低温脂肪酶基因在毕赤酵母中的表达研究

将已有的低温脂肪酶基因克隆至表达载体pPICZα上,并转化毕赤酵母X-33。在低温条件下诱导培养6d后,发酵液中可检测到低温脂肪酶活力。重组酵母发酵液加入二硫苏糖醇后单位酶活力达到32U/mL,与未加入该试剂的原始菌株相比提高4倍以上。对重组脂肪酶的酶学性质研究表明,其最适pH为8.0,最适温度为35℃,50℃温浴30min后,仍有30.04%的活性。以不同碳链长度的4-Nitrophenyl Ester为底物检测其底物特异性,结果显示其较适合作用于短链底物。重组酵母能够在BMMY培养基中胞外分泌表达带有His标签的重组脂肪酶,经镍柱纯化后,SDS-PAGE检测可得到大约35kDa的单一蛋白质条带。

Expression of the Cold-adapted Lipase Gene in Pichia pastoris

The cold-adapted lipase(CAL) gene was inserted into expression vector pPICZα,and transferred into Pichia pastoris X-33.After 6 d cultivation under low temperature,lipase activity was detected in the fermentation broth.The lipase activity in the cultures of recombined Pichia pastoris reached to 32 U/mL after inducement with DTT.The enzyme activity increased more than four times compared to those without adding DTT.The optimal temperature and pH for lipase activity were 35 ℃ and pH8,and 30.04% of lipase activity still retained after incubating at 50 ℃ for 30 min.The recombinant CAL showed a preference to shorter carbon chain than the medium and long chain when tested with 4-nitrophenyl esters.The CAL with His tag was secreted extracellularly by the recombined Pichia pastoris in the BMMY medium and being purified by Ni2+-NTA affinity chromatography.The purified protein showed a single band of about 35 kDa on SDS-PAGE.