Department of Chemical Engineering and Bioengineering, Zhejiang University, Hangzhou 310027, China.
Bioresour Technol. 2011 Mar;102(6):4568-72. doi: 10.1016/j.biortech.2010.12.099. Epub 2011 Jan 1.
The cellobiase gene from Aspergillus niger was cloned and connected with the strong promoter Pcbh1 from Trichoderma reesei to construct a recombinant plasmid pHB9 with the hygromycin B resistance marker. The plasmid was transformed into conidia of T. reesei using the modified PEG-CaCl(2) method. Main factors effecting the transformation were discussed and about 99-113 transformants/μg DNA could be obtained under optimal conditions. It was found that the molecular mass of the recombinant cellobiase was about 120 kDa by SDS-PAGE analysis. The activity of cellobiase could reach 5.3 IU/ml after 48 h fermentation, which was as high as 106 times compared with that of the host strain. Meanwhile, the filter paper activity of recombinant T. reesei was 1.44-fold of the host strain. Saccharification of corncob residue with the crude enzyme showed that the hydrolysis yield (84.2%) of recombinant T. reesei was 21% higher than that (69.5%) of the host strain.
从黑曲霉中克隆了纤维二糖酶基因，并与里氏木霉的强启动子 Pcbh1 连接，构建了带有潮霉素 B 抗性标记的重组质粒 pHB9。使用改良的 PEG-CaCl2 方法将质粒转化到里氏木霉的分生孢子中。讨论了影响转化的主要因素，在最佳条件下可获得约 99-113 个转化体/μg DNA。通过 SDS-PAGE 分析发现，重组纤维二糖酶的分子量约为 120 kDa。发酵 48 h 后，纤维二糖酶的活力可达 5.3 IU/ml，比出发菌株高 106 倍。同时，重组里氏木霉的滤纸酶活力是出发菌株的 1.44 倍。用粗酶对玉米芯残渣进行糖化，结果表明重组里氏木霉的水解产率（84.2%）比出发菌株（69.5%）高 21%。
