One-pot synthesis of alkyl caffeates from chlorogenic acid using a heterogeneous biocatalyst generated by glycan oxidation-mediated immobilization (Gloxi)

Biotransformation of chlorogenic acid into alkyl caffeates is of great interest due to their valuable biological activities. Aiming to achieve one-pot synthesis of alkyl caffeates from chlorogenic acid, we perform an enzyme and reaction media screening, using the butyl caffeate synthesis as a model reaction. Employing the feruloyl esterase B from Aspergillus terreus (rAtFAEB) in butanol/water (6:1, v/v) mixture, we reached 41% of conversion after 4 h. To achieve higher yields and access biocatalyst recyclability, we decided to fabricate a heterogeneous biocatalyst. After screening 15 immobilization strategies, we concluded that hyper-glycosylation of recombinant rAtFAEB did not allow the proper binding of the enzyme to the supports. Our strategy involved oxidizing the glycosylation of the enzyme to transform the cis-diols of the glycans into aldehydes. The modified enzyme was then immobilized through a covalent bond with aminated supports (MANAE-Agarose and MANAE-Methacrylate) or aminated polycationic matrices. Gloxi-MANAE-Agarose biocatalyst allowed us to access 86% of butyl caffeate conversion after 24 h of reaction. Under the same reaction conditions, further scope studies for various alkyl caffeates showed conversions ranging from 15 to 94%. Testing recyclability, conversion declined to 13% after 4 cycles. This is the first report of a one-pot alkyl caffeate synthesis in solvent-free media using a heterogeneous biocatalyst. Furthermore, our immobilization approach enables the use of hyper-glycosylated enzymes incompatible with standard protocols, overcoming hyper-glycosylation challenges for enzyme immobilization.