节杆菌(Arthrobacter sp.) EpRS66 β-呋喃果糖苷酶重组表达及酶促合成低聚乳果糖

Recombinant expression of β-fructofuranosidase from Arthrobacter sp. EpRS66 and enzymatic synthesis of lactosucrose

摘要:

【背景】 低聚乳果糖是一种新型的功能性低聚糖，具有改善肠道菌群及提高免疫力等功能，被应用于食品、饲料等行业，市场需求量逐年增加。β-呋喃果糖苷酶对乳糖的受体特异性较高，工业上常用来制备低聚乳果糖，但现有酶种转化率较低、生产成本较高。【目的】 挖掘新型 β-呋喃果糖苷酶基因，探究其酶学性质及制备低聚乳果糖的能力。【方法】 克隆节杆菌(Arthrobacter sp.) EpRS66的β-呋喃果糖苷酶基因，将其在大肠杆菌(Escherichia coli)中重组表达，研究重组酶的酶学性质，优化其催化制备低聚乳果糖的反应条件。【结果】 重组酶的最适温度为40 ℃，最适pH值为6.0，在pH 5.0-8.0范围内稳定性良好，在40 ℃的半衰期为20 min。利用重组酶制备低聚乳果糖，以15%蔗糖和15%乳糖为底物，在40 ℃、pH 6.0及加酶量25 μg/mL的条件下反应3 h后转化率最高达到32.2%；而在30%蔗糖和30%乳糖的高底物浓度下，转化率也可达到30.8%。【结论】 本研究获得了一种能有效催化形成低聚乳果糖的新型β-呋喃果糖苷酶，为工业化生产低聚乳果糖提供了新的选择。

Abstract:

[Background] Lactosucrose, a novel functional oligosaccharide, has been demonstrated capable of improving the gut microbiota and boosting the immunity of humans and animals. Since it is increasingly utilized in food and feed industries, the market demand for lactosucrose has been increasing year by year. The industrial production of lactosucrose mainly uses β-fructofuranosidases due to the high product specificity. However, the conversion rates of existing enzymes were generally low, which made the production cost remaining high. [Objective] To identify new β-fructofuranosidases for the enzymatic properties and industrial production of lactosucrose. [Methods] We identified a β-fructofuranosidase gene from Arthrobacter sp. EpRS66 and expressed it in Escherichia coli. Furthermore, we characterized the enzymatic properties of the recombinant β-fructofuranosidase and optimized the optimum reaction conditions for the production of lactosucrose. [Results] The recombinant enzyme showed the optimal performance at 40 ℃ and pH 6.0. It maintained good stability within the range of pH 5.0-8.0 and had a half-life of 20 min at 40 ℃. Under the reaction conditions of 40 ℃ and pH 6.0, the conversion rate of lactosucrose reached 32.2% at the time point of 3 h with substrates of 15% sucrose and 15% lactose and an enzyme addition of 25 μg/mL. Moreover, with high concentrations of substrates (30% sucrose and 30% lactose), the conversion rate reached 30.8%. [Conclusion] This study provides a novel β-fructofuranosidase that can effectively catalyze the formation of lactosucrose, offering a new choice for industrial production of lactosucrose.