Improving phospholipase activity of PLA(1) by protein engineering and its effects on oil degumming

卷:119

An, Qun ; Wang, Fanghua ; Lan, Dongming ; Khan, Faez Iqbal ; Durrani, Rabia ; Yang, Bo ; Wang, Yonghua

South China Univ Technol

关键词: Lid domain;    Phospholipase activity;    PLA(1);    Protein engineering;    Oil degumming;   

DOI  :  10.1002/ejlt.201600110

学科分类：食品科学和技术

【 摘 要 】

Enzymatic activity is important characteristic of enzymes for industrial application, which can be improved by protein engineering. PLA(1) 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 degrees C and the k(cat)/K-M was improved. Degumming soybean oil, three of five mutants, R81A, R84M, and E87Q, decreased phosphorous content lower than 8.3mg/kg within 3h, which was highly improved compared with wild-type. R84M decrease phosphorus content less than 5mg/kg within 5h. 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 PLA(1) 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, PLA(1) can be applied in oil degumming. PLA(1) with higher phospholipase activity is more likely to suitable for oil degumming since it could decrease the phosphorous content of oil lower than 10mg/kg within shorter reaction, which showed great potencial for degumming application.