Preparation of Bio‐Orthogonal Self‐Cross‐Linked Arabinose Isomerase for Enzymatic Synthesis of <scp>ʟ</scp>‐Ribulose

Abstract ʟ ‐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.