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

Abstract

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.