Integrating lignocellulosic liquor valorization and enzymatic biodiesel production through Eversa Transform 2.0 immobilization

Enzymatic biodiesel production is a promising route for obtaining fatty acid esters under mild reaction conditions and with high selectivity. In this work, the catalytic performance of Eversa Transform 2.0 was evaluated in both free and immobilized forms to produce fatty acid ethyl esters from soybean oil. The main novelty of the proposed system lies in the immobilization of the enzyme in calcium alginate containing, within its matrix, the liquor obtained from the pretreatment of sugarcane straw, a stream rich in ionic liquid and lignin. Thus, besides acting as a structural and functional component of the immobilization matrix, the liquor is redirected to a new biotechnological application, adding value to a by-product from the lignocellulosic biomass processing chain. The reactions were carried out using an ethanol: oil molar ratio of 4.48:1, 3.41% of water and were standardized based on 1 mg of protein, either from the free or immobilized enzyme. Reactions were conducted for 24, 36, and 48 h, and the lipid fraction composition was evaluated based on the relative mass percentages of TAG, DAG, MAG, FFA, and FAEE. For the free enzyme, high FAEE formation was observed, reaching 81.4%, 72.7%, and 93.3% at 24, 36, and 48 h, respectively, indicating advanced glyceride conversion into esters. The immobilized enzyme also promoted FAEE formation, reaching 25.1%, 18.1%, and 40.1% at 24, 36, and 48 h, respectively. The relative catalytic performance of the immobilized enzyme, calculated from FAEE production compared with the free enzyme after 48 h, was approximately 43%. Overall, the results demonstrate the potential of the developed immobilization system, which combines biocatalyst recovery with the reuse of a biomass pretreatment stream, integrating biocatalysis, by-product valorization, and lignocellulosic biomass utilization for more sustainable enzymatic biodiesel production.