https://doi.org/10.17113/ftb.63.03.25.8940

Article in press

Evaluating Pre-Immobilization and Post-Immobilization Bioimprinting Strategies for the Activation of Lipases: A Case Study of LipC12

Leonardo Pellizzari Wielewski¹, Maria Lujan Ferreira^2,3, Robson Carlos Alnoch⁴, David Alexander Mitchell^1,5 and Nadia Krieger^1,6*

¹Postgraduate Program in Science - Biochemistry, Federal University of Paraná, P.O. Box 19046, Polytechnic Center, Curitiba 81531-980, Paraná, Brazil

²Department of Chemistry, National University of the South (UNS), Avda Alem 1253, Bahía Blanca 8000, Argentina

³Pilot Plant of Chemical Engineering - PLAPIQUI (UNS-CONICET), Bahía Blanca 8000, Argentina

⁴Department of Biology, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, 14040-901, Brazil

⁵Department of Biochemistry and Molecular Biology, Federal University of Paraná, P.O. Box 19046, Polytechnic Center, Curitiba 81531-980, Paraná, Brazil

⁶Department of Chemistry, Federal University of Paraná, P.O. Box 19032, Polytechnic Center, Curitiba 81531-980, Paraná, Brazil

Copyright © 2024 This is a Diamond Open Access article published under CC-BY licence. Copyright remains with the authors, who grant third parties the unrestricted right to use, copy, distribute and reproduce the article as long as the original author(s) and source are acknowledged.

Article history:

Received: 6 November 2024

Accepted: 21 May 2025

Keywords:

bioimprinting; lipases; immobilization; activation; enhancement of enzymatic properties

Summary:

Research background. Although there are many studies of the bioimprinting of lipases, there is no study comparing the strategies of bioimprinting prior to immobilization (pre-immobilization) and bioimprinting after immobilization (post-immobilization). Likewise, there is no study that compares bioimprinting of lipases immobilized from a pure lipase preparation and lipases immobilized from a crude extract. We therefore investigated these strategies, using the metagenomic lipase LipC12.

Experimental approach. We immobilized LipC12 covalently on the commercial support Immobead 150P and treated it with various bioimprinting agents, either pre-immobilization or post-immobilization. We also compared immobilization from a pure LipC12 preparation and immobilization from a crude cell-free extract.

Results and conclusions. The best improvements in triolein-hydrolyzing-activity in n-hexane, compared to a non-bioimprinted control, were obtained with post-immobilization bioimprinting, using oleic acid dissolved in t-butanol: a 12-fold improvement for immobilization from a pure LipC12 preparation and an almost 14-fold improvement for immobilization from the crude cell-free extract. This bioimprinting agent also gave a 3.5-fold increase in activity for the synthesis of ethyl oleate in n-hexane, this result being obtained for pre-immobilization bioimprinting and immobilization from the cell-free extract.

Novelty and scientific contribution. This study is the first to compare pre-immobilization and post-immobilization bioimprinting strategies, as well as bioimprinting of enzymes immobilized from both pure enzyme preparations and crude cell-free extracts. These results encourage further investigation into bioimprinting strategies.