Substrate bio-imprinted CLEAs of type B feruloyl esterase from Aspergillus terreus: A selective heterogeneous biocatalyst towards butyl caffeate

Abstract

The biological properties of caffeic acid esters, compel the search for selective biocatalysts for its synthesis since caffeic acid is generally mixed with ferulic acid in nature. Here, we employed a type B feruloyl esterase from Aspergillus terreus bio-imprinted and crosslinked (AtFAEB-CLEAs) to increase its selectivity towards the synthesis of butyl caffeate in caffeic/ferulic acid equimolar mixtures. The best AtFAEB-CLEAs were prepared using n-butanol/caffeic acid (50 mM) mixture for enzyme precipitation/bio-imprinting, polyethyleneimine as co-feeder, and glutaraldehyde as a crosslinker. Selected AtFAEB-CLEAs showed an expressed activity 2.6-fold higher (218 U·mg⁻¹) than free enzyme, and a 13.5-fold enhancement in synthesis selectivity on a hexane: n-butanol: water system, inverting the initial preference for ferulic acid towards caffeic acid. Intrinsic protein fluorescence profiles strongly suggest a freeze-up of conformational changes induced by bioimprinting in the AtFAEB. Additionally, immobilization increased the thermal and solvent stability of AtFAEB-CLEAS 7.9 and 5.7-fold, respectively. Final reaction tests (0.1 mg/mL) achieved 88 % conversion in 6 hours and demonstrated a strong preference for butyl caffeate synthesis of 26-fold in the first hour of reaction. Improvement through CLEA fabrication of selectivity and stability of AtFAEB could potentially lead to the valorization of agro-industrial by-products into relevant molecules for the cosmetic and pharmaceutic industries.