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采用RT-PCR方法克隆到里氏木霉Rut C-30木聚糖酶(XYN II)的cDNA序列。测序结果表明,XYN II的cDNA基因开放阅读框长度为669 bp,编码223个氨基酸,N端1~19个氨基酸为潜在的信号肽序列,删去潜在信号肽序列,将里氏木霉木聚糖酶的基因(xynII)构建到巴斯德毕赤酵母分泌表达载体pPIC9K上,线性化后电击转化到巴斯德毕赤酵母中,经G418筛选和PCR鉴定后的转化子用1%的甲醇进行诱导,对重组木聚糖酶活检测显示该基因能在毕赤酵母中表达有生物活性的XYN II并分泌到胞外。发酵液中的酶活在诱导培养60 h达到1.45 IU/mL,最适酶解温度为50℃,最适pH值为6.0。
The cDNA sequence of Trichoderma reesei Rut C-30 xylanase (XYN II) was cloned by RT-PCR. The sequencing results showed that the open reading frame of XYN II cDNA was 669 bp, encoding 223 amino acids and the potential amino acid sequence of 1-19 amino acids was the potential signal peptide sequence. The potential signal peptide sequence was deleted and the Trichoderma reesei The gene of xylanase (xynII) was constructed into the Pichia pastoris secretion expression vector pPIC9K, transformed into Pichia pastoris after linearization, transformed with Pichia pastoris by G418 selection and PCR identification with 1% methanol The recombinant Xylanase activity assay showed that the gene could express XYN II in Pichia pastoris and was secreted extracellularly. The activity of enzyme in the fermentation broth reached 1.45 IU / mL at 60 h after induction, the optimum temperature was 50 ℃ and the optimum pH was 6.0.