Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
J Ind Microbiol Biotechnol. 2013 Jul;40(7):773-9. doi: 10.1007/s10295-013-1267-5. Epub 2013 Apr 26.
The neutral endo-β-glucanase gene cel5A from Humicola insolens was cloned and connected with the cellobiohydrolase 1 promoter from Trichoderma reesei to construct a recombinant plasmid pCB-hEG with the hygromycin B resistance marker. The plasmid was introduced into conidia of T. reesei using the Agrobacterium tumefaciens mediated transformation method. Eight transformants were obtained on screening plates with sodium carboxymethyl cellulose as the sole carbon source. Stable integration of the cel5A gene into the chromosomal DNA of T. reesei was confirmed by PCR. An obvious protein band (approximately 52 kDa) was detected by SDS-PAGE from fermentation broth, which showed that the cel5A gene in recombinant T. reesei successfully fulfilled efficient expression and extracellular secretion. After 96 h shaking-flask fermentation, the endo-β-glucanase activity at pH 6.5 from recombinant T. reesei reached 3,068 U/ml, which was 11 times higher than that of the host strain. In a 2 m³ fermenter, the endo-β-glucanase activity could be further increased to 8,012 U/ml after 96 h fermentation. The results showed a good prospect for application of neutral endo-β-glucanase in the textile industry.
从康宁木霉中克隆了内切-β-葡聚糖酶基因 cel5A，并将其与里氏木霉的纤维二糖水解酶 1 启动子连接，构建了带有潮霉素 B 抗性标记的重组质粒 pCB-hEG。通过根癌农杆菌介导的转化方法将质粒导入里氏木霉的分生孢子中。在以羧甲基纤维素钠为唯一碳源的筛选平板上获得了 8 个转化子。通过 PCR 证实 cel5A 基因已稳定整合到里氏木霉的染色体 DNA 中。通过 SDS-PAGE 从发酵液中检测到明显的蛋白条带（约 52 kDa），表明重组里氏木霉中的 cel5A 基因成功实现了高效表达和细胞外分泌。在 96 h 摇瓶发酵后，重组里氏木霉在 pH 6.5 下的内切-β-葡聚糖酶活性达到 3068 U/ml，比出发菌株高 11 倍。在 2 m³发酵罐中，96 h 发酵后，内切-β-葡聚糖酶活性可进一步提高到 8012 U/ml。结果表明，中性内切-β-葡聚糖酶在纺织工业中有很好的应用前景。
