栓孔菌属真菌物种亲缘关系与产漆酶能力的相关性及所产漆酶对环境激素降解的研究

Endocrine disruptor compounds (EDCs), as exogenous toxic agents, would interfere with human and animals’ health and reproduction when released to the environment. Since laccase is found to be able to catalyze the oxidation of an array of aromatic compounds, it is extensively involved in EDCs bioremediation and detoxification. Although the protein is widely distributed in nature, the majority of laccase described in the literature have been isolated from white rot fungi, and there is no doubt that Trametes species are the most attractive fungi. Recent years, studies on fungal phylogeny and biological evolution revealed that species with close relationship usually share similar characters. Whether laccase-producing capability among Trametes species has similar features? In this project, the Trametes strain with higher laccase-producing capability will be screened by systematic resources collection and enzyme assays. The correlations of Trametes species genetic relationships and their laccase-secreting abilities will be analyzed by phylogenetic tree establishment. Furthermore, the laccase from the screened strain will be purified to homogeneity by a three-step method, and characterized for unusual properties (molecular weight, pH stability, thermostability, substrate specificity, metal ion effect, ect.). Immobilization of the purified enzyme will be performed by entrapment of chitosan beads with crosslinker genipin. Influences of conditions on the immobilization process will be explored including crosslinker concentration, enzyme solution, activation time, and contact time. The biodegradable capabilities of the free and immobilized laccase for EDCs will be assessed. The results of the current study will facilitate a better application of pollution-free treatment of EDCs and support the exploitation and utilization of new fungal resources in many industrial fields.

环境激素是一类外源性有毒化合物，释放到环境中会对人类及动物的健康和繁衍造成严重危害。漆酶可降解包括环境激素在内的多种芳香族化合物，在环境激素的降解与去毒等领域备受关注。目前大量关于漆酶的研究均集中于白腐真菌，而栓孔菌更是研究的热点。随着系统发育学和生物进化研究的不断深入，发现亲缘关系较近的真菌往往具有相近的特性。栓孔菌属真菌产漆酶能力是否也存在类似的特征？本项目拟对中国栓孔菌属真菌展开系统性的资源收集和酶活检测，筛选出高产漆酶菌株，同时通过构建系统发育树分析栓孔菌不同种类亲缘关系与其产漆酶能力的相关性。此外，对筛选得到的菌株所分泌的漆酶进行分离纯化及酶学性质分析，采用包埋交联法对上述纯化的酶蛋白进行固定化处理及条件优化，并最终应用于环境激素降解中，从而为环境激素的无污染化处理奠定基础，同时也为白腐真菌新资源的开发利用提供相应的理论依据和技术手段。

漆酶是一种性质优良的多酚氧化酶，由于在分子氧的协助下可将酚类、羧酸类、芳胺类化合物等多种底物氧化，最终得到水及终产物，符合当代环保工业要求，因而在降解与去毒释放到环境会对人类及动物的健康和繁衍造成严重危害的环境激素等领域有着巨大的应用潜力。许多具有价值的漆酶来源于真菌，特别是白腐真菌，而栓孔菌漆酶更是研究的热点。因此充分收集、挖掘和利用栓孔菌资源、获取耐受力突出且稳定的酶蛋白就显得极为关键。通常亲缘关系较近的真菌往往具有相近的特性，而栓孔菌属真菌产漆酶能力是否也存在类似的特征？通过本项目的实施，对栓孔菌展开了资源调查、标本采集和菌株分离纯化培养及分子生物学鉴定，共采集标本300余号，包含栓孔菌标本100余号，分离纯化菌株50株，基于ITS序列准确鉴定其分属于9个种。利用愈创木酚平板显色法定性初筛和ABTS液体发酵法定量复筛对其产漆酶活性进行了系统性研究，最终筛选获得高产漆酶菌株绒毛栓孔菌、东方栓孔菌和毛栓孔菌。并发现栓孔菌具有较普遍的产漆酶特性，同一种类不同菌株的产漆酶能力有所不同，而亲缘关系相近的菌株其产酶能力也存在差异，这可能与遗传多样性、地理分布、寄主、实验条件等密切相关。通过三步法对上述筛选菌株分泌的漆酶进行了分离纯化并测定其性质，得到了3种对极端pH、温度、存在金属离子、抑制剂、表面活性剂或有机溶剂等环境条件耐受力强、稳定性高的酶蛋白。分别通过壳聚糖作为载体、戊二醛或京尼平作为交联剂对纯化酶进行了固定化处理，显著提高了酶的pH适应能力、抗热变性能力、贮存稳定性及回收利用效率。最终将经纯化及固定化处理的漆酶应用于降解环境激素中，发现2种形式的漆酶催化的降解途径是一致的，主要是通过氧化、羟基化、羧基化、脱氢、去羟基化、去甲基化和甲氧基化等作用完成；且被降解后的环境激素毒性明显减小。该研究为环境激素的无污染化处理奠定了基础，同时也为白腐真菌新资源的开发利用提供了相应的理论依据和技术手段。