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

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• 作者：Qun An ; Fanghua Wang ; Dongming Lan ; Faez Iqbal Khan ; Rabia Durrani ; Bo Yang and Yonghua Wang
• 刊名：European Journal of Lipid Science and Technology
• 出版年：2017
• 出版时间：February 2017
• 年：2017
• 卷：119
• 期：2
• 全文大小：1540K
• ISSN：1438-9312

文摘

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 k_cat/K_M 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.