Abstract：
L-ribulose, a kind of high-value rare sugar, could be utilized to manufacture L-form sugars and antiviral drugs, generally produced from L-arabinose as a substrate. However, the production of L-ribulose from L-arabinose is limited by the equilibrium ratio of the catalytic reaction, hence, it is necessary to explore a new biological enzymatic method to produce L-ribulose. Ribose-5-phosphate isomerase (Rpi) is an enzyme that can catalyze the reversible isomerization between L-ribose and L-ribulose, which is of great significance for the preparation of L-ribulose. In order to obtain highly active ribose-5-phosphate isomerase to manufacture L-ribulose, ribose-5-phosphate isomerase A (OsRpiA) from Ochrobactrum sp. CSL1 was engineered based on structural and sequence analyses. Through a rational design strategy, a triple-mutant strain A10T/T32S/G101N with 160% activity was acquired. The enzymatic properties of the mutant were systematically investigated, and the optimum conditions were characterized to achieve the maximum yield of L-ribulose. Kinetic analysis clarified that the A10T/T32S/G101N mutant had a stronger affinity for the substrate and increased catalytic efficiency. Furthermore, molecular dynamics simulations indicated that the binding of the substrate to A10T/T32S/G101N was more stable than that of wild type. The shorter distance between the catalytic residues of A10T/T32S/G101N and L-ribose illuminated the increased activity. Overall, the present study provided a solid basis for demonstrating the complex functions of crucial residues in RpiAs as well as in rare sugar preparation.
摘要：
L-核糖,一种高价值的稀有糖，可以用来制造L型糖和抗病毒药物，通常由L-阿拉伯糖作为底物产生。然而,从L-阿拉伯糖生产L-核酮糖受到催化反应平衡比的限制，因此，有必要探索一种新的生物酶法生产L-核酮糖。核糖-5-磷酸异构酶（Rpi）是一种可以催化L-核糖和L-核酮糖之间可逆异构化的酶，这对L-核酮糖的制备具有重要意义。为了获得高活性的核糖-5-磷酸异构酶来制造L-核酮糖,来自苍白杆菌属的核糖-5-磷酸异构酶A（OsRpiA）。基于结构和序列分析对CSL1进行工程改造。通过合理的设计策略，获得具有160%活性的三突变菌株A10T/T32S/G101N。系统研究了突变体的酶学性质,并对最佳条件进行了表征，以实现L-核酮糖的最大产量。动力学分析阐明了A10T/T32S/G101N突变体对底物具有更强的亲和力并提高了催化效率。此外，分子动力学模拟表明，底物与A10T/T32S/G101N的结合比野生型更稳定。A10T/T32S/G101N和L-核糖的催化残基之间的较短距离表明活性增加。总的来说,本研究为证明RpiAs以及稀有糖制剂中关键残基的复杂功能提供了坚实的基础。
