原油高效降解真菌的筛选、鉴定及降解酶活性  

Screening and identifying highly efficient crude oil-degrading and degrading enzymes-producing fungi

作　　者：王秋颖 张国庆[2] 陈青君[2] 张娇[1] 李小凤 徐诗毅 WANG Qiuying;ZHANG Guoqing;CHEN Qingjun;ZHANG Jiao;LI Xiaofeng;XU Shiyi(Key Laboratory of Urban Agriculture(North China)of Ministry of Agriculture and Rural Affairs,College of Bioscience and Resource Environment,Beijing University of Agriculture,Beijing 102206,China;Beijing Advanced Innovation Center for Tree Breeding by Molecular Design,Beijing Key Laboratory for Agricultural Application and New Technique,College of Plant Science and Technology,Beijing University of Agriculture,Beijing 102206,China)

机构地区：[1]北京农学院生物与资源环境学院,农业农村部华北都市农业重点实验室,北京102206 [2]北京农学院植物科学技术学院,北京农学院林木分子设计育种高精尖创新中心,农业应用新技术北京市重点实验室,北京102206

出　　处：《应用与环境生物学报》2021年第5期1289-1296,共8页Chinese Journal of Applied and Environmental Biology

基　　金：北京市科技计划项目(Z181100009818007);现代农业产业技术体系北京市食用菌创新团队(BAIC05-2019);北京农学院学位与研究生教育改革与发展项目(2020YJS040)资助。

摘　　要：原油开采、加工和运输过程中会造成环境污染.为获得原油高效降解真菌,以采集自内蒙古、广西和北京的56株真菌为材料,开展高效降解菌筛选、鉴定及降解酶研究.通过原油耐受性初筛和愈创木酚、α-萘酚、苯胺蓝平板复筛,获得目标菌株JY-11.结合形态与分子鉴定,确定该菌株为硬毛香菇(Lentinus strigosus),其菌丝体最适培养条件为:可溶性淀粉作为碳源,酵母浸粉作为氮源,最适温度为36℃,最适C/N为20/1、最适生长pH为7.0.JY-11菌株在添加0.5%原油的降解培养基30℃、150 r/min振荡培养10 d,原油降解率为70.88%±2.48%.在添加0%-2%原油的原油降解培养基30℃、150 r/min振荡培养20 d,各处理发酵液中均具有漆酶活性,而缺乏锰过氧化物酶、木质素过氧化物酶活性;Native-PAGE漆酶同工酶电泳显示,原油的添加对漆酶具有诱导作用.JY-11菌株发酵液具有较高的抗氧化活性,原油降解培养基不添加原油组,48 h时抗氧化活性为7.57±0.01 mmol/L(FeSO4),添加0.5%原油组表现出最高的抗氧化活性,为11.83±0.01 mmol/L(FeSO4).本研究获得了具有高效降解原油活性的真菌,可以通过分泌胞外酶降解原油,具有潜在的环境修复应用前景.Crude oil is called“industrial blood”,as it causes environmental pollution while it is extracted,processed,and transported.Therefore,56 fungal strains collected from Inner Mongolia,Guangxi,and Beijing of China were screened and identified to determine which of their enzymes were highly efficient at degrading crude oil.The target strain JY-11 was obtained via a preliminary screening of crude oil tolerance followed by further screening with guaiacol,α-naphthol,and aniline blue plates.The strain was identified as Lentinus strigosus using morphological and molecular analyses.The optimum mycelial culture conditions were as follows:soluble starch and yeast extract as the carbon and nitrogen sources,respectively,in addition to a temperature of 36℃,C/N ratio of 20/1,and pH of 7.0.Strain JY-11 had a degradation rate of 70.88%±2.48%for crude oil after 10 d of shaking incubation at 30℃and 150 r/min in a degradation medium with 0.5%crude oil.Subsequently,the JY-11 strain was shakingcultured in a crude oil degradation medium supplemented with 0%-2%crude oil for 20 d at 30℃and 150 r/min.The fermentation broth in each treatment showed laccase activity but no manganese peroxidase or lignin peroxidase activity.Native-PAGE results showed that crude oil induced laccase expression.The fermentation broth of the JY-11 strain also displayed high antioxidant properties.The antioxidant activity was 7.57±0.01 mmol/L(FeSO4)at 48 h in the fermentation broth for the crude oil degradation medium in which crude oil had not been added.The 0.5%crude oil treatment showed the highest antioxidant activity at 11.83±0.01 mmol/L(FeSO4).The present study identified a degrading fungus that could degrade crude oil highly efficiently via the secretion of extracellular enzymes.It has the potential to be applied in environmental remediation projects.

关 键 词：原油降解 菌株鉴定 培养条件 降解酶 

分 类 号：X172[环境科学与工程—环境科学]