It is of great significance to study the effect of multi-enzyme aggregation behavior at the interface on the formation of multi-enzyme complexes and their co-catalytic characteristics, which is helpful for us to design and construct immobilized multi-enzyme complex systems for in vitro synthetic biology. Here, a magnetic microsphere with chelated Ni 2+ , was prepared to explore the self-assembly characteristics of PduQ-SpyTag (P-T) and Nox-SpyCatcher (N C) on its surface, based on the mixed interaction mode consisting the affinity of His-tag/Ni 2+ and covalent binding of SpyTag/SpyCatcher. After studying the effect of saturated or unsaturated adsorption of P-T on the covalent binding between P-T and N C at the interface, a possible multienzyme interaction mechanism for the affinity-assisted covalent self-assembly on the Ni 2+ chelating surface was proposed. The time evolution of NADH showed that the immobilized P-T/N-C complex formed by this method and the free P-T/N-C complex exhibited similar synergistic catalytic properties, and presented higher catalytic efficiency than the simple mixing of P-T and N C. The optimal catalytic conditions, stability and reusability of the immobilized multi-enzyme complexes prepared in this study were also discussed by comparing them with free enzymes. In this study, we demonstrate a simple and effective strategy for self-assembling SpyTag/SpyCatcher fusion proteins on the surface of magnetic beads, which is inspiring for the construction of more cascade enzyme systems at the interface. It provides a new method for facilitating the rapid construction of immobilized multi-enzyme complexes in vitro from the crude cell lysis .
Background Vanillin is a widely utilized flavor compound of significant value in the food and pharmaceutical sectors, which can be obtained through natural extraction, chemical synthesis, or biotechnological processes. However, the yield from vanilla pods is insufficient to meet market demand, and chemically synthesized vanillin not only encounters limitations in its application within the food and pharmaceutical industries but also needs to address environmental concerns and unsustainable raw material sources. Hence, it is imperative to explore alternative approaches to develop an efficient and cost-effective green vanillin. To address the challenges encountered in vanillin biosynthesis, such as substrate uptake limitations and product-induced inhibition of cell growth,we leveraged the advantages of surface display technology and artificial multi-enzyme scaffolds to construct a hybrid surface-display biocatalytic system by assembling Eugenol oxidase (EUGO) and dioxygenase (NOV1), which can convert lignin biowaste 4- n -propylguaiacol (4-PG) into vanillin on the surface of Escherichia coli BL21(DE3). Results To assemble bioactive macromolecules of EUGO and NOV1 on the surface of E. coli BL21(DE3), we utilized Lpp-OmpA-SpyCatcher (LOAS) as an anchoring motif and displayed EUGO-linker-NOV1-SpyTag (ELNS) by covalent interaction between SpyTag andSpyCatcher to allow their spatial proximity. After optimization of the reaction system, our self-assembly display system exhibited highly efficiency in converting 4-PG into vanillin and reached a final concentration of vanillin at 12.58 g/L, 2.5 times higher than that achieved by thewhole-cell biocatalytic system. The LOAS-ELNS display system was applied to the sustainable biosynthesis of vanillin from lignin-derived 4-n-propylguaiacol at least 10 times. Conclusions This work provided a generalized approach to co-expressing proteins and offered an efficient, eco-friendly, and renewable method for the biosynthesis of vanillin from 4-PG.
Xiao-Qing Gong,Chuan-Wan Wei,Jia-Kun Xu,Xiao-Juan Wang,Shu-Qin Gao,Ying-Wu Lin
The encapsulated enzyme system by metal-organic frameworks (MOFs) exhibits great potential in biofuel cells, pharmaceuticals, and biocatalysis. However, the catalytic efficiency and the enzymatic activity are severely hampered due to enzyme leaching and deficiency of storage stability. In this study, we immobilized cytochromec(Cytc) into dimethylimidazole-copper (Cu(Im)2) by biomimetic mineralization, and constructed a bioinorganic hybrid material, termed Cytc@Cu(Im)2. Encapsulated Cytcin Cu(Im)2with a nanosheet structure exhibited significantly improved catalytic efficiency, enzymatic activity and kinetic performance. The catalytic efficiency (kcat/Km) for Cytc@Cu(Im)2was ~20-fold higher compared to that of free Cytc. Moreover, the increased activity was not affected by long-term storage. Based on this system, we further constructed a multi-enzyme composite with glucose-oxidase (GOx), termed GOx-Cytc@Cu(Im)2, which exhibited greatly improved enzymatic activity, stability, and excellent selectivity for the detection of low concentrations of glucose. This strategy may provide new insights into the design of enzymes with high activity and stability, as well as the construction of multi-enzyme systems.Keywords:enzymatic immobilization;biomimetic mineralization;multi-enzyme;bioinorganic hybrid material;biosensors
危险品化学品经营许可证（带存储）