运用定点突变提高重组木聚糖酶在毕赤氏酵母中的表达

Improvement or Recombinant Xylanase XynF1 From Aspergillus oryzae Expressed in Pichia pastoris by Site-directed Mutagenesis

运用PCR介导的定点突变对米曲霉 (Aspergillusoryzae)来源的木聚糖酶在毕赤酵母中的重组表达进行了研究 ,获得一表达量远远高于亲本的突变株I1 5 6A ,对其进行了提纯并研究其酶学特性 ,除热稳定性外其余与亲本基本一致。突变株I1 5 6A所产木聚糖酶XynFl的分子量为 3 5kD ,在pH 4～ 9范围内稳定 ,最适pH为 7 0 ,最适温度为 45℃ ,在 5 0℃以下稳定性略高于亲本。

The recombinant xylanase XynF1 from Aspergillus oryzae expressed in the methylotrophic yeast Pichia pastoris was improved by site\|directed mutagenesis.The mutant I156A secreted about 47 7U/mL xylanase XynF1 in the BMMY medium.The mutant xylanase XynF1 was purified by ion exchange and gel filtration chromatographies.The molecular weight of purified XynF1 was 35kD.XynF1 was stable between pH 4～9 and its optimum pH was 7.0.The optimum temperature of XynF1 was 45℃ and it was stable below 50℃.The properties of mutant were same as the parent's except the stability of temperature.