鼠李糖乳杆菌碱性磷酸酶的提取条件优化及其降解有机磷农药作用

Optimization of the extraction of alkaline phosphatase from Lactobacillus rhamnosus and the mechanism underlying the degradation of organophosphorus pesticides

摘要:

【背景】我国作为农业大国，对农药的大量使用是不可避免的，但是农药的超范围使用、超标及高检出率对于环境的污染与人体健康的威胁日趋严重。【目的】碱性磷酸酶(alkaline phosphatase，ALP)对有机磷农药具有积极的降解作用，因此，本文对鼠李糖乳杆菌(Lactobacillus rhamnosus) Z23(LGG Z23)所产碱性磷酸酶的提取条件进行优化，并研究其对有机磷农药的降解作用。【方法】使用单因素试验和正交试验优化ALP的提取条件；使用对硝基苯酚法测定酶活力；使用分级沉淀和层析法提纯ALP；使用乙酰胆碱酯酶抑制法测定ALP对有机磷农药的降解率。【结果】LGG Z23所产ALP的最优提取条件为：细胞破碎时间15 min，破碎功率450 W，料液比(质量体积比)1:6，提取液pH 10.0，此条件下酶活力为(4.95±0.26) U/mL，比优化前提高2.11倍；对6种有机磷农药的降解率效果为敌敌畏(95.79%±0.01%)>甲基对硫磷(90.69%±0.03%)>毒死蜱(88.90%±0.02%)>敌百虫(86.07%±0.03%)>马拉硫磷(85.31%±0.02%)>乐果(83.18%±0.03%)，其中对敌敌畏和甲基对硫磷的降解效果最好，可达90%以上，并且降解作用差异显著(P<0.05)。【结论】本研究为LGG Z23所产ALP的应用研究提供了理论依据和实验数据。

关键词:鼠李糖乳杆菌;碱性磷酸酶;提取条件;有机磷农药;降解率

Abstract:

[Background] China, one of the world's largest agricultural economy, has seen the large-scale use of pesticides. However, the overuse of pesticides, which has led to high detection rate, has threatened the environment and human health.[Objective] Alkaline phosphatase (ALP) can degrade organophosphorus pesticides. Therefore, we optimized the extraction of ALP from Lactobacillus rhamnosus Z23 (LGG Z23) and explored the mechanism underlying the degradation of organophosphorus pesticides. [Methods] Single factor test and orthogonal test were used to optimize the extraction. The enzyme activity was determined by detecting the amount of p-nitrophenol released. Fractional precipitation and chromatography were employed for the purification of ALP. The degradation rate of organophosphorus pesticides was determined based on the inhibition of acetylcholinesterase. [Results] The optimum conditions for extracting ALP from LGG Z23 were as follows:disruption of cells for 15 min at 450 W, material-liquid ratio (mass to volume ratio) of 1:6, and pH 10.0. Under the conditions, the activity of ALP was (4.95±0.26) U/mL, 2.11 times higher than that before optimization. The degradation rate of 6 organophosphorus pesticides was in the order of DDVP (95.79%±0.01%)>methyl parathion (90.69%±0.03%)>chlorpyrifos (88.90%±0.02%)>trichlorfon (86.07%±0.03%)>malathion (85.31%±0.02%)>dimethoate (83.18%±0.03%). Among them, the activity of degrading DDVP and methyl parathion was the highest (over 90%), and the difference was significant (P<0.05). [Conclusion] The result lays a theoretical basis and provides data for the application of ALP from LGG Z23.

Key words:Lactobacillus rhamnosus;alkaline phosphatase;extraction conditions;organophosphorus pesticides;degradation rate