Research

1. Enzyme Immobilization and Catalysis

Enzymes play not only a central role in physiological processes, but also essential catalysts in industrial biotransformation, laundry detergents, clinical diagnosis, etc. Recent developments in materials science, nanotechnology and synthetic biology are advancing the applications of enzymes from the aqueous condition to non-aqueous conditions, from a single enzymatic reaction to multienzyme reactions, and from catalysis to smart, programmable and controllable applications. Engineering enzymes for practical applications will reshape many traditional processes toward greener, smarter and more efficient processes.

Stimuli-responsive Enzyme Catalysts

The integration of enzymes with stimuli-responsive polymers results in smart enzyme catalysts, which possess both catalytic activity and environmental responsiveness. These smart systems hold great promise for wide range of applications in controllable biocatalysis, drug delivery, biosensing and also intelligent devices. In the past, we have demonstrated smart enzyme catalysts with controllable activity in response to pH, ionic strength, temperature and mechanical stretching.

Select publications:

Yifei Zhang, Qile Chen, Jun Ge, Zheng Liu. Controlled display of enzyme activity with a stretchable hydrogel. Chemical Communications, 2013, 49, 9815−9817. [link]
Yifei Zhang, Kehang Han, Diannan Lu and Zheng Liu, Reversible encapsulation of lysozyme within mPEG-b-PMAA: experimental observation and molecular dynamics simulation. Soft Matter, 2013, 9, 8723−8729. [link]
Jingying Zhu, Yifei Zhang, Diannan Lu, Richard N. Zare, Jun Ge, and Zheng Liu, Temperature-responsive enzyme-polymer nanoconjugates with enhanced catalytic activities in organic media. Chemical Communications, 2013, 49, 6090−6092. [link]

Nanostructured Enzyme Catalysts

The rapid development of nanotechnology offers new opportunities to construct novel enzyme catalysts with improved stability, activity and reusability. Generally, the introduction of nanomaterials in enzymatic catalysis will influence the reaction process by interfering with mass transfer and enzyme catalytic property. In addition, the immobilization of enzymes on nanomaterials often helps to improve the thermo-stability and solvent tolerance of enzymes.

Select publications:

Yifei Zhang, Jun Ge and Zheng Liu. Enhanced activity of immobilized or chemically modified enzymes. ACS Catalysis, 2015, 5, 4503−4513. [link]
Fengjiao Lyu, Yifei Zhang, Richard N. Zare, Jun Ge, and Zheng Liu. One-pot synthesis of protein-embedded metal-organic frameworks with enhanced biological activities. Nano Letters, 2014, 14, 5761−5765. [link]

Non-aqueous Enzymatic Catalysis

Enzymes have been demonstrated their ability to catalyze reaction in non-aqueous solvents. However, the insoluble enzymes in organic solvents often exhibit 10³ to 10⁵ lower activity than in water due to the poor accessibility. To overcome this, we improved the dispersibility of enzymes into organic solvents by immobilizing them in nanogels or conjugating them with amphiphilic polymers. The obtained enzyme catalysts have been successfully used in the synthesis of multiple chemicals including anticancer drugs, antibiotics, superplasticizers and others.

Select publications:

Yifei Zhang, Yang Dai, Miao Hou, Tian Li, Jun Ge, Zheng Liu. Chemo-enzymatic synthesis of valrubicin using pluronic conjugated lipase with temperature responsiveness in organic media. RSC Advances, 2013, 3, 22963−22966. [link]
Rui Wang, Yifei Zhang, Jinhai Huang, Diannan Lu, Jun Ge and Zheng Liu, Substrate imprinted lipase nanogel for one-step synthesis of chloramphenicol palmitate. Green Chemistry, 2013, 15, 1155−1158. [link]

2. Rational Design of High-efficiency Enzyme Cascades

Cascade reactions catalyzed by multiple enzymes hold new opportunities in biotransformation and diagnostics. They have attracted interest from both academia and industry in the past decade. My research focuses on both understanding of cascade reaction kinetics and the construction of high-efficiency multienzyme systems.

The Origin of the Activity Enhancement in Enzyme Cascades

Activity enhancements in enzyme cascades on scaffolds were frequently reported in the past decade and have attracted tremendous attentions. The observed activity enhancements were attributed to the proximity channeling that facilitated transport of intermediate substrate. However, our experiments experimentally demonstrated that the diffusion time was negligible even in the free enzyme cascade and the overall activity must be limited by the slower enzyme. The possible mechanism for the observed enhancements is that the charged scaffolds like DNA origami structures altered the local pH and thereby increased the individual enzyme activity. This concept was then applied to optimize the overall activity of an enzyme cascade with enzymes have different pH optimums.

