The State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China; School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China; Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, Jiangnan University, Wuxi 214122, China.
The State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China; School of Bioengineering, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China.
Int J Biol Macromol. 2024 Jan;255:128010. doi: 10.1016/j.ijbiomac.2023.128010. Epub 2023 Nov 16.
In practical applications, the gelatinisation temperature of starch is high. Most current glycogen branching enzymes (GBEs, EC 2.4.1.18) exhibit optimum activity at moderate or low temperatures and quickly lose their activity at higher temperatures, limiting the application of GBEs in starch modification. Therefore, we used the PROSS strategy combined with PDBePISA analysis of the dimer interface to further improve the heat resistance of hyperthermophilic bacteria Pyrococcus horikoshii OT3 GBE. The results showed that the melting temperature of mutant T508K increased by 3.1 °C compared to wild-type (WT), and the optimum reaction temperature increased by 10 °C for all mutants except V140I. WT almost completely lost its activity after incubation at 95 °C for 60 h, while all of the combined mutants maintained >40 % of their residual activity. Further, the content of the α-1,6 glycosidic bond of corn starch modified by H415W and V140I/H415W was approximately 2.68-fold and 1.92-fold higher than that of unmodified corn starch and corn starch modified by WT, respectively. Additionally, the glucan chains of DP < 13 were significantly increased in mutant modified corn starch. This method has potential for improving the thermal stability of GBE, which can be applied in starch branching in the food industry.
在实际应用中，淀粉的胶凝温度较高。目前大多数糖原分支酶（GBE，EC 2.4.1.18）在中温和低温下表现出最佳活性，在较高温度下迅速失活，限制了 GBE 在淀粉改性中的应用。因此，我们使用 PROSS 策略结合 PDBePISA 分析二聚体界面，进一步提高了嗜热细菌 Pyrococcus horikoshii OT3 GBE 的耐热性。结果表明，与野生型（WT）相比，突变体 T508K 的熔点提高了 3.1°C，除 V140I 外，所有突变体的最适反应温度都提高了 10°C。WT 在 95°C孵育 60 h 后几乎完全失去活性，而所有组合突变体仍保持>40%的剩余活性。此外，H415W 和 V140I/H415W 修饰的玉米淀粉中α-1,6 糖苷键的含量分别比未修饰的玉米淀粉和 WT 修饰的玉米淀粉高约 2.68 倍和 1.92 倍。此外，突变体修饰的玉米淀粉中 DP < 13 的葡聚糖链显著增加。该方法有望提高 GBE 的热稳定性，可应用于食品工业中的淀粉分支。
