Expression,Purification and Enzymatic Characterization of Thermophilic Xylose Isomerase in Escherichia coli

To obtain thermostable xylose isomerase,the gene xylA from an extreme thermophilic bacterium,namely Thermus thermophilus HB8,was cloned and its product was overexpressed in Escherichia coli BL21(DE3) with expression vector pET22b(+).The sequence of PCR product was compared with T.thermophilus HB8 xylA gene(GenBank accession number: D90256),and N91D mutation in the TtXI was identified by DNA sequencing.The overexpressed xylose isomerase was purified by heat precipitation and anion-exchange chromatography.The property of xylose isomerase was determined by the coupled sorbitol dehydrogenase assay with D-xylose as a substrate.Results showed that the optimum temperature was 80℃,and the optimum pH was 8.0.The half life of recombinant enzyme on 80℃ was 225 min.At 60℃ and pH 7.5,the Km was 15.20 mmol/L,Vmax was 69.54 μmol/min,kcat was 50.62/s,kcat/Km was 3.33 L/s·mmol.The results can lay a foundation for application in industry of xylose isomerase.