Select publications:

Yifei Zhang, Stanislav Tsitkov, Henry Hess. Proximity does not contribute to activity enhancement in the glucose oxidase-horseradish peroxidase cascade. Nature Communications, 2016, 7, 13982. [link]
Yifei Zhang, and Henry Hess. Toward rational design of high-efficiency enzyme cascade. ACS Catalysis, 2017, 7, 6018−6027. [link]
Yifei Zhang, Qin Wang, Henry Hess. Increasing enzyme cascade throughput by pH-engineering the microenvironment of individual enzymes. ACS Catalysis, 2017, 7, 2047–2051. [link]

Preparation of Multienzyme Systems

We also constructed multienzyme systems through various strategies and applied these systems in catalysis and biosensing. In the past, we showed spatial immobilization of multiple enzymes on porous materials, selectively clustering glycoenzymes with lectins, and patterned deposition of enzymes by inkjet printing.

Select publications:

Yifei Zhang, Fengjiao Lyu, Jun Ge, Zheng Liu. Ink-jet printing an optimal multi-enzyme system. Chemical Communications, 2014, 50, 12919−12922. [link]
Yifei Zhang, You Yong, Jun Ge, Zheng Liu. Lectin agglutinated multienzyme catalyst with enhanced substrate affinity and activity. ACS Catalysis, 2016, 6, 3789−3795. [link]
Zhixian Li, Yifei Zhang, Yechao Su, Pingkai Ouyang, Jun Ge, Zheng Liu. Spatial co-localization of multi-enzymes by inorganic nanocrystal-protein complexes. Chemical Communications, 2014, 50, 12465−12468. [link]

3. Enzymatic Reaction Networks

Enzymatic reaction networks capable of generating complex spatiotemporal dynamics are not only the basis of vital biological processes but also the basic units of synthetic systems with autonomous, adaptive and programmable behaviors. The construction of enzymatic reaction networks in vitro with predictable dynamics of interest is an emerging area of synthetic and systems biology, and will pave the way to biorobotics, and hopefully, will shed some light on the origin of life.

Complex Dynamics of Enzymatic Reaction Networks

The aim of this research is to construct enzyme-based circuits and artificial cells with life-like functionality and adaptivity. To achieve that, deep understanding of the biochemical functions of basic building blocks and their interactions is desired. We plan to document typical key node metabolites and regulatory enzymes, and to create artificial reaction networks by both modelings and experiments. For instance, we recently discovered a new reaction in the glucose oxidase (GOx) and horseradish peroxidase (HRP) cascade. Taking advantage of this, we presented a two-enzyme reaction network which can demonstrate multiple tunable pulses and a green bottle experiment.

Green bottle experiment

Enzymatic Reaction Driven Rayleigh–Bénard Convection

This GOx-HRP reaction network will lead to visible spatiotemporal patterns from Rayleigh-Benard convection in a petri dish when the system is exposed to air. The convective flow is driven by the GOx-catalyzed reaction and visualized by the HRP-catalyzed reaction.

Depth = 2.6 mm

Depth = 7.0 mm

Depth = 8.8 mm

Select publications:

Yifei Zhang, Stanislav Tsitkov, Henry Hess. Complex dynamics in a two-enzyme reaction network with substrate competition, Nature Catalysis, 2018, 1, 276–281.

4. Can Enzymes diffuse faster during catalysis?

Recent experimental studies have measured a 30–80% increase of the diffusion coefficient when various enzymes are catalytically active. It implies that any enzyme can in principle be considered as a molecular engine. If this effect is physically valid, it would refigure our current understanding of enzymatic catalysis and enable us to construct nano- or micro-machines by harnessing the power of enzymatic reactions. However, theoretical understanding of the apparent enhanced diffusion of active enzymes remains controversy and the current experiments need more rigorous controls since most observations were based solely on fluorescence correlation spectroscopy (FCS).

By using a different technique, dynamic light scattering (DLS), my measurement of the most controversial enzyme, aldolase, showed that this enzyme did not show enhanced diffusion in the presence of its substrate. It is the first experiment that clearly states the failure of enhanced diffusion for an enzyme (aldolase).

Later, we chronologically summarize and discuss the experimental observations and theoretical interpretations and emphasize the potential contradictions in this topic. We point out that the existing multimeric forms of enzymes or isozymes may cause artifacts in measurements and that the conformational changes upon substrate binding are usually not sufficient to give rise to a diffusion enhancement greater than 30%.

Select publications:

1. Yifei Zhang, Megan J. Armstrong, Neda M. Bassir Kazeruni, Henry Hess. Aldolase does not show enhanced diffusion in dynamic light scattering experiments. Nano Letters, 2018, 18, 8025–8029. [link]

2. Yifei Zhang, Henry Hess. Enhanced diffusion of enzymes induced by catalytic reactions. ACS Central Science, 2019, 5, 939-948. [link]

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