特异腐质酶Humicola insolens Y1耐热型木聚糖酶基因的克隆及酶学性质分析

利用简并PCR和TAIL PCR从嗜热特异腐质霉Humicola insolens Y1中克隆得到一个新的耐热型木聚糖酶基因xynD。该基因全长1 104 bp, 共编码367个氨基酸,不含有内含子,N端的22个氨基酸为信号肽。成熟蛋白在毕赤酵母中进行了重组表达,重组蛋白经超滤和离子交换柱纯化后达到电泳纯。对其酶学性质分析表明,XynD的最适pH为6.0,在pH 4.0～7.0可以保持80%以上的酶活性,在pH 5.0～10.0具有良好的pH稳定性。最适作用温度为70℃,在60℃条件下保持稳定。多数金属离子对XynD酶活力没有明显的影响。麦芽汁降解实验表明,XynD与商用β 葡聚糖共同作用可以提高麦芽的过滤速率(45.7％)并降低粘度(81％)。因此XynD在酿酒业,特别是啤酒制造业具有潜在的应用价值。

Gene Cloning and Analysis of Enzymology Property of an Thermophilic Xylanase from Humicola insolens Y1

An novel thermophilic xylanase encoding gene was cloned from Humicola insolens Y1 by degenerate PCR and TAIL PCR．The complete gene sequence consisted of 1 104 bp and encoded a protein with 367 amino acid. There was no intron and only a signal peptide from the 22 residues of the N terminus．The matured xylanase was expressed in Pichia pastoris. The recombinant enzyme XynD was purified to homogeneity by ultrafiltration and ion exchange chromatography, which showed optimal activity at pH 6.0 and retained over 80% of the maximal activity at pH 4.0～7.0. XynD showed high stability at pH 5.0～10.0. The optimum temperature of XynD was 70℃, and XynD was stable at 60℃. XynD had strong resistance to most metal ions. Brewery mashing studies indicated that the combination of XynD and β glucanase could make the filtration rate of brewery mash increased by 41.7% and the viscosity of brewery mash reduced by 8.1%. These result indicated that XynD had potential application value in brewing industry, especially in beer brewing.