Abstract

Enzyme-mediated peptide synthesis is a promising alternative for current industrial requirements, mainly due to its potential for increasing process sustainability and efficiency. In this work, peptide synthesis using lipases was studied, and the peptide synthesis activity of a previously reported Bacillus subtilis lipase A (BSLA) variant, P5F3_BSLA (F41L-?W42-?D43-R44P) was evaluated. P5F3_BSLA was immobilized on a hydrophilic mesoporous silica support functionalized with glyoxyl groups. A rapid and direct immobilization approach was used from cell culture supernatant in a single vessel without prior protein purification. The effect of immobilization on the performance of P5F3_BSLA regarding substrate specificity, peptide synthesis, and thermal and solvent resistance was evaluated and compared to the WT_BSLA enzyme. The variant showed a similar immobilization yield compared to Wt_BSLA; however, it showed different immobilization kinetics. The P5F3 variant showed an increased solvent resistance in 100 % isopropyl alcohol and an increased activity against long-chain fatty acid substrates. Besides, the P5F3_BSLA variant showed a 1.2-fold increase in glycine polymerization compared to WT_BSLA. The simple, efficient, and successful immobilization of this lipase, its resistance to solvents, and its potential use in peptide synthesis opens an exciting possibility of applications for the industry, highlighting the combination of protein engineering and enzyme immobilization tools to develop biocatalysts with novel properties from widely studied enzymes. © 2025 Elsevier Ltd