Synergizing Enzyme and Encapsulation Engineering to Develop Superoptimal Biocatalysts

酶是促进化学反应的蛋白质。它们可以生产生物燃料、化学品和药品。它们可以被设计成在非自然条件下表现良好。设计工作通常会导致房产之间的权衡。例如，增加酶的稳定性通常会导致活性降低。该项目的主要目标是评估一种新的设计策略。这一策略可能会在不降低其他属性的情况下改善某些属性。该策略将计算方法与一种新颖的起始材料相结合。这项工作还将通过实践研究、研讨会和讲座来支持本科生研究人员和未被充分代表的群体的招募和参与。这项工作的总体目标是开发一种“超最佳”生物催化系统，其中酶可以在其特征的最佳温度、周转频率(TOF，kCAT)和转化率之外工作。要做到这一点，需要酶工程直接修改它们的催化性能和包封性，以改变稳定性以及底物和产物的传输速率和分配。该项目将使用含有4-亚甲基咪唑-5-酮(MIO)的氨基酸解氨酶(XAL)作为模型酶。我们将检验三种不同的假设。首先，在工程设计过程中，放宽稳定性标准可以获得新的酶活性。第二，含有MIO的XAL酶可以接受非芳香族底物。第三，工程化的包埋系统可以提高酶的稳定性、反应速度和转化率。定向进化工作流程将识别具有高活跃度的XAL变体。在定向进化过程中，稳定性要求将会放宽。鉴定在非同源氨基酸底物上具有活性的变异体将是另一个目标。XAL酶以前从未被设计成作用于非芳香族底物。酶工程工作将使用深度突变扫描(DMS)指导的实验工作流程以及计算建模。将设计和评估用于提高酶的稳定性、反应速度和产品产量的包裹体。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Enzymes are proteins that promote chemical reactions. They can produce biofuels, chemicals, and medicines. They can be designed to perform well under unnatural conditions. Design efforts usually result in trade-offs between properties. For example, increasing enzyme stability often results in reduced activity. A primary objective of the project is to evaluate a new design strategy. This strategy might improve certain properties without degrading others. The strategy combines a computational approach with a novel starting material. This work will also support recruitment and engagement of undergraduate researchers and underrepresented groups through hands-on research, workshops, and lectures.The overall goal of this work is to develop a “super-optimal” biocatalytic system where enzymes can operate beyond their characterized optimal temperatures, turnover frequency (TOF, kcat), and conversion. Achieving this requires enzyme engineering to directly modify their catalytic properties and encapsulation to alter stability and substrate and product transport rates and partitioning. The project will use a 4-methylideneimidazole-5-one (MIO)-containing amino acid ammonia-lyases (XAL) as a model enzyme. Three different hypotheses will be tested. The first is that novel enzyme activity can be achieved by relaxing stability criteria during engineering. The second is that MIO-containing XAL enzymes can accept non-aromatic substrates. The third is that an engineered encapsulation system can enhance enzyme stability, reaction rates, and conversion. A directed evolution workflow will identify XAL variants with high activity. Stability requirements will be relaxed during directed evolution. Identifying variants with activity on non-cognate amino acid substrates will be another objective. XAL enzymes have never previously been engineered to act on non-aromatic substrates. The enzyme engineering work will use a deep mutational scanning (DMS)-guided experimental workflow as well as computational modeling. Encapsulations for enzymes to enhance their stability, reaction rate, and product yield will be designed and evaluated.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.