类芽孢杆菌碱性果胶裂解酶Pel在毕赤酵母中的高效表达

Expression of the Alkaline Pectate Lyase Gene pel from Paenibacillus campinasensis BL-11 in Pichia pastoris

本研究全基因合成了来源于类芽孢杆菌的碱性果胶酶基因pel,克隆至p HKA载体上,成功实现其在毕赤酵母GS115中的分泌表达;并对信号肽和启动子进行优化,使得摇瓶发酵诱导168 h果胶裂解酶酶活达到432.36 U/m L。初步测定了果胶裂解酶Pel的基本酶学性质,其最适反应温度和p H分别为60℃、10.0;在50℃下处理300 min裂解酶活力保留90%以上,而在60℃下处理210 min,酶活力剩余约50%;在p H 9~12的50 m M各缓冲液中处理酶液16 h,酶活力仍然保留90%以上,该酶耐碱性强,但在偏酸性缓冲液中处理该酶,酶活力有所下降;同时研究了二价金属离子对果胶裂解酶活性力的影响,Ca^(2+)对Pel发挥裂解作用是必需的,Fe^(2+)、Mg^(2+)、Ni^(2+)对Pel均有激活作用,而金属离子螯合物EDTA的存在则使酶活力完全丧失;该酶在碱性条件下较好的稳定性决定了其潜在的工业应用前景。

The alkaline pectate lyase gene pel from Paenibacillus campinasensis BL-11 was synthesized by total gene synthesis, cloned on a pHKA carrier, and actively expressed in Pichia pastoris GS115. The signal peptide and promoter were optimized, so that the pectate lyase activity reached 432.36 U/mL after induction was carried out by 168-h shake flask fermentation. The basic enzymatic properties of the recombinant pectate lyase were preliminarily measured, and the optimal pH and temperature were 10.0 and 60, respectively. The residual enzyme activity was still higher than 90% when it was incubated at 50 for 300 min, while the residual enzyme activity was only about 50% after the treatment was carried out at 60 for 210 min. The enzyme activity remained more than 90% after the recombinant pectate lyase was treated with different buffer solutions ranging from pH 9~12 for 16 h. This enzyme had strong alkali resistance, but the enzyme activity was slightly decreased after being treated in slightly acidic buffers. Meanwhile, the effects of divalent metal ions on pel expression were also determined. The results showed that Ca2+was essential for the trans-elimination reaction, and Fe2+, Mg2+and Ni2+had activating effects on pel activity, while the enzyme was completely inactivated with metal ion-ethylenediaminetetraacetic acid complexes. In summary, pectate lyase exhibits potential industrial application owing to its good stability under alkali conditions. © 2015, Editorial Board of Modern Food Science and Technology. All right reserved.