低温脂肪酶基因在毕赤酵母中的表达研究  

Expression of the Cold-adapted Lipase Gene in Pichia pastoris

作　　者：刘羽[1,2] 王能飞[1,2] 黄亦钧 林学政[2] 沈继红[2] 

机构地区：[1]中国海洋大学,山东青岛266003 [2]国家海洋局第一海洋研究所,山东青岛266061 [3]青岛康地恩生物科技有限公司,山东青岛266061

出　　处：《海洋科学进展》2012年第1期155-162,共8页Advances in Marine Science

基　　金：国家高技术发展研究计划项目--极地特殊海洋微生物资源利用的关键技术研究(2007AA091905);海洋公益性行业科研专项--盐生植物生产纤维素乙醇与电厂废气联产生物柴油研究与示范(201005031);利用极地微生物生产低温纤维素酶关键工程化技术开发与应用研究(201005020-8);绿潮藻纤维素乙醇的制备技术研究(201105028-02);国家海洋局海洋生物活性物质重点实验室开放基金--南极冰藻L4Psbo基因在盐度和光照胁迫的表达差异研究(MBSMAT-2009-02);"十二五"国家科技计划课题--微藻减排燃煤烟气及二氧化碳和炼制生物柴油的关键技术和产业化示范(2011BAD14B04)

摘　　要：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。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.

关 键 词：低温脂肪酶 重组蛋白 毕赤酵母 组氨酸标签 

分 类 号：Q933[生物学—微生物学]