嗜热古菌Sulfolobus tokodaii strain 7中麦芽寡糖基海藻糖合酶的酶学性质

Characterization of a maltooligosyl trehalose synthase from hyperthermophilic archaeon Sulfolobus tokodaii strain 7

【目的】克隆表达嗜热古菌Sulfolobus tokodaii strain 7中的ST0929基因,并测定其酶活性。【方法】根据ST0929基因设计引物进行PCR扩增,将这段基因克隆到p ET-15b质粒上,重组质粒导入大肠杆菌BL21细胞中表达。亲和层析纯化酶蛋白,并测定其酶活性。【结果】SDS-PAGE分析表明其分子量大约为83 k D。酶学性质研究表明该酶的最适温度为75°C,最适p H为5.0,具有很强的热稳定性和p H稳定性。该酶还能对多种金属离子和有机溶剂具有一定的耐受性。底物特异性研究发现该酶能够利用麦芽糊精作底物,而不能利用壳寡糖、麦芽糖等。【结论】通过以上酶学性质的研究,说明这种来源于超嗜热古菌的麦芽寡糖基海藻糖合酶在工业生产海藻糖领域具有一定的应用前景。

[Objective] To clone the gene ST0929 encoding maltooligosyl trehalose synthase from the hyperthermophilic archaeon Sulfolobus tokodaii strain 7 and characterize the enzyme. [Methods] The ST0929 gene was amplified by PCR based on Sulfolobus tokodaii strain 7 ST0929 gene sequence and cloned into the expression vector p ET-15 b. The recombinant plasmid was transformed into E. coli BL21. After induced by IPTG（isopropyl-β-D-1-thiogalactoside）, the bacterial pellet was sonicated and purified by affinity chromatography. The enzymatic properties were then measured. [Results] SDS-PAGE analysis showed that the molecular mass of the enzyme was about 83 k D. The optimal temperature was at 75 °C and p H at 5.0. Moreover, the enzyme exhibited notable p H and thermal stability and was resistant to additives and mental ions. Substrate specificity analysis showed that the enzyme could use maltodextrin and maltooligosaccharide as substrates but could not use maltose, chitooligosaccharide. [Conclusion] The recombinant enzyme described in this study suggest its potential in industrial production of trehalose.