Enzyme immobilization is an effective method for improving the stability and reusability. However, linking at random sites on the enzyme results in low catalytic efficiency due to blockage of the active site or conformational changes. Therefore, controlling the orientation of enzymes on the carrier has been developed. Here, the site-specific mutation and the SpyTag/SpyCatcher systems were used to prepare a site-directed immobilized enzyme. The thermal stability of the immobilized enzyme was better than that of the free enzyme, and ≥80 % of the catalytic activity was retained after 30 days of storage. Furthermore, the Michaelis constant (Km) and the turnover number (kcat) of the immobilized enzyme were 5.23-fold lower and 6.11-fold higher than those of the free enzyme, respectively, which appeared to be related to changes in secondary structure after immobilization. These findings provide a new and effective option for enzyme-directed immobilization.
Keywords: Glutamate decarboxylase; Self-assembly; Site-directed immobilization; SpyTag/SpyCatcher.
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