嗜热真菌木聚糖酶1YNA及其双硫键突变体在毕赤酵母中的表达  

Expression of Thermomyces lanuginosus Xylanase 1YNA and Its Disulphide Bridge Mutant in Pichia Pastoris

作　　者：李思佳[1] 王亚伟[1] 付正[1] 汪文俊[1] Ossi Turunen 熊海容[1] 

机构地区：[1]中南民族大学生命科学学院南方民族地区生物资源保护与综合利用工程中心,武汉430074 [2]Aalto University,Finland,FI-00076

出　　处：《中国生物工程杂志》2013年第3期74-79,共6页China Biotechnology

摘　　要：将前期已经获得的大肠杆菌表达的木聚糖酶1YNA的野生型(BL1)及定点突变后的双硫键型(C24)基因,分别克隆到表达载体pPIC9中,获得的重组质粒转入毕赤酵母GS115中,构建成功毕赤酵母工程菌并表达获得木聚糖酶野生型(TLX)及木聚糖酶双硫键型(DSB)。在对表达量和酶特性进行分析后的结果证实,2株重组毕赤酵母均能高效表达,在摇床培养水平上,野生型与突变型均表达出具有高活力的酶蛋白,表达量分别为1.75mg/ml(TLX)与1.82mg/ml(DSB),且在培养液中目标产物外的杂蛋白含量非常少。DSB显示出的热稳定性明显高于TLX。DSB的最适反应温度为75℃,TLX的最适反应温度为68℃。DSB在75℃下处理30 min分钟后仍能保留50%的残余酶活力,同等处理条件下TLX的残余酶活力则低于20%。本项实验是首次报道利用毕赤酵母表达木聚糖酶1YNA及其突变体。The wild type xylanase gene （1yna） and its disulfide bridge mutant gene were linked to vectors pPIC9, and then transformed into Pichia pastoris GS115 to construct the recombinant strains for expression the wild type xylanase （TLX） and its disulfide bridge mutant （DSB）, respectively. The recombinants express TLX and DSB at high level, with electrophoresis pure protein products. In shake flask cultivation, the protein expression levels by P. pastoris were 1.75 mg/ml （TLX） and 1.82 mg/ml （DSB）, respectively. The enzymatic properties showed that DSB performed the optimum temperature at 75℃, and the TLX performed the optimum temperature at 68℃. DSB showed a better thermostabllity than TLX. After 30 min inactivation at 75℃, DSB still remained 50% of the residual activity, whereas TLX only remained less than 20% of its activity.

关 键 词：木聚糖酶1YNA 毕赤酵母 异源表达 酶特性 耐热性 

分 类 号：Q78[生物学—分子生物学]