Song Yafeng, Fu Gang, Dong Huina, Li Jianjun, Du Yuguang, Zhang Dawei
Tianjin Institute of Industrial Biotechnology and ‡Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences , Tianjin 300308, P. R. China.
Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen , 9713 AV, Groningen, The Netherlands.
J Agric Food Chem. 2017 Mar 29;65(12):2540-2548. doi: 10.1021/acs.jafc.6b05528. Epub 2017 Mar 15.
The manno endo-1,4-mannosidase (β-mannanase, EC. 3.2.1.78) catalyzes the random hydrolysis of internal (1 → 4)-β-mannosidic linkages in the mannan polymers. A codon optimized β-mannanase gene from Bacillus licheniformis DSM13 was expressed in Bacillus subtilis. When four Sec-dependent and two Tat-dependent signal peptide sequences cloned from B. subtilis were placed upstream of the target gene, the highest activity of β-mannanase was observed using SP as a signal peptide. Then a 1.25-fold activity of β-mannanase was obtained when another copy of groESL operon was inserted into the genome of host strain. Finally, five different promoters were separately used to enhance the synthesis of the target protein. The results showed that promoter P, a modified maltose-inducible promoter, significantly elevated the production of β-mannanase. After 72 h of flask fermentation, the enzyme activity of β-mannanase in the supernatant when using locust bean gum as substrate reached 2207 U/mL. This work provided a promising β-mannanase production strain in industrial application.
甘露聚糖内切 -1,4 -甘露糖苷酶（β -甘露聚糖酶，EC. 3.2.1.78）催化甘露聚糖聚合物中内部（1→4）-β -甘露糖苷键的随机水解。来自地衣芽孢杆菌DSM13的密码子优化β -甘露聚糖酶基因在枯草芽孢杆菌中表达。当从枯草芽孢杆菌克隆的四个依赖Sec和两个依赖Tat的信号肽序列置于靶基因上游时，使用SP作为信号肽观察到β -甘露聚糖酶的最高活性。然后，当将groESL操纵子的另一个拷贝插入宿主菌株基因组时，获得了1.25倍活性的β -甘露聚糖酶。最后，分别使用五个不同的启动子来增强靶蛋白的合成。结果表明，启动子P（一种修饰的麦芽糖诱导型启动子）显著提高了β -甘露聚糖酶的产量。摇瓶发酵72小时后，以上刺槐豆胶为底物时，上清液中β -甘露聚糖酶的酶活性达到2207 U/mL。这项工作为工业应用提供了一种有前景的β -甘露聚糖酶生产菌株。
