黑曲霉β-木糖苷酶An-xyl的重组表达与木糖耐受性表征

Expression of β-xylosidase An-xyl from Aspergillus niger and characterization of its xylose tolerance

摘要:

β-木糖苷酶催化低聚木糖水解在木质纤维素降解中起重要作用，但该酶活性易被产物木糖抑制，严重限制了其应用。基于分子对接，本文研究了茶梗发酵培养基差异表达显著的黑曲霉(Aspergillus niger) β-木糖苷酶An-xyl与木糖的亲和性，并对其进行克隆表达和性质表征，进一步探讨了该酶与纤维素酶对茶梗中木质纤维素的降解作用。分子对接结果表明，An-xyl与木糖的亲和性低于木糖耐受性较差的米曲霉β-木糖苷酶xylA。重组表达的An-xyl木糖抑制常数K_i值为433.2 mmol/L，与同为GH3家族的β-木糖苷酶相比木糖耐受性较高。以pNPX为底物时，K_m和V_max分别为3.6 mmol/L和10 000 μmol/(min·mL)。An-xyl最适温度65 °C，最适pH 4.0，65 °C处理300 min能保持约61%的酶活力，在pH 2.0-8.0的范围内处理24 h后酶活力仍能维持80%左右。添加An-xyl与纤维素酶共同水解茶梗，反应2 h和4 h产生还原糖含量比单独使用纤维素酶水解分别提高了19.3%和38.6%。本研究表明，通过差异表达挖掘的An-xyl具有高木糖耐受性和较好的催化活性和稳定性，可协同作用水解茶梗类木质纤维素，丰富了具有高木糖耐受性β-木糖苷酶的资源，为其应用提供了实验依据。

Abstract:

The hydrolysis of xylo-oligosaccharides catalyzed by β-xylosidase plays an important role in the degradation of lignocellulose. However, the enzyme is easily inhibited by its catalytic product xylose, which severely limits its application. Based on molecular docking, this paper studied the xylose affinity of Aspergillus niger β-xylosidase An-xyl, which was significantly differentially expressed in the fermentation medium of tea stalks, through cloning, expression and characterization. The synergistic degradation effect of this enzyme and cellulase on lignocellulose in tea stems was investigated. Molecular docking showed that the affinity of An-xyl to xylose was lower than that of Aspergillus oryzae β-xylosidase with poor xylose tolerance. The K_i value of xylose inhibition constant of recombinant-expressed An-xyl was 433.2 mmol/L, higher than that of most β-xylosidases of the GH3 family. The K_m and V_max towards pNPX were 3.6 mmol/L and 10 000 μmol/(min·mL), respectively. The optimum temperature of An-xyl was 65 °C, the optimum pH was 4.0, 61% of the An-xyl activity could be retained upon treatment at 65°C for 300 min, and 80% of the An-xyl activity could be retained upon treatment at pH 2.0-8.0 for 24 h. The hydrolysis of tea stem by An-xyl and cellulase produced 19.3% and 38.6% higher reducing sugar content at 2 h and 4 h, respectively, than that of using cellulase alone. This study showed that the An-xyl mined from differential expression exhibited high xylose tolerance and higher catalytic activity and stability, and could hydrolyze tea stem lignocellulose synergistically, which enriched the resource of β-xylosidase with high xylose tolerance, thus may facilitate the advanced experimental research and its application.