Carrier-bound and carrier-free immobilization of type A feruloyl esterase from Aspergillus niger: Searching for an operationally stable heterogeneous biocatalyst for the synthesis of butyl hydroxycinnamates.

• Feruloyl esterases (FAE) low stability in solvents challenges its use in synthesis. • FAE A from A. niger was successfully immobilized in epoxy activated supports and CLEA. • CLEA with BSA as cofeeder and 15 mM glutaraldehyde was the most active biocatalyst. • CLEA attained complete butyl sinapate solvent free esterification within 24 h. • CLEA performed 10 esterification cycles without significant loss of activity. Feruloyl esterases synthesize butyl hydroxycinnamates, molecules possessing interesting biological properties, nonetheless, they exhibit a low stability under synthesis conditions in organic solvents, restricting its use. To enhance its operational stability in synthesis, we immobilized type A feruloyl esterase from Aspergillus niger (AnFAEA) using several carrier-bound and carrier-free strategies. The most active biocatalysts were: 1) AnFAEA immobilized on epoxy-activated carriers (protein load of 0.6 mg enzyme x mg−1 carrier) that recovered 91 % of the initial hydrolytic activity, and 2) AnFAEA aggregated and cross-linked in the presence of 5 mg of BSA and 15 mM of glutaraldehyde (AnFAEA-amino-CLEAs), which exhibited 385 % of its initial hydrolytic activity; both using 4-nitrophenyl butyrate as substrate. The AnFAEA-amino-CLEAs were 12.7 times more thermostable at 60 °C than the AnFAEA immobilized on epoxy-activated carrier, thus AnFAEA-amino-CLEAs were selected for further characterization. Interestingly, during methyl sinapate hydrolysis (pH 7.2 and 30 °C), AnFAEA-amino-CLEAs K M was 15 % higher, while during butyl sinapate synthesis the K M was reduced in 63 %, both compared with the soluble enzyme. The direct esterification of butyl sinapate at solvent free conditions using sinapic acid 50 mM, reached 95 % conversion after 24 h employing AnFAEA-amino-CLEAs, which could be used for 10 cycles without significant activity losses, demonstrating their outstanding operational stability. [ABSTRACT FROM AUTHOR]