High-Efficiency Secretion of beta-Mannanase in Bacillus subtilis through Protein Synthesis and Secretion Optimization
High-Efficiency Secretion of beta-Mannanase in Bacillus subtilis through Protein Synthesis and Secretion Optimization
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通过蛋白质合成和分泌优化在枯草芽孢杆菌中高效分泌β-甘露聚糖酶
DOI：
10.1021/acsofc.6b05528
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发表时间：
2017
影响因子：
6.1
通讯作者：
Zhang Dawei
中科院分区：
文献类型：
--
作者：
Song Yafeng;Fu Gang;Dong Huina;Li Jianjun;Du Yuguang;Zhang Dawei
甘露内切-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-甘露糖苷键的无规水解。密码子优化的地衣芽孢杆菌DSM 13 β-甘露聚糖酶基因在枯草芽孢杆菌中得到表达。从B.在目的基因的上游插入枯草杆菌，以SPlipA为信号肽的β-甘露聚糖酶活性最高。将另一拷贝的groESLoperon插入到宿主菌基因组中，β-甘露聚糖酶活力提高了1.25倍。最后，分别使用五种不同的启动子来增强目的蛋白的合成。结果表明，麦芽糖诱导型启动子Pmglv能显著提高β-甘露聚糖酶的产量。摇瓶发酵72 h后，以刺槐豆胶为底物时，上清液中β-甘露聚糖酶的酶活达到2207 U/mL。本研究为β-甘露聚糖酶的工业化生产提供了一株有前途的菌株。
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 fromBacillus licheniformis DSM13 was expressed inBacillus subtilis. When four Sec-dependent and two Tat-dependent signal peptide sequences cloned fromB. subtiliswere placed upstream of the target gene, the highest activity of β-mannanase was observed using SPlipAas a signal peptide. Then a 1.25-fold activity of β-mannanase was obtained when another copy ofgroESLoperon 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 Pmglv, 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.