植物乳杆菌植酸酶理性设计及表达鉴定

Rational design and expression identification of phytase from Lactobacillus plantarum

植酸酶能将植酸水解成肌醇衍生物,并释放出无机磷酸,是一种重要的生物催化剂。植酸酶的热稳定性和比活性是决定其潜在应用价值的2个关键因素。该研究从植物乳杆菌中克隆到了具有233个氨基酸残基、高活性的植酸酶基因(LpPHY233),并在大肠杆菌中实现了高水平的异源表达,其表达水平是植物乳杆菌的800倍。基于理性设计的结果,通过二硫键工程显著提高了Lp PHY233的温度特性和催化性能。突变体Lp PHY233S58C/K61C的最适反应温度较Lp PHY233提高了15℃。此外,它还具有良好的pH特性和动力学参数,在食品加工和饲料添加剂领域具有较大的应用前景。该研究结果为植酸酶的进一步分子修饰和工业应用提供了坚实的理论基础。

Phytase is an important biocatalyst that can dissociate phytate into inositol derivatives and release inorganic phosphoric acid.It can be used as feed additive to reduce phytate anti-nutrient factor in feed.The thermal stability and specific activity of phytase are two key factors that determine its application potential.In this study,a phytase gene with 233 amino acid residues(LpPHY 233)was cloned from Lactobacillus plantarum,and the highly heterologous expression was achieved in Escherichia coli,which was 800 times higher than that of L.plantarum.Then,based on the results of in silico design,the temperature characteristics and catalytic performance of Lp PHY233 were significantly improved by disulfide bond engineering.The optimal reaction temperature of Lp PHY233S58C/K61C was 15℃higher than that of Lp PHY233.In addition,it also has good pH characteristics and kinetic parameters,which has a broad application potential in the feed additives field.This study provides a solid theoretical basis for further molecular modification and industrial application of phytases.