The increasing demand pressures the vegetable oil industry to develop novel refining methods. Degumming with type C phospholipases (PLCs) is a green technology and provides extra oil. However, natural PLCs are not active under the harsh conditions used in oil refining plants, requiring additional unit operations. These upfront capital expenditures and the associated operational costs hinder the adoption of this method. Here, we present a process based on ChPLC, a synthetic PLC obtained by consensus sequence design, possessing superior thermal stability and catalytic properties. Using ChPLC, crude soybean oil degumming was completed at 80 °C in 30 min, the temperature and residence time imposed by the design of existing oil refining plants. Remarkably, an extra yield of oil of 2% was obtained using 60% of the dose recommended for PLCs marketed today, saving upfront investments and reducing the operational cost of degumming. A techno-economic analysis indicates that, for medium size plants, ChPLC reduces the overall cost of soybean oil enzymatic degumming by 58%. The process presented here facilitates the implementation of enzymatic technologies to oil producers, regardless of their processing capacity, bringing potential annual benefits in the billion-dollar range for the global economy.

No abstract

Proteins' extraordinary performance in recognition and catalysis have led their use in a range of applications. But proteins obtained from natural sources are oftentimes not suitable for direct use in industrial or diagnostic setups. Natural proteins, evolved to optimally perform a task in physiological conditions, usually lack the stability required to be used in harsher conditions. Therefore, the alteration of the stability of proteins is commonly pursued in protein engineering studies. Here we achieved a substantial thermal stabilization of a bacterial Zn(II) dependent phospholipase C by consensus sequence design. We retrieved and analyzed sequenced homologs from different sources, selecting a subset of examples for expression and characterization. A non-natural consensus sequence showed the highest stability and activity among those tested. Comparison of activity and stability parameters between this stabilized mutant with other natural variants bearing similar mutations allow us to pinpoint the sites most likely to be responsible for the enhancement. We show that the stabilized version of the protein retains full activity even in the harsh oil degumming conditions.

Enzymes are extraordinary catalysts and a preferred tool for green chemistry, but they have not evolved to tolerate the harsh conditions required in large-scale chemical processes, making the replacement of inorganic catalysts extremely expensive in most cases. However, the continuous knowledge generated by protein engineers helps to realize the promise of using biocatalysts to address critical environmental and industrial challenges. The increasing demand for food and biofuels pressures the vegetable oil industry to continuously improve its manufacturing efficiency and at the same time mitigate the environmental pollution caused by the refineries. Enzymatic degumming is a key enabling technology to obtain cleaner and more efficient methods, but advanced enzymes are needed for widespread adoption. Here, we present an enzymatic degumming process based on chPLC, a synthetic phospholipase C with remarkable thermal stability and catalytic properties. The process developed does not require modifications in current soybean oil factories, avoiding upfront investments, and is four times faster than the enzymatic processes available in the market. A techno-economic analysis predicts a ~ 60% cost reduction for a base-case scenario, establishing the largely expected conditions to promote the global adoption of enzymatic oil degumming by refineries of all sizes. To the best of our knowledge, this is the first report showing the application of a synthetic enzyme designed to address an industrial need having an impact on both the global economy and the environment, in the billion-dollar range.