黄瓜膨胀素的重组表达及活性分析

目的:提高纤维素的酶水解效率和开发高效的纤维素酶水解过程。方法:采用RT-PCR方法从黄瓜胚轴细胞中分离了膨胀素S1的cDNA,并使之与毕赤酵母表达质粒pPICZ(A连接,形成重组质粒pPICZ(A-exs1。通过电转化方法,用质粒pPICZ(A-exs1转化巴氏毕赤酵母GS115,得到重组菌株P.pastoris-exs1。在该重组菌株中,膨胀素的基因通过同源重组整合在毕赤酵母的染色体上,并处于毕赤酵母甲醇氧化酶启动子的下游。重组菌株P.pastoris-exs1在甲醇诱导下可合成并分泌膨胀素。结果:培养上清液没有纤维素酶活性,但具有破坏滤纸纤维素结晶结构的能力。培养上清液与里氏木霉纤维素酶等量混合后,可使纤维素酶的滤纸酶活力提高50%。结论:采用巴氏毕赤酵母GS115重组成功表达了黄瓜膨胀素,其表达产物可以促进纤维素酶对滤纸的水解。

Expression of the Cucumis Sativus Expansin S1 and Analysis of Its Activity

Objectives:Improve the efficiency of enzymatic hydrolysis of cellulose and exploit efficient process for cellulose hydrolysis by applying the botanical protein expansin that was capable of destroying the hydrogen bonds between cellulose fibers.Methods: The cDNA of Cucumis Sativus expansin S1 was isolated from cucumber hypocotyl through RT-PCR,and was ligated with the Pichia expression vector pPICZ(A,resulting in the recombinant plasmid pPICZ(A-exs1.The recombinant plasmid was transformed into P.pastoris GS115 through electroporation.The expansin S1 gene was in frame integrated into the Pichia genome through homologous recombination and the resultant recombinant strain was named P.pastoris-exs1.In the recombinant strain,the cDNA of expansin S1 was located downstream of the strong alcohol oxidase promoter.With methanol induction,strain P.pastoris-exs1 expressed and secreted protein CuExS1 into the cultivation supernatant. Results: The expressed product was able to destruct the crystal structure of cellulose in filter paper,but did not exhibit cellulase activity.When mixed with equal volume of Trichoderma reesei cellulase,the culture supernatant enhanced the filter paper activity of cellulase by 50%.Conclusion: Cucumis Sativus expansin S1 was successfully expressed in P.pastoris-exs1,and the heterologous expressed expansin S1 was able to enhance cellulolytic activity of T.reesei cellulase.