马克斯克鲁维酵母菌菊粉酶的异源表达及低聚果糖制备工艺优化

Heterelogous expression of Kluyveromyces marxianus inulinase gene and optimization of reaction parameters for oligofructose preparation from inulin

摘要:

【目的】低聚果糖是新型的食品和保健品原料，具有广阔的市场需求。以菊粉酶水解菊粉制备低聚果糖的酶法工艺是先进的绿色制造。本研究旨在获得高产的菊粉酶菌株及以菊粉为原料酶法制备低聚果糖的优化工艺。【方法】采用基因工程手段克隆马克斯克鲁维酵母菌(Kluyveromyces marxianus)的菊粉酶基因，实现其在毕赤酵母中的高效表达；测定菊粉酶在不同pH、温度、金属离子和底物浓度等条件下的酶活变化趋势，获得最佳的反应参数；通过高效液相色谱法检测水解产物，获得不同酶量水解产物各组分分布。【结果】菊粉酶工程菌株在10 L发酵罐中的产菊粉酶活达1 570 U/mL、蛋白质含量为2.75 g/L发酵液；菊粉酶最适反应参数为：在体积为1 L的反应体系中，pH 5.0、反应温度50 °C、含0.2 mmol/L Mg2+以及菊粉浓度为8%。在该条件下，酶量为10 U时菊粉被完全水解。水解产物中单糖和二糖含量仅为9.25%，而低聚果糖(C3?C8)含量为90.75%，且C3?C5低聚果糖含量高达72.92%。【结论】克隆了K. marxianus菊粉酶基因并实现了高效表达，获得了水解菊粉制备低聚果糖的最佳工艺条件。为菊粉酶的大量生产及低聚果糖的酶法制备奠定了良好的基础。

Abstract:

[Objective] Oligofructose is a promising food additive and health product. Enzymatically hydrolyzing the inulin by inulinase to produce oligofructoses was regarded as a promising, green and environment friendly technics. The aim of this study is to achieve an efficient production of Kluyveromyces marxianus inulinase and optimize the reaction parameters for oligofructose production from inulin. [Methods] The inulinase gene was cloned from K. marxianus strain CICC 1726 and expressed in Pichia pastoris cells. The reaction parameters such as pH, temperature, metal ions, substrate concentration and the amount of enzyme on the hydrolyzing of inulin to produce oligofructose were optimized. [Results] The inulinase activity of Pichia recombinants in 10 L fermenter reached 1 570 U/mL and the protein content in fermentation broth reached 2.75 g/L. In a 1 L reaction volume, when the parameters were pH 5.0, tempreture 50 °C, 0.2 mmol/L Mg2+ and 8% substrate, the inulin was properly hydrolyzed, with the content of C1 and C2 sugar was 9.25% and the oilgofructose (C3?C8) was 90.75% in the hydrolysates, among which the content of C3–C5 fraction was 72.92%. [Conclusion] This study has realized high-level heterologous expression of inulinase and facilitated the bulk preparation of oligofructose from inulin.