D-阿洛酮糖是一种在自然界中存在极少的酮糖,因其具有与蔗糖相近的甜度但热量更低而备受关注。然而,D-阿洛酮糖的高效生物合成仍然面临挑战,是由于用于生产的酮糖3-差向异构酶的热稳定性较差。该研究通过理性设计,引入二硫键和属性嫁接策略,成功获得了热稳定性更高的最佳突变体M03。最佳突变体M03的Tm值从52.7 ℃提高到69.4 ℃;在无需外源添加金属离子的条件下,55 ℃时半衰期延长至254.3 min,是野生型的54.1倍;通过分子动力学模拟和结构分析揭示了性能提升的详细机制。该研究为工业化生产D-阿洛酮糖提供了一个有潜力的突变菌株。
D-allulose is a rare ketose found in nature, which has attracted significant attention for its similar sweetness to sucrose but lower caloric content.However, the efficient biosynthesis of D-allulose still poses a challenge, primarily attributed to the low thermostability of the ketose 3-epimerase utilized in production.In this study, the optimal mutant M03 with higher thermostability was developed by rational design, through the introduction of a disulfide bond and attribute grafting strategy.The Tm value of the best mutant M03 increased from 52.7 ℃ to 69.4 ℃.At 55 ℃, the half-life was extended to 254.3 min without metal ions, which was 54.1 times that of the wild-type.The detailed mechanism of performance improvement was analyzed by molecular dynamics simulation and structure analysis.This study provided a potential mutant strain for the industrial production of D-allulose.
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