One-step direct immobilization of engineered prenyltransferase on polystyrene for prenylated flavonoid production

Enzymes are highly efficient and specific biocatalysts. However, they are unstable and prone to deactivation, which limits their practical applications. To address these challenges, immobilization technology has been developed. Traditional immobilization methods typically require purified enzymes, but enzyme purification is often time-consuming and labor-intensive. Therefore, this study aims to develop a one-step method that directly purifies, adsorbs, and immobilizes enzymes from crude extracts, while simultaneously enhancing their activity. In this work, we examined the importance of the location of anchoring peptide (LCI) in fused enzyme functionality and binding affinity across various materials. LCI was fused with Aspergillus fumigatiaffinis prenyltransferase (AfPT) at the N-terminus to obtain the fusion enzyme LCI-AfPT, which displayed maximum load (38.9 mg/g) and highest activity recovery (92.7%) on polystyrene (PS). The free enzyme LCI-AfPT exhibited highest activity at pH 8.0 and 40°C. The highest activity of LCI-AfPT@PS was achieved at pH 7.5 and 55°C, with over 70% residual activity after 4 hours of incubation at 55°C. The tolerance of LCI-AfPT@PS to ethanol and methanol was significantly higher than that of LCI-AfPT. Immobilization significantly improved substrate affinity (Km reduced from 0.273 mM to 0.09 mM) and organic solvent tolerance, with no activity loss in ≤10% ethanol/methanol, highlighting its potential for industrial biocatalysis of hydrophobic flavonoids. The Kcat/Km value of LCI-AfPT@PS reached 0.072 s^-1· mM^-1, which was higher than that of LCI-AfPT and AfPT. LCI-AfPT was directly immobilized on PS from crude enzymes without purification, with a high adsorption selectivity of 80.5%, which was about 8-fold higher than that of AfPT. LCI-AfPT@PS retained 70% of its original activity after 10 cycles of reuse, resulting in a total of 4.4 mM of 3’-C-prenylnaringenin. This study represents the first investigation on the one-step purification and immobilization of prenyltransferase using an anchoring peptide, and its application in the efficient production of prenylated flavonoids.