Engineering high-activity crosslinked enzyme aggregates via SpyCatcher/SpyTag-mediated self-assembly

Area of Science:

• Biocatalysis and Enzyme Engineering
• Protein Chemistry
• Biotechnology

Background:

• Crosslinked Enzyme Aggregates (CLEAs) offer advantages in operational stability and recyclability.
• Traditional CLEA preparation methods can negatively impact enzyme activity by distorting active sites.
• There is a need for improved methods to create stable and active CLEAs.

Purpose of the Study:

• To develop a universally applicable crosslinked SpyCatcher scaffold system for facile CLEA preparation.
• To enhance enzyme activity and stability in CLEAs through a novel scaffold approach.
• To demonstrate the effectiveness of the system in constructing specific enzyme complexes like xylanase-CLEAs and cellulase-CLEAs.

Main Methods:

• Engineered SpyCatcher with N-terminal lysine residues for selective glutaraldehyde crosslinking.
• Enzyme assembly via SpyTag-SpyCatcher specific binding.
• Optimization of SpyCatcher precipitation and glutaraldehyde crosslinking conditions (e.g., (NH4)2SO4 precipitation, temperature, time, speed).
• Immobilization of SpyTagged enzymes (xylanase, cellulase) onto the SpyCatcher scaffold to form CLEAs.

Main Results:

• The SpyCatcher scaffold achieved over 90% crosslinking efficiency.
• Constructed xylanase-CLEAs (X-CLEAs) and cellulase-CLEAs (C-CLEAs) demonstrated accurate enzyme complex formation.
• C-CLEAs maintained ~90% activity after 3 cycles and >50% after 10 cycles, indicating excellent stability and reusability.
• C-CLEAs enhanced corn stover hydrolysis by 1.6-fold compared to free enzymes, suggesting improved cascade reaction efficiency.

Conclusions:

• The developed SpyCatcher scaffold system provides a facile and effective method for preparing high-activity and stable CLEAs.
• This approach overcomes limitations of traditional CLEA preparation, preserving enzyme active sites.
• The enhanced performance of C-CLEAs in biomass hydrolysis highlights the potential of this system for various industrial biocatalytic applications.