Lipase B from Candida antarctica (CAL-B) is largely employed as a biocatalyst for hydrolysis, esterification, and transesterification reactions. CAL-B is a good model enzyme to study factors affecting the enzymatic structure, activity and/or stability after an immobilization process. In this study, we analyzed the immobilization of CAL-B enzyme on different magnetic nanoparticles, synthesized by the coprecipitation method inside inverse micelles made of zwitterionic surfactants, with distinct carbon chain length: 4 (ImS4), 10 (ImS10) and 18 (ImS18) carbons. Magnetic nanoparticles ImS4 and ImS10 were shown to cross-link to CAL-B enzyme via a Michael-type addition, whereas particles with ImS18 were bond via pyridine formation after glutaraldehyde cross-coupling. Interestingly, the Michael-type cross-linking generated less stable immobilized CAL-B, revealing the influence of a cross-linking mode on the resulting biocatalyst behavior. Curiously, a direct correlation between nanoparticle agglomerate sizes and CAL-B enzyme reuse stability was observed. Moreover, free CAL-B enzyme was not able to catalyze transesterification due to the high methanol concentration; however, the immobilized CAL-B enzyme reached yields from 79.7 to 90% at the same conditions. In addition, the transesterification of lipids isolated from oleaginous yeasts achieved 89% yield, which confirmed the potential of immobilized CAL-B enzyme in microbial production of biodiesel.

PlsX is the first enzyme in the pathway that produces phosphatidic acid in Gram-positive bacteria. It makes acylphosphate from acyl-acyl carrier protein (acyl-ACP) and is also involved in coordinating phospholipid and fatty acid biosyntheses. PlsX is a peripheral membrane enzyme in Bacillus subtilis, but how it associates with the membrane remains largely unknown. In the present study, using fluorescence microscopy, liposome sedimentation, differential scanning calorimetry, and acyltransferase assays, we determined that PlsX binds directly to lipid bilayers and identified its membrane anchoring moiety, consisting of a hydrophobic loop located at the tip of two amphipathic dimerization helices. To establish the role of the membrane association of PlsX in acylphosphate synthesis and in the flux through the phosphatidic acid pathway, we then created mutations and gene fusions that prevent PlsX's interaction with the membrane. Interestingly, phospholipid synthesis was severely hampered in cells in which PlsX was detached from the membrane, and results from metabolic labeling indicated that these cells accumulated free fatty acids. Because the same mutations did not affect PlsX transacylase activity, we conclude that membrane association is required for the proper delivery of PlsX's product to PlsY, the next enzyme in the phosphatidic acid pathway. We conclude that PlsX plays a dual role in phospholipid synthesis, acting both as a catalyst and as a chaperone protein that mediates substrate channeling into the pathway.