菌株Bacillus subtilis GW-01跨膜运输β-氯氰菊酯的特性研究

The degradation of β-cypermethrin (β-CY) by microorganisms has been regarded as a potential eco-friendly approach because it is safe and applicable. Nonetheless, the microorganisms could not efficiently degrade β-CY in food production due to they could not effectively touched with the pesticide, which was adsorbed on particle surface of food. Our previous researches indicated that some surfactants could improve the degradation through increasing the touch between β-CY and microbial cells and extracellular degrading enzyme. When the microorganisms degraded β-CY through intracellular enzyme, β-CY would touch the degrading enzyme only after it transported across the selective cell membrane. However, there is no research on trans-membrane transport of contaminants such as β-CY residues in food by microorganisms. Therefore, we tested the hypothesis that, based on studying the mechanism and regulations of trans-membrane transport of β-CY by microorganism, the pesticide residues in food would be efficiently degraded by the intracellular enzyme. In this research, strain Bacillus subtilis GW-01 as the study subject, the mechanism and regularities of trans-membrane transport of β-CY would be studied; further, nutritional and environmental factors would be used to regulate β-CY degradation. The objectives of this study were to establish the theoretical basis for mechanism of trans-membrane transport of hydrophobic organic compounds, and to provide an effective pathway for efficient degradation of pyrethroid pesticide in food.

微生物降解β-氯氰菊酯（β-CY）是一种潜在的环境友好型方法，其具有较好的安全性和适用性。但是，由于β-CY通常会吸附于食物颗粒表面，使其与降解酶难以有效接触，导致微生物在食品生产中很难实现高效降解。我们前期研究发现，一些表面活性剂能增加β-CY与微生物细胞和胞外降解酶的接触，促进降解。当降解酶位于细胞内时，β-CY需跨越具有高度选择性的细胞膜，才能与降解酶接触。然而，目前没有微生物跨膜运输β-CY等食品污染物的相关研究。因此，提出“研究微生物跨膜运输β-CY的机制和调控，能促进微生物通过胞内酶降解食品中残留的β-CY”的假说。本研究以Bacillus subtilis GW-01为对象，研究菌株GW-01跨膜运输β-CY的规律和机制，并对其降解β-CY进行营养和环境调控，为微生物跨膜运输疏水性有机污染物的机制研究奠定了理论基础，为微生物降解消除食品中拟除虫菊酯类农药残留提供了有效途径。

食品中残留的化学农药是威胁人体健康的重要因素。β-氯氰菊酯（β-CY）是一种广泛使用的拟除虫菊酯类农药，具有环保、安全和适用性好等特点的微生物降解法被广泛用于农残消除。然而，当降解β-CY的酶位于细胞内时，微生物跨膜运输β-CY的机制成为制约农药降解的关键因素。此外，微生物彻底降解β-CY的途径及降解酶编码基因、可降解农药微生物的在食品中应用的安全性及其在体内消除β-CY残留的可行性和机制也是限制微生物在食品领域消除农残的瓶颈性问题。本项目研究结果表明：（1）通过测定蜡样芽胞杆菌（Bacillus cereus）GW-01降解β-CY时生物量、氧化应激相关酶、细胞表面疏水性、生物膜、细胞膜脂肪酸和细胞膜完整性和通透性，并利用转录组和蛋白组分析了其在分子层面的变化。结果表明，β-CY通过诱发氧化应激反应对菌株GW-01造成毒害，抑制菌株GW-01生长、细胞膜脂肪酸代谢、生物膜合成、细胞表面疏水性增强等，导致细胞膜通透性增强，促进β-CY进入细胞；同时，β-CY还导致细胞膜上ABC转运蛋白及其编码基因发生显著变化，但转运β-CY的编码基因需要进一步验证。添加茶多酚等抗氧化剂能显著促进菌株GW-01降解β-CY。（2）利用GC-MS、HPLC、转录组、蛋白组和异源表达结果表明，菌株GW-01通过α/β-水解酶、细胞色素aa3喹啉氧化酶、氯水解酶、苯甲酸降解酶和儿茶酚-2,3-双加氧化酶分别降解β-CY及其中间代谢产物3-苯氧基苯甲酸、苯酚、苯甲酸和儿茶酚。（3）对菌株GW-01进行全基因组测序，利用比较基因组分析和小鼠实验对其安全性进行分析结果表明，菌株GW-01与致病性B. cereus中合成毒素的相关基因有差异，灌胃2.0×109 CFU/g菌株GW-01，小鼠体重和脏器指数未发生明显变化。（4）制备菌株GW-01口服菌剂，研究其安全性及其消除体内β-CY的效果与机制。结果表明，菌株GW-01可明显降低小鼠体内β-CY的含量和减轻β-CY对小鼠的伤害，菌株GW-01可在胃肠模拟液中吸附和降解β-CY。本项目的实施为消除食品中拟除虫菊酯类农药提供了分子基础，为可降解农药细菌食品领域应用的安全性分析建立了方法，为口服菌剂消除和减轻体内农残与伤害提供了参考。