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首页> 美国卫生研究院文献>Computational and Structural Biotechnology Journal >Computational design of noncanonical amino acid-based thioether staples at N/C-terminal domains of multi-modular pullulanase for thermostabilization in enzyme catalysis
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Computational design of noncanonical amino acid-based thioether staples at N/C-terminal domains of multi-modular pullulanase for thermostabilization in enzyme catalysis

机译：在酶催化中的多模淀粉酶N / C末端域基于非甘露氨基酸基硫醚钉的计算设计

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Enzyme thermostabilization is considered a critical and often obligatory step in biosynthesis, because thermostability is a significant property of enzymes that can be used to evaluate their feasibility for industrial applications. However, conventional strategies for thermostabilizing enzymes generally introduce non-covalent interactions and/or natural covalent bonds caused by natural amino acid substitutions, and the trade-off between the activity and stability of enzymes remains a challenge. Here, we developed a computationally guided strategy for constructing thioether staples by incorporating noncanonical amino acid (ncAA) into the more flexible N/C-terminal domains of the multi-modular pullulanase from Bacillus thermoleovorans (BtPul) to enhance its thermostability. First, potential thioether staples located in the N/C-terminal domains of BtPul were predicted using RosettaMatch. Next, eight variants involving stable thioether staples were precisely predicted using FoldX and Rosetta ddg_monomer. Six positive variants were obtained, of which T73(O2beY)-171C had a 157% longer half-life at 70 °C and an increase of 7.0 °C in Tm, when compared with the wild-type (WT). T73(O2beY)-171C/T126F/A72R exhibited an even more improved thermostability, with a 211% increase in half-life at 70 °C and a 44% enhancement in enzyme activity compared with the WT, which was attributed to further optimization of the local interaction network. This work introduces and validates an efficient strategy for enhancing the thermostability and activity of multi-modular enzymes.
机译：酶热稳定化被认为是生物合成中的关键和通常是义务步骤，因为热稳定性是酶的显着性，可用于评估其用于工业应用的可行性。然而，热稳定酶的常规策略通常引入由天然氨基酸取代引起的非共价相互作用和/或天然共价键，并且酶活性与稳定性之间的权衡仍然是一个挑战。在这里，我们通过将非甘露糖苷氨基酸（NCAA）掺入来自Bacillus Thermoleovorans（BTPUL）的多模块化蛋白酶（BTPUL）的更柔性N / C末端结构粒子来构建硫醚钉，以提高其热稳定性的计算引导策略。首先，使用玫瑰甜雀预测位于BTPUL的N / C末端结构域中的潜在硫醚钉。接下来，使用FOLDX和ROSETTTA DDG_MOROMORER精确地预测涉及稳定硫醚钉的八个变体。获得了六个阳性变体，其中T73（O2BEY）-171C在与野生型（WT）相比时，在70℃下在70℃下的半衰期增加了157％的半衰期和7.0℃。 T73（O2BEY）-171C / T126F / A72R表现出更高的热稳定性，在70°C下半衰期增加211％，与WT相比，酶活性增强44％，归因于进一步优化本地交互网络。该工作引入并验证了提高多模块化酶的热稳定性和活性的有效策略。

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