Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, Israel.
Technion Center for Structural Biology, Lorry I. Lokey Center for Life Sciences and Engineering, Technion-Israel Institute of Technology, Haifa, Israel.
Appl Environ Microbiol. 2018 Nov 15;84(23). doi: 10.1128/AEM.02143-18. Print 2018 Dec 1.
An enhanced stability of enzymes in organic solvents is desirable under industrial conditions. The potential of lipases as biocatalysts is mainly limited by their denaturation in polar alcohols. In this study, we focused on selected solvent tunnels in lipase from T6 to improve its stability in methanol during biodiesel synthesis. Using rational mutagenesis, bulky aromatic residues were incorporated to occupy solvent channels and induce aromatic interactions leading to a better inner core packing. The chemical and structural characteristics of each solvent tunnel were systematically analyzed. Selected residues were replaced with Phe, Tyr, or Trp. Overall, 16 mutants were generated and screened in 60% methanol, from which 3 variants showed an enhanced stability up to 81-fold compared with that of the wild type. All stabilizing mutations were found in the longest tunnel detected in the "closed-lid" X-ray structure. The combination of Phe substitutions in an A187F/L360F double mutant resulted in an increase in unfolding temperature ( ) of 7°C in methanol and a 3-fold increase in biodiesel synthesis yield from waste chicken oil. A kinetic analysis with -nitrophenyl laurate revealed that all mutants displayed lower hydrolysis rates (), though their stability properties mostly determined the transesterification capability. Seven crystal structures of different variants were solved, disclosing new π-π or CH/π intramolecular interactions and emphasizing the significance of aromatic interactions for improved solvent stability. This rational approach could be implemented for the stabilization of other enzymes in organic solvents. Enzymatic synthesis in organic solvents holds increasing industrial opportunities in many fields; however, one major obstacle is the limited stability of biocatalysts in such a denaturing environment. Aromatic interactions play a major role in protein folding and stability, and we were inspired by this to redesign enzyme voids. The rational protein engineering of solvent tunnels of lipase from is presented here, offering a promising approach to introduce new aromatic interactions within the enzyme core. We discovered that longer tunnels leading from the surface to the enzyme active site were more beneficial targets for mutagenesis for improving lipase stability in methanol during biodiesel biosynthesis. A structural analysis of the variants confirmed the generation of new interactions involving aromatic residues. This work provides insights into stability-driven enzyme design by targeting the solvent channel void.
在工业条件下，希望酶在有机溶剂中的稳定性得到增强。脂肪酶作为生物催化剂的潜力主要受到其在极性醇中变性的限制。在这项研究中，我们专注于脂肪酶 T6 中的选定溶剂隧道，以提高其在生物柴油合成过程中甲醇中的稳定性。通过合理的诱变，引入大的芳基残基占据溶剂通道并诱导芳族相互作用，从而实现更好的内部核心堆积。系统地分析了每个溶剂隧道的化学和结构特征。选择的残基被苯丙氨酸、酪氨酸或色氨酸取代。总的来说，生成了 16 个突变体，并在 60%甲醇中进行了筛选，其中 3 个变体的稳定性比野生型提高了 81 倍。所有稳定化突变都发生在“闭盖”X 射线结构中检测到的最长隧道中。在 A187F/L360F 双突变体中引入苯丙氨酸取代的组合导致在甲醇中解折叠温度 ( ) 升高 7°C，并且从废鸡油中生物柴油合成产率提高 3 倍。用 -硝基苯棕榈酸酯进行的动力学分析表明，所有突变体的水解速率 () 都较低，尽管它们的稳定性特性主要决定了转酯化能力。解决了不同变体的七个晶体结构，揭示了新的 π-π 或 CH/π 分子内相互作用，并强调了芳香相互作用对提高溶剂稳定性的重要性。这种合理的方法可用于在有机溶剂中稳定其他酶。在许多领域，有机溶剂中的酶合成具有越来越多的工业机会；然而，一个主要障碍是生物催化剂在这种变性环境中的稳定性有限。芳香相互作用在蛋白质折叠和稳定性中起着重要作用，我们受到这一启发来重新设计酶的空隙。这里介绍了来自 T6 的脂肪酶溶剂隧道的合理蛋白质工程，为在生物柴油生物合成过程中引入新的酶核心内芳香相互作用提供了一种有前途的方法。我们发现，从表面到酶活性位点的较长隧道是突变的更有利目标，可提高甲醇中脂肪酶在生物柴油合成过程中的稳定性。变体的结构分析证实了涉及芳香族残基的新相互作用的产生。这项工作通过针对溶剂通道空隙提供了稳定性驱动的酶设计的见解。
