Improving phospholipase activity of PLA(1) by protein engineering and its effects on oil degumming
Improving phospholipase activity of PLA(1) by protein engineering and its effects on oil degumming
复制标题
蛋白质工程提高PLA(1)磷脂酶活性及其对油脂脱胶的影响
DOI：
10.1002/ejlt.201600110
复制
发表时间：
2017
影响因子：
2.7
通讯作者：
Wang Yonghua
中科院分区：
文献类型：
--
作者：
An Qun;Wang Fanghua;Lan Dongming;Khan Faez Iqbal;Durrani Rabia;Yang Bo;Wang Yonghua
酶的活性是工业用酶的重要特性，通过蛋白质工程可以提高酶的活性。磷脂酶A1是一种具有磷脂酶活性的磷脂改性生物催化剂，在油脂脱胶中的应用受到广泛关注。生物信息学分析表明，位于该蛋白盖区的R81、R84和E87三个带电荷残基可能影响盖区的构象变化，从而影响酶的活性。在目前的研究中，这些残基的诱变与蛋白质工程进行。筛选出具有较高磷脂酶活性的R81 A、R84 A、R84 M、R84 K和E87 Q等5个突变体。生化特性分析表明，它们的最适pH值与野生型相同，但最适温度降低到50°C，kcat/Km提高。脱胶大豆油，五个突变体中的三个，R81 A，R84 M和E87 Q，在3小时内将磷含量降低到低于8.3 mg/kg，这与野生型相比有很大改善。R84 M在5 h内使土壤磷含量降低到5 mg/kg以下。这些结果不仅可以优化酶在脱胶中的性能，而且为生物信息学技术和蛋白质工程技术在工业应用中的应用提供了理论依据。实际应用：研究结果表明，根据生物信息学和蛋白质工程设计蛋白质是可行的，高活性的磷脂酶更适合油脂脱胶，基于生物信息学分析的蛋白质工程可以提高磷脂酶PLA 1的磷脂酶活性。利用其水解磷脂sn-1位酯键的特性，将非水化磷脂转化为水化磷脂，可应用于油脂脱胶。磷脂酶活性较高的PLA 1更适合油脂脱胶，在较短的反应时间内可使油脂中磷含量降至10 mg/kg以下，具有很大的应用潜力。
Enzymatic activity is important characteristic of enzymes for industrial application, which can be improved by protein engineering. PLA1is 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 thekcat/KMwas 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 PLA1can 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, PLA1can be applied in oil degumming. PLA1with 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.