School of Biological Engineering, Henan University of Technology, Zhengzhou 450001, China.
Food Laboratory of Zhongyuan, Luohe 462333, China.
Biomolecules. 2024 Sep 20;14(9):1188. doi: 10.3390/biom14091188.
This study identified a salt-tolerant GH11 xylanase, Xyn, which was isolated from a soil bacterium sp. SC1 and can resist as high as 4 M NaCl. After rational design and high-throughput screening of site-directed mutant libraries, a double mutant W6F/Q7H with a 244% increase in catalytic activity and a 10 °C increment in optimal temperature was obtained. Both Xyn and W6F/Q7H xylanases were stimulated by high concentrations of salts. In particular, the activity of W6F/Q7H was more than eight times that of Xyn in the presence of 2 M NaCl at 65 °C. Kinetic parameters indicated they have the highest affinity for beechwood xylan ( = 0.30 mg mL for Xyn and 0.18 mg mL for W6F/Q7H), and W6F/Q7H has very high catalytic efficiency (/ = 15483.33 mL mg s). Molecular dynamic simulation suggested that W6F/Q7H has a more compact overall structure, improved rigidity of the active pocket edge, and a flexible upper-end alpha helix. Hydrolysis of different xylans by W6F/Q7H released more xylooligosaccharides and yielded higher proportions of xylobiose and xylotriose than Xyn did. The conversion efficiencies of Xyn and W6F/Q7H on all tested xylans exceeded 20%, suggesting potential applications in the agricultural and food industries.
本研究从土壤细菌 sp. SC1 中分离到一种耐盐 GH11 木聚糖酶 Xyn，它能耐受高达 4 M 的 NaCl。通过对定点突变文库进行合理设计和高通量筛选，得到了一个双突变体 W6F/Q7H，其催化活性提高了 244%，最适温度提高了 10°C。Xyn 和 W6F/Q7H 木聚糖酶都受到高浓度盐的刺激。特别是在 65°C 下，W6F/Q7H 在 2 M NaCl 存在时的活性超过 Xyn 的 8 倍。动力学参数表明，它们对桦木木聚糖具有最高的亲和力（Xyn 为 0.30 mg mL，W6F/Q7H 为 0.18 mg mL），且 W6F/Q7H 具有非常高的催化效率（/ = 15483.33 mL mg s）。分子动力学模拟表明，W6F/Q7H 具有更紧凑的整体结构、活性口袋边缘的刚性提高以及灵活的上端α螺旋。W6F/Q7H 水解不同的木聚糖释放出更多的低聚木糖，生成的木二糖和木三糖比例高于 Xyn。Xyn 和 W6F/Q7H 对所有测试的木聚糖的转化率均超过 20%，这表明它们在农业和食品工业中有潜在的应用。
