嗜热木糖异构酶在大肠杆菌中的表达、纯化及性质研究

为获得具有高热稳定性的木糖异构酶，运用基因工程技术，从嗜热栖热菌Thermus thermophilus HB8中克隆到嗜热木糖异构酶基因xylA。测序结果表明，该基因与GenBank数据库中相比271位的碱基A突变为G，导致氨基酸序列中N91D突变。将该基因克隆到载体pET22b(+)，并在E. coli BL21(DE3)中进行高效表达。通过热变性和强阴离子交换两步对该酶进行纯化，并对酶学性质进行了研究。结果表明，该酶最适温度为80 °C，最适pH为8.0，80 °C下半衰期为225 min。在60 °C，pH 7.5该酶的Km为15.20 mmol·L-1，Vmax为69.54 μmol·min-1，kcat为50.62 s-1，kcat/Km为3.33 L·s-1·mmol -1。研究结果为嗜热木糖异构酶的进一步工业应用奠定了基础。

Expression ,Purification and Enzymatic Characterization of Thermophilic Xylose Isomerase In Escherichia coil

To obtain thermostable xylose isomerase, the gene xylA from an extremely thermophilic bacterium,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 °C, and the optimum pH was 8.0. The half life of recombinant enzyme on 80 °C was 225 min. At 60 °C and pH 7.5, the Km was 15.20 mmol·L-1, Vmax was 69.54 μmol·min-1，kcat was 50.62 s-1，kcat/Km was 3.33 L·s-1·mmol –1. The results can lay a foundation for application in industry of xylose isomerase.