棘孢曲霉β-葡萄糖苷酶I的毕赤酵母多拷贝体系构建与在其高密度发酵表达.pdfVIP

现代食品科技 Modern Food Science and Technology 2014, Vol.30, No.6 棘孢曲霉β-葡萄糖苷酶 I的毕赤酵母多拷贝体系 构建及其在高密度发酵中的表达 陈文勇，胡霞艳，康雅君，郑穗平 （华南理工大学生物科学与工程学院，广东广州510006） 摘要：β- 葡萄糖苷酶目前广泛应用于合成烷基糖苷、芳基糖苷和辅助纤维素酶水解纤维素。本研究将来自棘孢曲霉（Aspergilus aculeatus）的β- 葡萄糖苷酶I (ABGL)在毕赤酵母中通过构建多拷贝表达盒的方法实现高效表达，利用同尾酶Bgl II/BamH I 反复酶切 和用DNA 连接酶连接的方法，成功构建了含多拷贝表达盒5’AOX-ABGL-TT 的pHKA-(ABGL) 的分泌重组质粒，并线性化重组质粒 3 转化毕赤酵母GS115，经过七叶苷显色筛选平板筛选获得高表达的GS115/pHKA-(ABGL)3 菌株，且在50 升发酵罐实现高密度发酵表 达。研究表明，以pNPG 为水解底物，在甲醇诱导 120h 时摇瓶发酵液上清酶活可达83.15 U/mL，较之前报道的酶活提高了3.35 倍； 同时在50 L 发酵罐实现高密度发酵，在甲醇诱导120 h 时发酵罐上清酶活可达979 U/mL，较对照菌株的发酵罐上清液酶活提高2.94 倍，且总蛋白表达量可以达到12 g/L。 关键词：棘孢曲霉NO.F-50 ；β- 葡萄糖苷酶；多拷贝表达盒；毕赤酵母；分泌表达；高密度发酵 文章篇号：1673-9078(2014)6-70-75 Construction and High Expression of β-glucosidase I Gene from Aspergillus aculeatus NO.F 50 in Pichia pastoris CHEN Wen-yong, HU Xia-yan, KANG Ya-jun, ZHENG Sui-ping (School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China) Abstract: β-Glucosidaseiswidely used in synthesis of alkyl glucosides and aryl glucosides,as well asdegradation of cellulose. The β-glucosidaseI gene from Aspergilus aculeatus No.F50 was cloned, andmultiple copies of expression cassette 5’AOX-ABGL-TT was inserted intosecreted expression vector pHKA, which could cut apart by the restriction enzymes Bgl II and BamH I . And thenthey were connected together by the use of DNA ligase. The constructed pHKA-(ABGL)3 recombinant plasmid was linearized and integrated into Pichia Pastoris GS115 strain by electroporation. Esculin high-throughput screening method was used to detect positive clones which were cultured in shake flask. In the methanol indu