Improving phospholipase activity of PLA₁ by protein engineering and its effects on oil degumming

Abstract

Enzymatic activity is important characteristic of enzymes for industrial application, which can be improved by protein engineering. PLA1 is a biocatalyst applied in phospholipid modification for its phospholipase activity and had obtained board attention for its application in oil degumming. Bioinformatics analysis suggested that three charged residues, R81, R84, and E87 located in the lid of the protein, may affect the conformational change of lid thus influence the activity of the enzyme. In the current study, mutagenesis of these residues was conducted with protein engineering. Five mutants such as R81A, R84A, R84M, R84K, and E87Q with higher phospholipase activity were screened out. Biochemical properties analysis showed that all of them had identical optimal pH value with wild‐type, while the optimal temperature was decreased to be 50°C and the kcat/KM was improved. Degumming soybean oil, three of five mutants, R81A, R84M, and E87Q, decreased phosphorous content lower than 8.3 mg/kg within 3 h, which was highly improved compared with wild‐type. R84M decrease phosphorus content less than 5 mg/kg within 5 h. These findings not only permit optimization of enzyme performance in degumming but also shed light on the application of bioinformatics techniques and protein engineering techniques on industry application. Practical applications: The results support that design proteins according to bioinformatics and protein engineering is desirable and the phospholipase with higher activity is more suitable for oil degumming. The phospholipase activity of PLA1 can be improved by protein engineering based on bioinformatics analysis. With its properties of hydrolyzing sn‐1 position ester bond of phospholipids, nonhydratable phospholipids were converted into their hydratable forms, PLA1 can be applied in oil degumming. PLA1 with higher phospholipase activity is more likely to suitable for oil degumming since it could decrease the phosphorous content of oil lower than 10 mg/kg within shorter reaction, which showed great potencial for degumming application.

Effective Expression of the Serratia marcescens Phospholipase A1 Gene in Escherichia coli BL21(DE3), Enzyme Characterization, and Crude Rapeseed Oil Degumming via a Free Enzyme Approach

“…The conditions of pH 7.5 and 50°C were, respectively, maintained to investigate the effect of temperature and pH on enzyme activity. The enzymatic characteristics of OM-PLA1 in terms of optimum temperature, optimum pH, temperature stability, pH tolerance, and kinetic parameters were determined by measuring enzymatic activities under different conditions (An et al, 2017). The Arrhenius plot method was used to analyze the effect of temperature on the stabilization rate of OM-PLA1 by drawing the relationship between the initial rate of enzymatic hydrolysis and reaction temperature (Fan et al, 2000).…”

Latest advances in degumming feedstock oils for large‐scale biodiesel production

“…As mentioned earlier, the PL enzyme degumming reaction takes place at the interface area of the aqueous phase containing enzyme and oil phases, which means that the reaction is very slow. By increasing the interfacial area the rate of this reaction can be increased. The kinetics of this reaction have also been studied taking interfacial area into account.…”