Advanced upcycling of spent hen lipids using engineered-enzymatic cascade

Abstract

A novel enzymatic epoxidation process has been carried out for the first time, utilizing lipid and fatty acid methyl esters (FAMEs) obtained from spent hens and olives. In this process, an engineered enzymatic cascade was designed to facilitate epoxidation by leveraging the catalytic activity of galactose oxidase and lipase B. The primary objective of the study was to develop a reusable enzyme cascade for the epoxidation of lipids extracted from spent hens employing microwave technology. First of all, the Strep-tag II-galactose oxidase-macaque histatin (StrepII-GalOx-MH) complex was successfully bound to commercial Immbodead 150 particles with pre-immobilized lipase B. Furthermore, our study demonstrated that the StrepII-GalOx-MH was stable in a two-phase system consisting of sodium-phosphate buffer (100 mM, pH 7) and toluene in a 1:2 mixture. Thus, this complex showed 10 % less activity after 72 h of incubation compared to the control incubated in sodium phosphate buffer. The structural analysis of the extracted lipids and FAMEs obtained from spent hens and olives after incubation was performed using ATR-FTIR and GC-MS. The results revealed a successful epoxidation evident from the decreased wavenumbers at ∼3010 cm⁻¹(double bond CH stretch) and ∼1654 cm⁻¹ (double bond C-C stretch), while an increase in the two oxirane regions at ∼1497-1432 cm⁻¹ (oxirane C-C stretching), and 862-762 cm⁻¹ (oxirane C-O-C stretching). After 48 h of incubation, the average conversion of all spent hen FAMEs in the Erlenmeyer flask was 77.0 ± 3.5 % and 59.1 ± 2.2 % when the glass vessel was used. Finally, we demonstrated the reusability and functionality of the enzyme cascade for another 24 h. The successful development of an alternative, biobased approach to produce epoxides from underutilized waste highlights the promising prospect of biotechnology to achieve a circular economy.