【Poster Presentation】Exploration of Cardinal Lipases and Their Producing Bacteria from Anaerobic Digester Sludge through Functional Metaproteomics

Anaerobic digestion offers fundamental benefits by converting various organic wastes into biogas.Lipids are commonly found in wastewater from sources such as food processing plants, slaughterhouses, and edible oil production facilities and are considered fundamental energy resources.However, the microorganisms responsible for lipid hydrolysis remain largely unknown.In this study, we aimed to identify the key lipase and its secreting microorganisms in anaerobic digestion sludge through functional metaproteomic approach.We performed 2D-gel zymography followed by two-dimensional polyacrylamide gel electrophoresis, which led to the identification of four protein spots exhibiting lipase activity.These spots were excised from the gel and analyzed using nanoLC-MS/MS to determine the partial amino acid sequences of the lipases.To facilitate protein identification, we constructed a custom metagenomic database through metagenomic analysis using a PacBio Sequel IIe and DNBSEQ sequencer.Using this database, we successfully identified six hypothetical proteins as potential lipase candidates.To assess their lipase activity, we cloned the corresponding genes into Escherichia coli for recombinant protein expression.One of these hypothetical proteins was successfully expressed, and its lipase activity was confirmed.The DNA sequence of this lipase was found within a metagenome-assembled genome classified as belonging to the Oscillospiraceae family, implying that unfamiliar bacterium involved in lipipolysis in anaerobic digestion process.We hope the presented approach will facilitate the understanding of lipid hydrolysis in anaerobic digestion, contributing to the optimization of this bioprocess.