From random to chemoselective cross-linking: Reorientation and enhancement of immobilized cross-linked enzyme aggregates of cyclodextrin glucanotransferase from Shouchella lehensis G1 via targeted domain modification

Uncontrolled, random cross-linking near the enzyme's active site may impede mass transfer and overall activity, which is one of the challenges in cross-linked enzyme aggregates (CLEAs) immobilization. Likewise, a thorough understanding of intermolecular interactions between cross-linkers and enzymes is crucial to yield robust and oriented CLEAs. Therefore, this study aims to chemoselectively cross-link cyclodextrin glucanotransferase G1 (CGTaseG1) with a glutaraldehyde via computational and rational design approaches. The domain D of CGTaseG1 was targeted for mutation, leading to the construction of four variants involved in substituting all solvent-exposed residues within the β-sheet or coil secondary structure with either Lys or Ser. The successful reorientation of CLEAs was assessed, and the possible interaction was proposed. The CLEAs of CGTaseG1 variants exhibited a 31.5% enhancement in activity recovery relative to wild-type CLEAs. Effective cross-linking was observed at an optimal pH of 6 for CLEAs of mSerCGTaseG1, whereas mLysCGTaseG1-CLEAs required a pH of 8 to attain successful reorientation. mSer and mLysCGTaseG1-CLEAs demonstrated an improvement in catalytic efficiency, with increases of 171.8% and 136.3%, respectively, compared to CGTaseG1-CLEAs (32.1 mM⁻¹ s⁻¹). Additionally, mSer and mLysCGTaseG1-CLEAs demonstrate enhanced mechanical and thermal stability, resulting in increased product formation and retained higher reusability recoveries of 53.8% and 39.7%, respectively, in contrast to wild-type CLEAs (35.3%) after five cycles of each two-hour reaction. This study has switched the focus from glutaraldehyde's typical amine target to the hydroxyl group, offering structural insights into the aldehyde-mediated cross-linking mechanism, facilitating the design and enhancement of immobilized biocatalysts for potential industrial applications.