Preparation of Bio‐Orthogonal Self‐Cross‐Linked Arabinose Isomerase for Enzymatic Synthesis of ʟ‐Ribulose

Abstract

Abstractʟ‐Ribulose is a bioactive molecule of significance that is present in various valuable chemical scaffolds. However, the synthesis of ʟ‐ribulose is limited by the high cost of enzyme biocatalyst. In this work, we leveraged the self‐coupling reactivity of SpyTag/SpyCatcher to aggregate the key arabinose isomerase (araA) for the enzymatic isomerization of ʟ‐arabinose to ʟ‐ribulose. The cross‐linking of SpyTag/SpyCatcher fused araA was simplified by performing in cell lysates without further purification. The resulting araA cross‐linked aggregates (araA‐CLEs) inherited their original activity well with 0.32 U·mg−1. Deep analysis of araA‐CLEs by Gaussian fitting indicated a little change in secondary structure, mainly focusing on α‐helix. The araA‐CLEs maintained 69.10% catalytic activity after six repeated uses in preparing ʟ‐ribulose. This one‐step cross‐linking and purification method contributed greatly to obtaining araA‐CLEs, thus, benefiting the full use of araA protein and saving costs for ʟ‐ribulose production.