Region-Directed Enzyme Immobilization through Engineering Protein Surface with Histidine Clusters

Area of Science:

• Biocatalysis and Enzyme Engineering
• Materials Science and Nanotechnology
• Chemical Engineering

Background:

• Enzyme immobilization is crucial for industrial applications but often lacks control over enzyme orientation, leading to reduced activity and stability.
• Conventional immobilization methods, like His-tags, offer limited orientation control, restricting enzyme positioning to termini.
• Developing general approaches for highly active and robust heterogeneous biocatalysts remains a challenge.

Purpose of the Study:

• To engineer surface histidine clusters on dehydrogenases for site-selective enzyme immobilization.
• To investigate the impact of histidine cluster position, density, and metal chelate type/density on immobilization efficiency and enzyme stability.
• To compare the enzyme orientation and performance of His-clusters with conventional His-tags.

Main Methods:

• Engineering two model dehydrogenases with surface histidine clusters in flexible, non-catalytic regions.
• Immobilizing enzyme variants on various carriers functionalized with different metal chelates (Co2+, Cu2+, Ni2+, Fe3+).
• Assessing immobilization yield, recovered activity, enzyme stability against denaturing agents, and operational stability in batch reactions.
• Utilizing proteomic studies to analyze enzyme orientation differences between His-clusters and His-tags.

Main Results:

• His-clusters demonstrated comparable or superior immobilization efficiency and enzyme activity compared to conventional His-tags.
• Engineered enzymes showed enhanced stability against certain denaturing agents.
• Enzyme orientation, influenced by His-cluster design and metal chelate properties, significantly impacted immobilization parameters and biocatalyst stability.
• Proteomic analysis confirmed His-clusters facilitate different enzyme orientations than His-tags.
• Optimized oriented biocatalysts exhibited increased operational stability in batch reactions.

Conclusions:

• Surface-engineered histidine clusters offer a versatile strategy for controlled enzyme immobilization, improving biocatalyst performance.
• This approach enables distinct enzyme orientations, leading to enhanced activity, stability, and operational robustness.
• The findings pave the way for more predictable and efficient design of heterogeneous biocatalysts for industrial applications.