阿魏酸酯酶A的基因克隆与表达及其水解产物的纯化

Gene cloning, expression of a feruloyl esterase A and purification of its hydrolysis products

摘要:

为在毕赤酵母中表达来源于米曲霉Aspergillus oryzae的A型阿魏酸酯酶并研究其水解功能，探讨大孔树脂对其水解产物阿魏酸的纯化条件及纯化效果，以米曲霉A. oryzae CICC 40186总RNA为模板，通过RT-PCR技术克隆出了米曲霉阿魏酸酯酶A (AorFaeA) 成熟肽的编码基因AorfaeA，并借助pPIC9K质粒实现了其在毕赤酵母GS115中的异源表达。SDS-PAGE分析结果显示纯化后的重组阿魏酸酯酶 (reAorFaeA) 为单一条带，其表观分子质量约39.0 kDa。以阿魏酸甲酯为底物，经高效液相色谱法测得该酶的最高酶活为58.35 U/mg。利用reAorFaeA和木聚糖酶复合酶水解去淀粉麦麸制备阿魏酸，用大孔树脂纯化小麦麸皮阿魏酸粗提液，所测树脂中HPD-300型大孔树脂的吸附量和解吸率较高，以50%的乙醇为洗脱液，当流速为1.0 mL/min时洗脱效果较好。在该纯化条件下，阿魏酸的回收率为92%，质量分数由原材料中的0.13％富集提高到10.55%。这些研究为阿魏酸的酶法“绿色生产”及应用奠定了坚实的理论基础。

Abstract:

To express feruloyl esterase A from Aspergillus oryzae in Pichia pastoris expression system and study its hydrolysis function, explore the conditions and effects of purification for ferulic acid extracts by macroporos resin. Using the total RNA from A. oryzae CICC 40186 as the template, we amplified coding sequence AorfaeA encoding a mature feruloyl esterase A (AorFaeA) by RT-PCR technique. Then, the coding sequence AorfaeA was successfully expressed in Pichia pastoris GS115 mediated by an expression plasmid pPIC9K. The purified recombinant AorFaeA (reAorFaeA) showed one single band on SDS-PAGE with an apparent molecular weight of 39.0 kDa. The maximum activity of reAorFaeA to methyl ferulate, measured by high-performance liquid chromatography (HPLC), was 58.35 U/mg. Then, reAorFaeA was used to release ferulic acid from de-starched wheat bran in the presence of xylanase. The purification tests for ferulic acid from the enzymatic hydrolysate were carried out with preselected macroporous resins. The results showed that macroporous resin HPD-300 had much higher adsorption and desorption capacities. Ferulic acid could be quantitatively recovered by 50% of the eluent concentration at a flow speed of 1 mL/min. Under the purification condition, the recovery ratio of ferulic acid was 92%, and the content of ferulic acid was increased from 0.13% in the raw material to 10.55%. This work exploits the breakdown of ferulic acid by recombinant enzymeand provids a good strategy to its “green production”.