Degree Type


Date of Award


Degree Name

Doctor of Philosophy


Chemical and Biological Engineering

First Advisor

Balaji Narasimhan

Second Advisor

Surya K. Mallapragada


The widely existing MECs in Nature have inspired researchers to design synthetic analogs to promote the overall catalytic efficiency in vitro by co-localizing multiple enzymes to mimic the MECs' unique functionalities. A number of efforts have been devoted to designing synthetic MECs in the past couples of decades. This thesis work has focused on developing novel strategies based on enzyme immobilization to design nano-carriers for multi-enzyme co-localization to realize kinetics enhancement and strong control of spatial arrangement of the enzymes. Three distinct approaches have been designed using different attachment methods and platforms.

First, the multifunctional polystyrene nanoparticles were designed for immobilization and sequential co-localization of GOX and SHRP enzymes using covalent binding and streptavidin-biotin coupling attachment techniques. The sequentially co-localized GOX and SHRP on B/C-PS nanoparticles were capable of enhancing the overall conversion rate by approximately two-fold compared to the equivalent concentration of free enzymes in solution. Secondly, amphiphilic Pluronic-QD micelles were designed to co-localize multiple enzymes to investigate the effect of a more flexible substrate compared to the rigid polystyrene particles. FRET was used to characterize the adsorption of single enzymes and co-localization of multiple enzymes on the micelles. The two co-localized enzymes enhanced the overall conversion rate by ~100% compared to the equivalent concentration of free enzymes in solution, which is very comparable to the findings in the first study. To further investigate the spatial arrangement impact on enzyme co-localization, the precise DNA hybridization was investigated to direct multi-enzyme co-localization on PS nanoparticles. It was found that the co-localized GOX and HRP via DNA hybridization significantly improved the overall reaction efficiency by close to 200% as compared to single enzyme immobilization mixture. The co-localized enzymes also exhibited well stability over time.

In summary, the current research has demonstrated the superior potential of co-localized multiple enzymes in terms of kinetically-driven benefits. The spatial arrangement plays a significant role in mimicking the MECs in vitro. Looking forward, the design of sustainable and re-usable multi-enzyme biocatalysts would lead to both scientifically exciting research as well as economically viable designs for next generation catalysts and biosensors.


Copyright Owner

Feng Jia



File Format


File Size

157 pages