核糖醇脱氢酶热稳定性改造及其在全细胞催化制备阿洛醇中的应用

Thermostability Engineering of Ribitol Dehydrogenase and Its Application in Whole-Cell Catalytic Production of Allitol

摘要:

阿洛醇是一种具有降血压、抗肥胖等生理活性的六碳稀有糖醇,在功能食品与医药领域具有应用价值。目前,全细胞催化制备阿洛醇仍受到反应温度偏低的限制。催化阿洛酮糖还原为阿洛醇的核糖醇脱氢酶(PaRDH)通常适宜在中低温条件下工作,这不仅降低了转化效率,还增加了体系染菌等工艺风险。为提升PaRDH的应用性能,采用PROSS网页预测、自由能分析及保守性位点分析等多种策略,对PaRDH的热稳定性进行了系统改造。第一轮突变得到11个性能提升的突变体,再经过一轮叠加突变得到10个热稳定性大幅度提升的突变体。其中突变体PaRDHVM-N7G-I93V的初始酶活是模板PaRDHVM的130.68%,半衰期由原来的7.66h提高到16.26h。将PaRDHVM-N7G-I93V带入三酶催化体系,所得工程菌命名为Ec/DRF-Therm-VMGV。以10 OD₆₀₀的细胞添加量在45℃条件下反应24h,阿洛醇转化率达到79.43%,比携带PaRDHVM的对照菌株的转化率高了4.10%。优化反应体系中甲酸钠的浓度,将其从0.75mol/L提高至1.00mol/L。以工程菌Ec/DRF-Therm-VMGV为催化剂,当反应细胞浓度为15 OD₆₀₀时,反应12h和24h的阿洛醇转化率分别达到83.10%和90.45%,为目前报道的最高水平。本研究旨在为提高阿洛醇的生产效率和推动其工业化应用提供理论参考。

Abstract:

Allitol is a six-carbon rare sugar alcohol with physiological activities such as antihypertensive and anti-obesity effects, holding application value in the functional food and pharmaceutical industries. Currently, whole-cell catalysis for allitol production is still limited by relatively low reaction temperatures. Ribitol dehydrogenase (PaRDH), which catalyzes the reduction of psicose to allitol, typically functions optimally under low to moderate temperature conditions, which not only reduces conversion efficiency but also increases process risks such as microbial contamination. To enhance the application performance of PaRDH, multiple strategies including PROSS web-based prediction, free energy analysis, and conserved site analysis were employed to systematically modify the thermostability of PaRDH. In the first round of mutagenesis, 11 mutants with improved performance were obtained. After another round of combinatorial mutagenesis, 10 mutants with significantly enhanced thermostability were generated. Among them, the initial enzyme activity of mutant PaRDHVM-N7G-I93V increased by 30.68% compared to the template PaRDHVM, and its half-life was extended from 7.66h to 16.26h. This mutant was then introduced into a three-enzyme cascade system, and the resulting engineered strain was designated as Ec/DRF-Therm-VMGV. Using a cell dosage of 10 OD₆₀₀ at 45℃ for 24h, the allitol conversion rate reached 79.43%, which was 4.10% higher than that of the control strain harboring PaRDHVM. Subsequently, the concentration of sodium formate in the reaction system was optimized and increased from 0.75mol/L to 1.00mol/L. Finally, using the engineered strain Ec/DRF-Therm-VMGV as the catalyst at a cell concentration of 15 OD₆₀₀, the allitol conversion rates reached 83.10% and 90.45% after 12h and 24h of reaction, respectively, representing the highest levels reported to date. This study aimed to provide a theoretical reference for improving the production efficiency of allitol and promoting its industrial application.