里氏木霉木聚糖酶基因(XynⅡ)在毕赤酵母中异源表达及重组蛋白酶学性质分析  

Expression of the Trichoderma reesei Rut C-30 Xylanase Gene 2(XynⅡ) in Pichia pastoris and Characterization of Its Recombinant Enzyme

作　　者：何军[1] 余冰[1] 张克英[1] 陈代文[1] 

机构地区：[1]四川农业大学动物营养研究所,教育部动物抗病营养重点开放实验室,雅安625014

出　　处：《农业生物技术学报》2009年第5期920-924,共5页Journal of Agricultural Biotechnology

基　　金：四川省科技攻关项目(No.2007Z06-050);教育部长江学者与创新团队计划(No.IRTO555-5)资助

摘　　要：利用RT-PCR技术从高产木聚糖酶里氏木霉(Trichoderma reesei)RutC-30中成功地扩增出木聚糖酶基因(XynⅡ),经序列分析证实,在该菌株诱变选育过程中其成熟肽序列有2处碱基发生突变;将不带原基因信号肽的XynⅡ克隆到毕赤酵母高效表达载体pPICZαA上,线性化后经电击转化到毕赤酵母(Pichia pastoris)中,经Zeocin及PCR筛选后得到的转化子用1%甲醇诱导。SDS-PAGE证实,重组子实现了分泌表达,且发酵上清中几乎无杂蛋白;重组酶酶学性质分析表明,最适反应温度为60℃,最适反应pH值为6.0,热稳定性较好,在50℃下保温30min仍能保留95%以上活性。The xylanase 2 gene（XynⅡ）, encoding the main Trichoderma reesei Rut C-30 endo-β-1, 4-xylanase, was successfully cloned by RT-PCR.Sequence analysis indicated that there was two-base mutations produced during its mutant selection program.The XynⅡgene was subsequently cloned into the pPICZаA vector and expressed in Pichia pastoris.The desired P.pastoris strains produced β-xylanase free of any side activities under the induction of 1% methanol, and the molecular mass was estimated by SDS-PAGE as 21 kD.The activity of the recombinant XynⅡ was highest at 60 ℃ which was 5 ℃ higher than that of native xylanse.In addition, the recombinant XynⅡ was active over a broad range of pH 3.0～8.0 with maximal activity at pH 6.0.The enzyme was quite stable at 50 ℃ and retained more than 95% of its activity after 30 min incubation.These properties shall make the enzyme an attractive candidate in various industrial applications.

关 键 词：里氏木霉 木聚糖酶 毕赤酵母 表达 

分 类 号：S188[农业科学—农业基础科学]