Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University, No 11 Fucheng Street, Haidian District, Beijing, 100084, China.
Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, No 11 Fucheng Street, Haidian District, Beijing, 100084, China.
BMC Biotechnol. 2019 Jul 25;19(1):51. doi: 10.1186/s12896-019-0541-7.
A mesophilic xylanase PjxA from Penicillium janthinellum MA21601 has high specific activity under acidic condition and holds great potential for applications in the animal feed industry. To enhance the thermostability of xylanase PjxA, two mutation strategies in the N-terminal region were examined and then integrated into the xylanase to further improvement. The recombinant xylanase PTxA-DB (The meaning of DB is disulfide-bridge.) was constructed by replacement of five residues in the mutated region in TfxA (T10Y, N11H, N12D, Y15F, N30 L), combined with an additional disulfide bridge in the N-terminal region.
The T value of mutant PTxA-DB was improved from 21.3 °C to 76.6 °C, and its half-life was found to be 53.6 min at 60 °C, 107-fold higher than the wild type strain. The location of the disulfide bridge (T2C-T29C) was between the irregular loop and the β-strand A2, accounting for most of the improvement in thermostability of PjxA. Further analysis indicated T2C, T29C, N30 L and Y15F lead to increase N-terminal hydrophobicity. Moreover, the specific activity and substrate affinity of PTxA-DB were also enhanced under the acidic pH values.
These results indicated PTxA-DB could be a prospective additive to industrial animal feeds.
来自青霉属（Penicillium）MA21601 的嗜温木聚糖酶 PjxA 在酸性条件下具有高比活性，在动物饲料工业中有很大的应用潜力。为了提高木聚糖酶 PjxA 的热稳定性，考察了其 N 端区域的两种突变策略，并将其整合到木聚糖酶中以进一步改进。通过在 TfxA 中的突变区域替换五个残基（T10Y、N11H、N12D、Y15F、N30L），并在 N 端区域增加一个二硫键，构建了重组木聚糖酶 PTxA-DB（DB 的含义是二硫键）。
突变体 PTxA-DB 的 T 值从 21.3°C提高到 76.6°C，在 60°C 时半衰期为 53.6 分钟，比野生型高 107 倍。二硫键的位置（T2C-T29C）位于不规则环和β-链 A2 之间，这是 PjxA 热稳定性提高的主要原因。进一步分析表明，T2C、T29C、N30L 和 Y15F 导致 N 端疏水性增加。此外，PTxA-DB 在酸性 pH 值下的比活性和底物亲和力也得到了增强。
这些结果表明，PTxA-DB 可以成为工业动物饲料的一种有前景的添加剂。
