K-xi、pGAP9K-xk into S. cerevisiae genome by electroporation technique and selecting the high gene copies recombinants from the 700μg/mL of G418 containing plate. The test of glycolysis proved that those Saccharomyces cerevisiaes expressed xylose isomerase show itself enzyme activity well.Our work of cloning xylose isomerase and xylulokinase genes, expressing recombinant isomerase and xylulokinase in P. pastoris and S. cerevisiaes and analyzing the bioactivity of the recombinant xylose isomerase has established foundation on construction of engineering S. cerevisiaes who can metabolize xylose directly to ethanol and production recombinant xylose isomerase and xylulokinase to using in the field of production hemicellulose ethanol in future.