深海太平洋火色杆菌琼胶酶Aga2660的克隆表达及发酵优化

Cloning, expression and production optimization of an agarase Aga2660 from a deep sea bacterium Flammeovirga pacifica WPAGA1

摘要:

[背景] 琼胶寡糖已成为化妆品、食品、医药等领域的研究热点，而生物酶法被认为是制备琼胶寡糖的高效方法。[目的] 从深海太平洋火色杆菌（Flammeovirga pacifica WPAGA1）筛选获得琼胶酶基因aga2660，对基因aga2660进行克隆转化，使其在大肠杆菌中进行表达，分析重组酶的性质以及酶解产物。[方法] 采用克隆表达和镍柱纯化方法获得aga2660基因表达的纯化产物；采用薄层层析和离子色谱法分析酶解产物；5 L发酵罐采用补料阶段指数流加、诱导阶段乳糖连续流加的策略进行产酶发酵条件的优化。[结果] 基因aga2660是具有GH50家族典型特征的基因，酶解产物为单一的新琼二糖。最适温度为30℃，最适pH为7.0，Mn²⁺、Ca²⁺和Mg²⁺能促进琼胶酶Aga2660的酶活。采用发酵优化策略后的酶活达到11.81 U/mL，比优化前提高了13.2倍。[结论] 琼胶酶Aga2660具有良好的热、酸、碱稳定性，其酶解产物为单一的新琼二糖，这为特定聚合度琼胶寡糖的制备奠定了良好基础。

Abstract:

[Background] Agaro-oligosaccharides (AOS) have become a research hotspot in cosmetics, food, medicine and other fields. Biological enzyme method is considered as an efficient method to prepare AOS. [Objective] The agarase gene aga2660 was obtained from Flammeovirga pacifica WPAGA1. The gene aga2660 was cloned and transformed to be expressed in Escherichia coli. The properties of recombinant enzyme and enzymatic hydrolysate were analyzed. [Methods] The pure expression product was obtained by clone expression and nickel column purification. Enzymatic products of the agarase were analyzed by thin-layer chromatography (TLC) and ion chromatography (IC). The optimization of enzyme production conditions was carried out in a 5 L fermentor using the strategies of exponential feeding in the feeding stage and continuous feeding of lactose in the induction stage. [Results] The elected gene aga2660 possessed typical sequence characteristics of glucoside hydrolase family 50 (GH50). The end-product of agar degradation by Aga2660 was neoagarobiose. The optimum temperature and pH of Aga2660 was 30℃ and 7.0, respectively. Meanwhile, Aga2660 showed outstanding temperature and pH stability. Activities of Aga2660 were enhanced by Mn²⁺, Ca²⁺ and Mg²⁺ (1 mmol/L). The enzyme activity reached 11.81 U/mL after fermentation optimization strategy, which was 13.2 times higher than that before optimization. [Conclusion] The agarase Aga2660 was an agarase of GH50 family and indentified with high agar-degrading activity and excellent stability against acid, alkali and thermo. Its enzymolysis product is neoagarobiose, which lays a good foundation for large-scale preparation of AOS with single polymerization degree.