黑曲霉阿魏酸酯酶在毕赤酵母中的组成型表达

Constitutive expression of feruloyl esterase from Aspergillus niger in Pichia pastoris

摘要:

【目的】实现黑曲霉来源的阿魏酸酯酶在毕赤酵母(Pichia pastoris GS115)中的组成型表达。【方法】以黑曲霉(Aspergillus niger)基因组为模板，经重叠延伸PCR扩增得到阿魏酸酯酶基因(AnfaeA)，将其与载体pGAP9K相连，构建重组表达载体pGAP9KAnfaeA，经Sal I线性化后电转入毕赤酵母GS115中，得到重组菌株。高效液相色谱法测定发酵液中阿魏酸酯酶活力，并对重组菌进行了发酵优化。【结果】克隆得到783 bp的阿魏酸酯酶编码基因并实现了其在毕赤酵母中的组成型表达。重组菌发酵84 h后，上清液中酶活达5.72±0.10 U/mL。重组酶(reAnfaeA)经分离纯化后比酶活为59.75 U/mg，大小约为40 kD。发酵优化结果为：葡萄糖40.0 g/L，蛋白胨10.0 g/L，酵母膏30.0 g/L，CaCO3 0.2 g/L，种龄28 h，接种量3% (体积比)，装液量50 mL/250 mL。在此条件下发酵培养，酶活达15.60±0.23 U/mL。【结论】阿魏酸酯酶在毕赤酵母中的组成型表达，对研究毕赤酵母组成型表达系统和阿魏酸酯酶的发酵生产具有一定的借鉴意义。

Abstract:

[Objective] This study is aimed to constitutively express feruloyl esterase gene (AnfaeA) from Aspergillus niger in Pichia pastoris GS115. [Methods] The AnfaeA gene was amplified from A. niger by overlap extension PCR and cloned into the expression vector pGAP9K.The recombinant expression vector (pGAP9KAnfaeA) was linearized by Sal I, then transformed into P. pastoris GS115. Feruloyl esterase activity of the recombinant strain was determined by high-performance liquid chromatography (HPLC) and we optimized the fermentation conditions. [Results] We cloned AnfaeA gene (783 bp) and constitutively expressed AnfaeA gene in P. pastoris GS115. Feruloyl esterase activity reached its peak (5.72±0.10 U/mL) after 84 h fermentation, its specific activity was 59.75 U/mg, and the molecular weight of reAnfaeA was about 40 kD. The optimal fermentation conditions were as follows: glucose 40.0 g/L, tryptone 10.0 g/L, yeast extract 30.0 g/L, CaCO3 0.2 g/L, inoculum age 28 hours, inoculation level 3% (v/v), medium volume 50 mL in 250 mL flask. Under these conditions, feruloyl esterase activity in fermentation supernatant was 15.60±0.23 U/mL. [Conclusion] We constitutively expressed feruloyl esterase in P. pastoris GS115. It was helpful to study the constitutive expression system in P. pastoris and improve the fermentation production of feruloyl esterase.