# Enzymatic Degumming of Soybean Oil for Raw Material Preparation in BioFuel Production
## 4 Discussion  
In this work, we focused on evaluating the effectiveness of the Purifine(r) 3G enzyme complex in the degumming of crude soybean oil, as well as determining the optimal process conditions, taking into account the enzyme dose, temperature, pH, reaction time, and the amount of water. The use of the Purifine(r) 3G enzyme complex, which includes phospholipases C and A2, provides a modern approach to the purification of soybean oil. This method allows reducing the phosphorus content to acceptable limits while maintaining a high yield of neutral oil. Purifine(r) 3G hydrolyzes all four phospholipids in crude oil, namely PC, PE, PI, and phosphatidic acid (Figure 2). Enzymatic degumming of soybean oil using the Purifine(r) 3G enzyme complex represents a modern approach that effectively reduces phospholipid content through the synergistic action of two key enzymes: PL-C and PL-A2. These enzymes perform specific functions aimed at the hydrolysis of phospholipids, which allows reducing the phosphorus content to the level required for biofuel production. PL-C is the first component in the Purifine(r) 3G pathway. It catalyzes the cleavage of the phosphodieser bond between the glycerol and phosphate groups in phospholipid molecules (Figure 2). This process leads to the conversion of phospholipids into diglycerides (DAG) and water-soluble phosphate residues (Figure 3a). The main substrates for PL-C are PI and PC, which constitute a significant proportion of phospholipids in crude soybean oil. During the reaction, the water-soluble phosphate residues migrate into the humic phase along with water, while the lipophilic diglycerides remain in the oil phase, increasing its yield (Figure 3a). This step results in a significant reduction in phosphorus, which is critical for further use of the oil. Figure 2: Phospholipases mode of action. A generalized depiction of a phospholipid, where X = H, choline, ethanolamine, inositol, etc. The various sites of attack for hydrolytic cleavage of ester bonds of the phospholipase types A2 and C are shown with arrows. The second step is the action of PL-A2, which cleaves the ester bond of glycerophospholipid molecules (Figure 2). This process results in the formation of lysophospholipids and FFA (Figure 3b). The main substrates for PL-A2 are PC and PE, which are also important components of the phospholipid composition of crude oil. The formed lysophospholipids have high surface activity and remain in the humic phase, facilitating the removal of residual phospholipids (Figure 3b). Free fatty acids are also removed during subsequent processing steps. The combined action of PL-C and PL-A2 allows for the effective hydrolyzation of phospholipids to water-soluble and lipophilic components. This provides a significant reduction in phosphorus content to <10 ppm, which meets international standards for biofuel feedstocks. It contributes to an increase in the yield of neutral oil due to the formation of diglycerides that remain in the product. It ensures minimization of oil losses due to the reduction of the formation of emulsions and humic phase and the environmental friendliness of the process due to the reduction of wastewater volumes and the reduction of the use of chemical reagents. The results of the study confirmed the high efficiency of using the Purifine(r) 3G enzyme complex for degumming soybean oil, which allows for significantly reducing the phosphorus content and ensuring the content of FFA to levels that meet industrial standards in the preparation of raw materials for biofuel production. Optimization of process parameters ensures cost-effectiveness and environmental friendliness of the technology, making it competitive in comparison with traditional degumming methods. The results of the experiments showed that the optimal dose of the enzyme is 100 ppm. At the same time, the phosphorus level decreased to 9.5 ppm, and FFA was 0.85 mg KOH/g oil.