食用菌菌渣修复有机氯农药污染土壤的酶学机制研究

Organochlorine pesticides (OCP) are persistent organic pollutants that are widely present in the soil and have risks on the environment and human health. Mushroom industries generate sufficient co-products called spent mushroom substrates (SMS). After use, these substrates that contain rich mushroom mycelium and other enzymes are generally considered as wastes. With the increasing production of mushrooms, the amount of SMS generated annually is been increasing. SMS containing active enzymes has great potential for biological treatments of the soils contaminated by OCP and offers an attractive technology. SMS is a type of promising bioremediation materials. It contains a number of microorganisms and enzymes and the compositions are relatively complex. The ability of SMS degrading organochlorine pesticides may be affected by various factors. The enzymatic mechanisms on soil remediation are not clear. Our previous research had confirmed that the laccase from edible fungi had demonstrated the ability to degrade pesticide residues. This protocol aims to select the SMS as a novel type of biodegradation materials to remediate the OCP polluted soils. SMS will be collected from selected substrates, including Lentinula edodes, Pleurotus ostreatus, Pleurotus eryngii, Agaricus bisporus, Flammulina velutipes and Volvariella volvacea. The contents of nutrients and organic matters will be analyzed. Then the activities and stabilities of key enzymes, such as laccases and manganese peroxidase, will be investigated. OCP pesticides, hexachloro-cyclohexane soprocide, and DDT will be selected as targeted pesticides, and the degradation capacities of the enzymes obtained from SMS before and after purification will be studied, and the degradation products will be identified. The process of the OCP adsorption and degradation in the SMS under simple/complex system conditions will be monitored. The key factors for remediation of soils polluted by OCP using the key enzymes will be explored and verified. The roles of key enzymes in SMS used for cleaning OCP contaminated soils will be revealed.

有机氯农药是一种广泛存在于土壤中的持久性有机污染物，具有潜在的环境健康风险。食用菌菌渣来源充足、价格低廉，可以有效降低污染土壤中的有机氯农药的含量，是极具应用前景的生物修复剂，但对有机氯农药的降解可能具有多方面的效应，其修复土壤的酶学机制尚不明确。本项目在前期食用菌漆酶能有效降解农药研究基础上，拟开展采用食用菌菌渣修复土壤有机氯农药污染研究。选择生产量大的香菇、糙皮侧耳、刺芹侧耳、双孢蘑菇、金针菇、草菇等的食用菌菌渣，分析其养分及有机质含量等性质，比较不同菌渣漆酶、锰过氧化物酶等主要酶系活性，对酶中具有降解功能基因进行鉴定与表达；以六六六、DDT等有机氯农药为研究对象，考察从菌渣中分离出的粗酶纯化前后降解有机氯农药的效果并鉴定降解产物；研究单纯/复杂体系条件下菌渣吸附和降解有机氯农药的过程。从而探明菌渣酶系修复有机氯农药污染土壤关键因子，揭示菌渣修复农田土壤有机氯农药污染中酶的作用机制。

随着食用菌产业的不断扩大，菌渣总量增长迅速，但利用率还相对较低。有机氯农药是一类高残留、难降解的农药，虽已禁止使用，但在多地土壤中仍可检测到，其污染农田，也使人类健康受到威胁。前期研究发现，食用菌菌渣对有机氯农药具有降解作用，基于此，本项目主要开展了以下方面的研究，研究结果可为食用菌菌渣作为环境生物修复材料的利用提供科学依据。.（一）选取了草菇、海鲜菇、金针菇、双孢菇、秀珍菇、杏鲍菇、平菇7种食用菌的菌渣为研究对象，对其理论性质和营养成分进行了分析。结果显示七种菌渣pH范围在5.34~8.89之间，草菇最高，杏鲍菇最低；可溶性蛋白含量在40.3~91.1mg/g之间，海鲜菇最高，金针菇最低；还原糖含量为0.05~1.12mg/ml之间，杏鲍菇最高，草菇、双孢菇最低；淀粉含量为0.12~0.91 mg/ml之间，海鲜菇最高，草菇最低。.（二）漆酶是食用菌菌渣中的一类重要降解酶系，不同菌渣中漆酶的酶学性质差异显著。上述7种菌渣中漆酶的最适pH为3~5之间，最适温度为20℃~40℃之间。在最适pH和温度下，杏鲍菇菌渣对六六六的降解效果最佳，72h时对α-HCH、γ-HCH和δ-HCH的降解率分别可达70.44%、75.69%和61.12%，并且在pH为4~6，温度为35℃~40℃范围内，杏鲍菇菌渣漆酶对六六六均具有较好的降解作用。.（三）鉴于杏鲍菇菌渣漆酶对六六六具有较好的降解作用，本研究通过GenBank进行搜素，共获得15条杏鲍菇漆酶基因序列。通过对基因序列进行分析后发现15条序列可分为5组，同组序列的相似度较高，可能来源于相同基因。.（四）食用菌菌渣受多因素影响，性质会缺乏均一性，经研究发现，阿魏侧耳产漆酶的最佳培养时间为9天，最佳碳源为可溶性淀粉，最佳氮源为蛋白胨，培养基最适的初始pH为6.0，0.1 mmol/L愈创木酚和1 mmol/L Mn2+对其产漆酶具有明显的促进作用。