Scalable Workflow for Green Manufacturing: Discovery of Bacterial Lipases for Biodiesel Production

Lipases are a group of enzymes capable of catalyzing the hydrolysis of triacylglycerides into free fatty acids during lipid metabolism. In the presence of short-chain alcohols, some of these enzymes can catalyze the transesterification of plant oils into biodiesel. Biodiesel has recently gained traction to be a green alternative to fossil fuel. Efficient production of biodiesel at an industrial scale is limited by the reusability of the enzyme and its tolerance toward high concentrations of organic solvents. We describe a scalable workflow that integrates web-based tools and automation to identify a group of 114 orthologous bacterial lipases for expression, purification, and characterization using a high-throughput platform at our biofoundry. The activity profile of these enzymes revealed many targets with different substrate specificities and an optimal pH range. Most of these enzymes are thermostable and can tolerate up to 40% methanol (v/v). Among the 114 lipases that were targeted, 22 were found to be able to produce methyl oleate from triolein in the presence of methanol. Our study demonstrates the utility of the workflow to identify lipase candidates for the industrial production of biodiesel.