普鲁兰酶突变体文库高通量筛选方法的建立及应用
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Establishment and Application of High-Throughput Screening Method for Pullulanase Mutant Library
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    摘要:

    在摇瓶水平实现普鲁兰酶基因(GenBank Accession No:AX203843)在大肠杆菌BL21 (DE3)的异源表达,并成功按规格缩小实现了基于深孔板的高通量细胞培养,同时建立了基于深孔板的普鲁兰酶酶活快速高效的高通量检测方法。利用易错PCR技术对普鲁兰酶基因进行定向进化,突变基因产物重组于表达载体pET-28a(+)-pelB中,并导入大肠杆菌BL21 (DE3)构建突变体文库,利用该高通量筛选方法实现了突变体文库的快速高效筛选并获得了一株突变菌株4A4,其表达量提高了46.5%。该方法不仅适用于淀粉酶、纤维素酶等酶类的高通量筛选,同时为菌株文库的筛选以及蛋白质的定向进化提供了一种新思路。

    Abstract:

    Pullulanase gene (GenBank Accession No:AX203843) was heterologous expression in BL21 (DE3) in shake flask and scale-down to the microtiter plate scale successfully. Based on microtiter plate,a high-throughput culture method and an efficient high-throughput detection method for pullulanase activity were established. Random mutagenesis on pullulanase gene was performed through error-prone PCR strategy. The error-prone PCR products were recombinated in expression vector pET-28a(+)-pelB and then introducted into BL21 (DE3) to construct mutant library. The High-throughput screening method for Pullulanase in study was used to screen the mutant library efficiently and optimum mutant 4A4 was screened,the pullulanase activity in supernatant improved 1.465 folds. This method not only is also applicable to high-throughput screening of amylase and cellulase,but also provides a new way for high-throughput screening of strain library and directed evolution of proteins.

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聂简琪,陈阿娜,刘秀霞,杨艳坤,白仲虎.普鲁兰酶突变体文库高通量筛选方法的建立及应用[J].食品与生物技术学报,2016,35(9):993-1000.

NIE Jianqi, CHEN Ana, LIU Xiuxia, YANG Yankun, BAI Zhonghu. Establishment and Application of High-Throughput Screening Method for Pullulanase Mutant Library[J]. Journal of Food Science and Biotechnology,2016,35(9):993-1000.

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  • 在线发布日期: 2016-11-01
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