ERI: Multi-Activity Proteins for Improved Carbohydrate Deconstruction

点击翻译按钮获取中文摘要

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.