重组枯草芽孢杆菌分泌表达缺陷假单胞菌磷酸三酯酶及其发酵优化

Recombinant expression and optimization of fermentation conditions of phosphotriesterase

磷酸三酯酶(phosphotriesterase, PTE, EC3.1.8.1)能够水解有机磷化合物,但其应用一直受限于酶表达量低的问题.为了获得高效表达的有机磷水解酶,本文构建了PTE基因来源于缺陷假单胞菌(Pseudomonas diminuta)的重组枯草芽孢杆菌(Bacillus subtilis WB600),并采用单因素实验和正交实验对培养基进行优化,确定了重组菌产酶的最佳发酵条件,同时检测重组酶对有机磷类化合物的降解作用.结果表明,最优的培养基组成为蔗糖(40 g/L)、酵母膏(40 g/L)、蛋白胨(20 g/L)、磷酸氢二钾(2 g/L)、硫酸锰(1 g/L)、硫酸镁(6 g/L).经测定,该酶4 h内对(5 mg/mL的甲基对硫磷、乐果以及神经毒剂模拟剂甲基磷酸二甲酯(dimethyl methyl phosphonate, DMMP)的降解率分别达到98%, 92%, 73%,且DMMP在12 h内也完全降解.本文实现了PTE的胞外分泌表达,为研制有机磷化合物的酶基消毒剂提供了技术支持.

Phosphotriesterase(PTE, EC 3.1.8.1) has been shown to hydrolyze organophosphorus compounds. However, its application has been limited by the low expression levels of enzymes. The purpose of the present study was to obtain highly expressed organophosphorus hydrolases. Recombinant Bacillus subtilis strains were developed to produce PTE from Pseudomonas diminuta. Single factor and orthogonal design experiments were used to optimize the broth. The enzymatic degradation efficiency of organophosphorus compounds with the recombinant enzyme was also determined. The results indicated that the optimal fermentation medium contained40 g/L sucrose, 40 g/L yeast extract, 20 g/L peptone, 2 g/L dipotassium hydrogen phosphate, 1 g/L manganese sulfate, and 6 g/L magnesium sulfate. As determined, the enzymatic degradation rate of parathion-methyl, dimethoate, and a nerve agent analogue(dimethyl methyl phosphonate, DMMP)(5 mg/mL) were 98%, 92%, and 73% under 4 h of biodegradation, respectively. In addition,the residual DMMP was completely degraded in 12 h. In the present study, we achieved the extracellular secretion of PTE for the first time, which lays a foundation for the development of bio-enzyme-based decontaminants for organophosphorus compounds.