ERI: Multi-Activity Proteins for Improved Carbohydrate Deconstruction

生物质是替代燃料和化学品的重要来源。增加生物质向有用产品的转化将改善环境中的碳平衡。多活性蛋白质(MAP)具有提高生物质转化率的潜力。在这个项目中，将制作并测试85张地图，以确定改进的生物质解构。将为研究生和本科生提供研究机会。此外，一系列讲座将向公众介绍酶学和蛋白质工程。项目重点是MAP的生产、纯化和表征。合成了具有两个催化区的85个新的多活性蛋白(MAP)的工程DNA序列，并将其克隆到pET151中进行异源生产。在两个催化区之间引入了TEV裂解位点。纯化的MAP的生化特性将集中在催化结构域之间的协同作用上，利用TEV-裂解位点。通过比较完整MAP和TEV消化产生的相应单个催化结构域的等摩尔混合物的动力学参数(Km和kcat)，将突出组合结构域之间的协同作用。通过邻近效应，根据连接的结构域，MAP有望显示复杂底物上活性的增加和产物的抑制作用的减少。除了确定改进碳水化合物解构的新催化剂外，该项目还应该提供对MAP中组合结构域之间相互作用的机械理解，并帮助指导定制应用的新蛋白质的工程。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。

Biomass is a critical source of alternative fuels and chemicals. Increasing the conversion of biomass to useful products will improve the carbon balance in the environment. Multi-activity proteins (MAPs) have the potential to be increase biomass conversion. In this project, 85 MAPs will be produced and tested to identify improved deconstruction of biomass. Research opportunities for graduate and undergraduate students will be provided. In addition, a series of lectures will inform the general public about enzymology and protein engineering.The project focuses on the production, purification, and characterization of MAPs. The engineered DNA sequences of 85 new multi-activity proteins (MAPs), with two catalytic domains, have been synthesized and cloned in the pET151 for heterologous production. A TEV-cleavage site was introduced between the two catalytic domains. The biochemical characterization of purified MAPs will focus on the synergy among the catalytic domains by taking advantage of the TEV-cleavage site. The synergistic interaction between the combined domains will be highlighted by the comparison of kinetic parameters (Km and kcat) for the complete MAP and for the equimolar mixture of the corresponding individual catalytic domains generated by TEV-digestion. Through proximity effects, and depending on the linked domains, MAPs are expected to display increased activity on complex substrates and reduced inhibition by the product. In addition to identifying new catalysts for improved carbohydrate deconstruction, this project should provide a mechanistic understanding of the interaction between the combined domains in MAPs and help guide the engineering of new proteins for custom applications.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.