Li Yuan, Tang Xiyu, Chen Liangqi, Xu Xinran, Li Jinyao
Institute of Materia Medica, Xinjiang University, Urumqi 830017, China.
Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830017, China.
Foods. 2022 Aug 10;11(16):2403. doi: 10.3390/foods11162403.
Nattokinase is a potential new thrombolytic drug because of its strong thrombolytic effect, high safety, and low cost. However, there is no research reporting on bile salt-tolerant nattokinase-producing probiotics. In this study, the bile salt-tolerant nattokinase-producing strain LY-06 was isolated from local Xinjiang douchi, and the fermentation yield of nattokinase of 1434.64 U/mL was obtained by both a single factor experiment and an orthogonal experiment. A gene responsible for fibrinolysis () was cloned from the genome of strain LY-06, and the soluble expression of this gene in (rAprY, fused with His-tag at C-terminus) was achieved; molecular docking elucidates the cause of insoluble expression of rAprY. The optimal pH and temperature for the fibrinolysis activity of nattokinase AprY fermented by LY-06 were determined to be pH 6.0 and 50 °C, respectively. However, the optimal pH of rAprY expressed in was 8, and its acid stability, thermal stability, and fibrinolytic activity were lower than those of AprY. Bioinformatics analysis found that the His-tag carried at the C-terminus of rAprY could affect its acidic stability by changing the isoelectric point and surface charge of the enzyme; in contrast to AprY, changes in the number of internal hydrogen bonds and the flexibility of the loop region in the structure of rAprY resulted in lower fibrinolytic activity and poorer thermal stability.
纳豆激酶因其强大的溶栓作用、高安全性和低成本而成为一种潜在的新型溶栓药物。然而，目前尚无关于耐胆盐产纳豆激酶益生菌的研究报道。在本研究中，从新疆当地豆豉中分离出耐胆盐产纳豆激酶菌株LY-06，并通过单因素实验和正交实验获得了1434.64 U/mL的纳豆激酶发酵产量。从菌株LY-06的基因组中克隆了一个负责纤维蛋白溶解的基因（），并在（rAprY，C末端融合His标签）中实现了该基因的可溶性表达；分子对接阐明了rAprY不溶性表达的原因。由LY-06发酵产生的纳豆激酶AprY的纤维蛋白溶解活性的最佳pH值和温度分别确定为pH 6.0和50°C。然而，在中表达的rAprY的最佳pH值为8，其酸稳定性、热稳定性和纤维蛋白溶解活性均低于AprY。生物信息学分析发现，rAprY的C末端携带的His标签可通过改变酶的等电点和表面电荷来影响其酸稳定性；与AprY相比，rAprY结构中内部氢键数量和环区柔韧性的变化导致其纤维蛋白溶解活性较低和热稳定性较差。
