Xu Ting, Zhu Tianhui, Li Shujiang
College of Forestry, Sichuan Agricultural University, Ya'an, 625014, China.
Key Laboratory of Forest Protection of Sichuan Province, Ya'an, 625014, China.
World J Microbiol Biotechnol. 2016 Feb;32(2):26. doi: 10.1007/s11274-015-1985-0. Epub 2016 Jan 8.
Bacillus velezensis is a known antifungal bacteria. To understand the role of β-1, 3-1, 4-glucanase played on B. velezensis about the mechanism which exerts effect on fungi, we isolated and cloned the β-1, 3-1, 4-glucanase gene (Bglu1) from B. velezensis ZJ20. The Bglu1 open reading frame was 732 bp that encoded a protein with 243 amino acids and a calculated molecular weight of 27.3 kDa. The same gene without the signal peptide, termed Bglu2, was also cloned and expressed in E. coli BL21. Among the two variants, only Bglu2 protein was expressed. Purified Bglu2 could be eluted with imidazole solution at concentrations ranging from 100 to 500 mM although the highest expression was observed at 150 and 200 mM and the purest was at 500 mM. In addition, activity of the crude enzyme was 1527 U ml(-1) and the highest activity of the purified enzyme was 1706 U ml(-1). The purified β-1, 3-1, 4-glucanase had activity on a wide range of pH and temperatures and displayed optimal activity at pH 5.0 and 35 °C. More importantly, the mycelial morphology of three pathogenic fungi was destroyed by the purified β-1, 3-1, 4-glucanase. In conclusion, β-1, 3-1, 4-glucanase from B. velezensis ZJ20 can be highly expressed in E. coli BL21 and the recombinant protein is pathogenic to fungi.
贝莱斯芽孢杆菌是一种已知的抗真菌细菌。为了了解β-1,3-1,4-葡聚糖酶在贝莱斯芽孢杆菌对真菌发挥作用的机制中所起的作用，我们从贝莱斯芽孢杆菌ZJ20中分离并克隆了β-1,3-1,4-葡聚糖酶基因（Bglu1）。Bglu1的开放阅读框为732 bp，编码一个含有243个氨基酸、计算分子量为27.3 kDa的蛋白质。同样不含信号肽的该基因，命名为Bglu2，也被克隆并在大肠杆菌BL21中表达。在这两个变体中，只有Bglu2蛋白得到了表达。纯化的Bglu2可以用浓度为100至500 mM的咪唑溶液洗脱，尽管在150和200 mM时观察到最高表达，而在500 mM时最纯。此外，粗酶的活性为1527 U ml(-1)，纯化酶的最高活性为1706 U ml(-1)。纯化的β-1,3-1,4-葡聚糖酶在广泛的pH和温度范围内具有活性，在pH 5.0和35°C时表现出最佳活性。更重要的是，纯化的β-1,3-1,4-葡聚糖酶破坏了三种致病真菌的菌丝形态。总之，来自贝莱斯芽孢杆菌ZJ20的β-1,3-1,4-葡聚糖酶可以在大肠杆菌BL21中高效表达，并且重组蛋白对真菌具有致病性。
