Enzymology & Molecular Biology

Biography

Biography: Zhenjun Liu

Abstract

Enzymes are economically viable catalysts in the production of useful compounds in biotechnology. Scaffolded assembly of the enzymes can potentially increase the processability, stability and reaction efficiency. Here, an efficient assembly strategy is presented based on the robust protein scaffolds, using Spy chemistry and some small size of protein linkers DDs. Spy chemistry is a genetically protein gelation strategy, which plays a role as a skeleton of the protein scaffolds. The DDs perform as protein linkers to immobilize enzymes on the protein scaffolds. Enzyme assemblies has been created by fusing the enzymes with the corresponding protein linkers and the strong and specific interactions between protein linkers improve the specificity of assembly. The DDs also provide a good condition for enzymatic reactions without excessive interference. The formation of protein scaffolds was first confirmed by SDS-PAGE and then screened out the suitable condition. Moreover, the reactivity and selectivity of using DDs to assemble enzymes are characterized by Florescence Resonance Energy Transfer (FRET). Two types of scaffold form were designed: One is crosslinked protein scaffold and another one is cyclic proteins scaffold. Both of the scaffolds can assemble fluorescent proteins as well as enzymes in menaquinone pathway. Importantly, the in vitro reaction efficiency increases on the cyclic protein scaffold. While, enzyme assembly on the crosslinked scaffold self-assembled together. The kinetic parameters and morphology in enzyme assembly was further assembled to explain these phenomena. The work paves the way for the assembly-based regulation of multienzyme biosynthetic pathways.