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

Crosslinked Enzyme Aggregates (CLEAs) are favored for their operational stability and recyclability. However, the traditional CLEAs preparation may distort the enzyme's active site and reduce activity. Therefore, we developed a universally applicable crosslinked SpyCatcher scaffold system designed for the facile preparation of CLEAs. Four lysine residues were introduced to the N-terminus of SpyCatcher to enhance the crosslinking selectivity with glutaraldehyde. This scaffold subsequently enables the assembly of enzymes through the specific binding affinity between SpyTag and SpyCatcher. By precipitating SpyCatcher with (NH)SO at a 1:2 (v/v) ratio at 4 °C for 1 h, followed by crosslinking with glutaraldehyde at 25 °C and 100 rpm for 3 h, the SpyCatcher scaffold achieved a crosslinking efficiency exceeding 90 %. Utilizing this approach for the immobilization of SpyTaged enzymes to construct xylanase-CLEAs (X-CLEAs) and cellulase-CLEAs (C-CLEAs) highlighted the accurate construction the enzyme complex. Furthermore, C-CLEAs retained approximately 90 % activity after 3 cycles and over 50 % activity after 10 cycles, demonstrating good operational stability and reusability. C-CLEAs achieved a 1.6-fold more reducing sugars in corn stover hydrolysis compared to free enzymes. This suggests that the close proximity within CLEAs provided an advantage for the enzymatic cascade reaction, highlighting the potential application of CLEAs in various fields.