里氏木霉纤维二糖水解酶II基因的高表达

摘要

纤维二糖水解酶II (CBH II)是纤维素酶的重要组分之一，对纤维素酶的水解性能有着重大影响，而里氏木霉(Trichoderma reesei)纤维素酶制剂中纤维二糖水解酶II明显不足，为了优化其酶系结构，采用了基因重组技术构建CBH II 高产菌株：将里氏木霉 CBH II 基因置于里氏木霉强启动子 Pcbh1(及其信号肽)和终止子 Tcbh1之间，并进一步以pCAMBIA1300为载体骨架，构建成含潮霉素B抗性标记的重组质粒pCAMBIA1300-hph-PsCT。以里氏木霉ZU-02为宿主，采用根瘤农杆菌介导转化技术将重组质粒转入宿主分生孢子。以潮霉素B为抗性标记初筛到324个阳性转化子，进一步通过复筛，在以微晶纤维素为唯一碳源的筛选培养基上获得8个生长较快的优良转化子。在摇瓶条件下，分别对8个转化子进行产酶试验，培养48 h时，纤维二糖水解酶活力最高可达18.24 U⋅mL-1，是出发菌株的2.51倍。本结果对于里氏木霉纤维素酶的定向进化、提高其对纤维素的协同糖化效率具有重要意义。%Cellobiohydolase II (CBH II) is an important cellulase component and has significant effects on the performance of cellulases in enzymatic hydrolysis. However, cellulases prepared from T. reesei are deficient in cellobiohydolase II. In this study, the cellobiohydrolase II gene was inserted between T. reesei strong promoter Pcbh1 (including secreting signal peptide sequence) and terminator Tcbh1, which was further linked to pCAMBIA1300 vector to construct a recombinant plasmid pCAMBIA1300-hph-PsCT with hygromycin B as the resistance marker. The recombinant plasmid was transformed into T. reesei ZU-02 via Agrobacterium tumefaciens mediated transformation. Hygromycin B was used as the resistance marker in the first screening step, which results in 324 positive transformants, and 8 fast-growing transformants were selected in the second screening step where microcrystalline cellulose was utilized as the sole carbon source. These transformants were tested in cellulases production under shaking flask fermentation conditions and after 48 h fermentation, the highest cellobiohydrolase activity is 18.24 U⋅mL-1 that is 2.51 times higher than original strain. These results should be useful in the study of directed evolution and saccharification efficiency improvement of T. reesei cellulases.