阴沟肠杆菌GX-3的中性蔗糖酶在大肠杆菌中的表达及其特性鉴定

Expression and characterization of a neutral Enterobacter cloacae GX-3 invertase

摘要:

摘要:【目的】在阴沟肠杆菌(Enterobacter cloacae) GX-3中发现了一个编码中性pH范围内起作用的蔗糖酶基因，在大肠杆菌(Escherichia coli)中进行克隆表达及纯化，并研究该蔗糖酶的酶学性质，为蔗糖酶的应用研究及开发利用奠定基础。【方法】通过查找GenBank数据库中来自阴沟肠杆菌同属中的编码蔗糖酶的基因序列，对这些序列进行比对分析设计简并引物扩增保守区;利用PCR扩增目的基因，以pQE30为表达载体构建重组质粒;镍亲和层析纯化重组蛋白，对重组酶的酶学性质进行详细研究。【结果】一个编码蔗糖酶的基因(Einv)被从阴沟肠杆菌GX-3中发现和克隆。生物化学特性鉴定表明这个蔗糖酶的活性最适温度为40℃和最适pH为6.5。凝胶渗透色谱法分析显示Einv是以二聚体的形式存在。Einv这个蔗糖酶在1170 mmol/L的蔗糖条件下仍保留有80%以上的水解活性。而在高浓度的蔗糖条件下，Einv只有水解活性而没有转糖基的副反应发生。它能够水解棉子糖、蔗果三糖、蔗果四糖、蔗果五糖和水苏糖。【结论】获得的Einv是一个在中性pH范围内起作用的β-呋喃果糖苷酶，只有水解功能而没有转糖基功能。这些特性表明这个中性蔗糖酶在食品工业具有重要的应用价值。

Abstract:

Abstract:［Objective］ To characterize a neutral invertase from Enterobacter cloacae GX-3.［Methods］ By searching GenBank database，we found the genes encoding invertase from the same genus Enterobacter. These sequences were aligned and analyzed. Then，a gene encoding neutral invertase was amplified by PCR. The recombinant plasmid pQE-Einv was constructed．We purified the expressed protein Einv with nickel-nitrilotriacetic acid chromatography．At last，the characterics of the recombinant protein Einv were studied in detail.［Results］ A gene encoding neutral invertase was discovered and cloned from E.cloacae GX-3. The recombinant enzyme Einv was characterized. Einv had an optimum pH of 6.5 and an optimum temperature of 40℃．The results of sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and gel permeation chromatography (GPC) showed that Einv was a homo-dimer protein．Einv retained 80% activity at sucrose concentrations up to 1170 mmol /L．But，Einv had no transglycosylation activity at high sucrose concentration．It could hydrolyze raffinose，1-kestose，nystose，fructofuranosylnystose and stachyose.［Conclusion］It is first reported that an invertase from Enterobacter cloacae is a β-fructofuranosidase at neutral pH range. It only has hydrolysis activity without tranglycosylation activity. These characteristics indicate that the neutral invertase Einv has important applications in food industry．