Enhancing the characteristics of phenolic acid decarboxylase via N-terminal substitution and investigating its immobilization

Phenolic acid decarboxylase (PAD) is an enzyme capable of catalyzing the nonoxidative decarboxylation of phenolic acids, yielding the corresponding 4-vinyl derivatives. This enzymatic process holds considerable promise for converting naturally abundant phenolic acid substrates into high-value compounds. The PAD gene from Bacillus subtilis J6 was cloned to yield the BJ6PAD enzyme, and its mutant BJ6PAD-N was generated by introducing an N-terminal substitution. Compared with BJ6PAD, BJ6PAD-N demonstrated not only higher specific enzyme activity but also increased alkaline resistance. The N-terminal region of BJ6PAD-N exhibited increased flexibility, leading to a looser structure. This change improved the catalytic efficiency for sinapic acid (SA) with bulky side chains. After its immobilization, the application potential of BJ6PAD-N was significantly enhanced, demonstrating reusability and storage stability that were superior to those of BJ6PAD. After 10 repeated uses, the residual enzyme activity remained above 80%. When stored at 4 °C for 60 days, 61.15% of the enzyme activity was retained. These characteristics are crucial for facilitating the industrial application of enzymes. Replacing the N-terminal of phenolic acid decarboxylase BJ6PAD (resulting in BJ6PAD-N) made the enzyme structure more flexible. While this reduced substrate binding stability, it increased specific enzyme activity. Notably, the enzyme showed improved catalytic efficiency for sinapic acid, which has a bulky side chain. After being immobilized, the performance stability of the enzyme has been further enhanced.