海栖热袍菌葡萄糖异构酶基因xylA的克隆、表达及酶学性质

Expression and Characterization of a Thermostable Glucose Isomerase from the Hyperthermophile, Thermotoga maritima

摘要:

海栖热袍菌(Thermotoga maritima)是嗜极端高温的厌氧细菌, 其产生的葡萄糖异构酶由于其出色的耐热性有着潜在的工业应用价值。由于海栖热袍菌苛刻的培养条件导致其葡萄糖异构酶产量较低。通过PCR方法克隆编码T. maritima MSB8葡萄糖异构酶基因xylA, 构建重组质粒pHsh-xylA, 转入Escherichia coli JM109, 通过热激诱导表达。通过热处理和离子交换层析纯化两步得到电泳纯的酶制品, 纯化倍数和回收率分别为8.02和49.02。对酶学性质研究表明, 该重组酶为金属离子激活性酶, Mg2+, Co2+对相对酶活有很强的激活作用, 其最适pH为7.0, 最适反应温度为95°C, 且在pH 6~8之间有着较好的稳定性, 在95°C下半衰期长达5 h以上。以葡萄糖为底物时的表观Km和Vmax分别为105 mmol/L和45.2 mol/min×mg。

Abstract:

The glucose isomerase produced from Thermotoga maritima, a hyperthermophilic anaerobic bacterium, has a large potential in industrial application because of its excellent thermostability. T. maritima can only produce small amount glucose isomerase since the rigorous cultivation conditions. The gene xylA encoding glucose isomerase from T. maritima MSB8 were cloned and expressed in Escherichia coli JM109 using a heat-shock expression vector pHsh. The recombinant glucose isomerase was purified 8.02-fold from the E. coli JM109 recombinant with a recovery of 49.02% using heat treatment and ion exchange chromatography, to give a single band on SDS-PAGE. The optimum pH and temperature of the enzyme were found to be pH 7.0 and 95°C. The enzyme was stable in the range pH 6~9, and showed a half-life of over 5 h at 95°C. The enzyme activity was activated with 5mmol/L Mg2+ and Co2+. The apparent Km and Vmax values for glucose were 105 mmol/L and 45.2 mol/(min×mg), respectively.