Recombinant Macrococcus caseolyticus Oleate hydratase (MCCL_0076) is produced in E. coli and includes an N-terminal 6xHis-tag that makes purification more straightforward. This protein spans amino acids 1-367 and comes as a partial form with purity above 85% based on SDS-PAGE analysis. The product is strictly for research purposes and should not be used in clinical or diagnostic settings.
Oleate hydratase from Macrococcus caseolyticus appears to play an important role in fatty acid metabolism, converting oleic acid into hydroxy fatty acids. Scientists have shown considerable interest in these enzymes, particularly for studying metabolic pathways that involve lipid modification and breakdown. Research into this hydratase's function and activity may help advance our understanding of microbial biochemistry and could potentially benefit industrial processes that transform fatty acids.
Potential Applications
Note: The applications listed below are based on what we know about this protein's biological functions, published research, and experience from experts in the field. However, we haven't fully tested all of these applications ourselves yet. We'd recommend running some preliminary tests first to make sure they work for your specific research goals.
Oleate hydratases from Macrococcus caseolyticus are strictly FAD-dependent enzymes. Catalytic activity is completely abolished without FAD, and specific residues (e.g., G29, G31, E56) are essential for FAD binding. While E. coli can express bacterial proteins, the formation of soluble, active enzyme is not guaranteed. Overexpression often leads to insoluble inclusion bodies or misfolded proteins, especially for enzymes requiring specific cofactors like FAD. The protein is expressed as a partial fragment (1-367aa). Although this may cover the functional core, the absence of the full-length sequence could impact proper folding or oligomerization. The N-terminal His-tag might also sterically interfere with the enzyme's active site or FAD-binding pocket.
Given the absolute requirement for FAD and the lack of validation data, this specific recombinant protein batch is highly likely to be incorrectly folded and lack bioactivity. It cannot be assumed to be functional for applications requiring enzymatic activity.
This recombinant protein can be used as an immunogen for antibody development. Antibodies often target linear epitopes, which may be present regardless of the protein's tertiary structure or FAD-binding status . The high purity (>85%) reduces the risk of generating antibodies against contaminants. However, antibodies produced against this potentially misfolded antigen might not recognize the native, FAD-bound, enzymatically active oleate hydratase in its physiological context, limiting their utility for techniques like immunoprecipitation or cellular localization that depend on native conformation.
Given the high probability that the protein is inactive due to the lack of confirmed FAD incorporation, the immediate priority is functional validation. First, perform a bioactivity assay to test for enzymatic activity using oleic acid as a substrate and monitor for the production of 10-hydroxystearic acid, ideally by GC-MS or HPLC. This assay must be conducted in the presence of FAD. Simultaneously, use biophysical methods like size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to assess oligomeric state (the active enzyme is typically a dimer ), and circular dichroism (CD) spectroscopy to analyze secondary structure. If the protein shows no activity, it should be considered misfolded/inactive. In this case, its use should be restricted to applications like linear-epitope antibody production, with all limitations clearly disclosed. If activity is confirmed after adding FAD, it may be suitable for limited biochemical characterization, but the potential impact of the partial sequence and N-terminal tag on full functionality should still be acknowledged.
