College of Food and Biological Engineering, Jimei University, Xiamen 361021, China.
College of Food and Biological Engineering, Jimei University, Xiamen 361021, China; Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China; Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, China.
Enzyme Microb Technol. 2024 Oct;180:110484. doi: 10.1016/j.enzmictec.2024.110484. Epub 2024 Jul 19.
There has been ongoing interest in improving the efficiency of glycoside hydrolase for synthesizing glycoside compounds through protein engineering, given the potential applications of glycoside compounds. In this study, a strategy of modifying the substrate access tunnel was proposed to enhance the efficiency of reverse hydrolysis catalyzed by Aspergillus niger α-L-rhamnosidase. Analysis of the tunnel dynamics identified Tyr299 as a key modifiable residue in the substrate access tunnel. The location of Tyr299 was near the enzyme surface and at the outermost end of the substrate access tunnel, suggested its role in substrate recognition and throughput. Based on the properties of side chains, six mutants were designed and expressed by Pichia pastoris. Compared to WT, the reverse hydrolysis efficiencies of mutants Y299P and Y299W were increased by 21.3 % and 11.1 %, respectively. The calculation results of binding free energy showed that the binding free energy was inversely proportional to the reverse hydrolysis efficiency. Further, when binding free energy levels were comparable, the mutants with shorter side chains displayed a higher reverse hydrolysis efficiency. These results proved that substrate access tunnel modification was an effective method to improve the reverse hydrolysis efficacy of α-L-rhamnosidase and also provided new insights for modifying other glycoside hydrolases.
鉴于糖苷化合物的潜在应用，通过蛋白质工程提高糖苷水解酶合成糖苷化合物的效率一直是人们关注的焦点。本研究提出了一种修饰底物进入隧道的策略，以提高黑曲霉 α-L-鼠李糖苷酶反向水解的效率。通过对隧道动力学的分析，鉴定出 Tyr299 是底物进入隧道中一个关键的可修饰残基。Tyr299 的位置靠近酶表面，处于底物进入隧道的最外端，提示其在底物识别和通量中起作用。根据侧链的性质，设计并通过毕赤酵母表达了六个突变体。与 WT 相比，突变体 Y299P 和 Y299W 的反向水解效率分别提高了 21.3%和 11.1%。结合自由能的计算结果表明，结合自由能与反向水解效率成反比。此外，当结合自由能水平相当时，具有较短侧链的突变体表现出更高的反向水解效率。这些结果证明了底物进入隧道修饰是提高 α-L-鼠李糖苷酶反向水解效率的有效方法，也为修饰其他糖苷水解酶提供了新的思路。
