一种黑曲霉α-L-阿拉伯呋喃糖苷酶克隆表达、性质分析和果汁澄清效果

Cloning, Expression and Characterization of α-L-Arabinofuranosidase from Aspergillus niger and Its Clarification Effect on Fruit Juice

利用基因重组技术克隆黑曲霉(Aspergillus niger)的1887 bpα-L-阿拉伯呋喃糖苷酶基因,在毕赤酵母SMD1168中诱导表达获得α-L-阿拉伯呋喃糖苷酶。结果表明,重组蛋白大小为85 kDa,纯化后其比活力为55.73 U/mg;最适反应温度和pH值分别为65℃和5.0;金属离子K^(+)对该酶活力有促进作用,而Cu^(2+)对酶活力有明显抑制作用;以4-硝基苯酚-α-L-阿拉伯呋喃糖苷为底物时测得K_(m)值和V_(max)值分别为2.31 mmol/L和625μmol/(mL·min);该酶对柑橘果汁具有显著澄清效果。本研究不仅丰富了α-L-阿拉伯呋喃糖苷酶资源库,同时为后续探究α-L-阿拉伯呋喃糖苷酶在果汁加工中的应用提供了参考。

The sequence of the 1887 bp longα-L-arabinofuranosidase gene in Aspergillus niger was cloned by using gene recombination,and was expressed in Pichia pastoris SMD1168.The recombinant protein had a molecular mass of 85 kDa and a specific activity of 55.73 U/mg.The optimal temperature and pH for the enzyme were 65℃and 5.0,respectively.The enzymatic activity was promoted by Mn^(2+),while it was significantly inhibited by K^(+).When p-nitrophenyl-α-Larabinofuranoside(pNPA)was used as the substrate,the K_(m) and V_(max) values ofα-L-arabinofuranosidase were 2.31 mmol/L and 625μmol/(mL·min),respectively.More interestingly,the purifiedα-L-arabinofuranosidase showed a significant clarification effect on citrus juice.This study not only enriches the resource library ofα-L-arabinofuranosidase,but also provides a reference for juice processing withα-L-arabinofuranosidase.