摘要
[目的]本研究旨在筛选油菜秸秆高效降解菌株并对其降解特性进行分析,为油菜秸秆的资源化利用提供科学依据。[方法]采用稀释涂布平板法、刚果红透明圈试验以及纤维素酶活性测定对菌株进行分离筛选;根据菌株的形态特征和ITS序列分析对菌株进行鉴定;结合扫描电镜技术,分析在降解过程中降解菌对油菜秸秆物理、化学性质的影响;设置盆栽试验模拟秸秆还田以综合探究菌株对油菜秸秆的降解特性;通过纤维素酶酶谱分析及质谱检测技术获得纤维素酶基因(celI)的序列信息,克隆该基因,再利用pPICZαA质粒和毕赤酵母X33进行异源表达;经镍柱纯化后对CelI的酶学性质进行分析。[结果]从腐烂油菜秸秆田土样中共分离纯化出8株真菌,其中菌株A15的透明圈直径/菌落直径比值最大,为3.0,且滤纸酶活性和内切葡聚糖酶活性均显著高于其他菌株,分别为16.13和8.16 U·g^(-1),经鉴定其为绿色木霉(Trichoderma viride);接种菌株A15固体发酵28 d后,油菜秸秆失重率达29.17%,纤维素及半纤维素含量分别下降至29.41%和17.18%,经过30 d的模拟秸秆还田盆栽试验,油菜秸秆失重率达57.50%;成功表达并纯化了菌株A15来源的GH7家族纤维素酶CelI,该蛋白相对分子质量约为72×10^(3),具有内、外切葡聚糖酶的双重活性,CelI的最适反应温度为50℃,且表现出良好的热稳定性,CelI的内切葡聚糖酶活性最适pH值为4,且Co^(2+)、Mn^(2+)、Ca^(2+)对其具有促进作用,而CelI的外切葡聚糖酶活性最适pH值为5,且Mn^(2+)对其具有显著激活作用,而Zn^(2+)、Fe^(3+)具有抑制作用。[结论]从腐烂油菜秸秆田土样中筛选到1株油菜秸秆降解真菌绿色木霉A15,其能高效产胞外纤维素酶,在油菜秸秆资源化利用方面具备良好的应用前景。
[Objectives]The purpose of this study was to screen and obtain strains with high degradation ability of rape straw,and analyze its degradation characteristics,so as to provide a scientific basis for the resource utilization of rape straw.[Methods]The strains were isolated and screened by dilution plating method,Congo red transparent circle test and determination of cellulase activity.The strain was identified according to its morphological characteristics and its sequence analysis.Combined with scanning electron microscopy,the effects of strain on the physical and chemical properties of rape straw were analyzed.A pot experiment was set up to simulate straw returning to the field to comprehensively explore the degradation characteristics of rape straw by this strain.The sequence information of cellulase gene(celI)was obtained by cellulase zymogram analysis and mass spectrometry,and this gene was cloned and heterologously expressed using pPICZαA plasmid and Pichia pastoris X33.The enzymatic properties of CelI were analyzed after purification by nickel column.[Results]Eight fungal strains were isolated and purified from rotten rape straw soils.The ratio of transparent circle diameter to colony diameter of strain A15 was the largest of 3.0,and the activities of filter paper enzyme and endoglucanase were significantly higher than those of other strains,which were 16.13 and 8.16 U·g-1 respectively.Strain A15 was identified as Trichoderma viride.After 28 days of solid fermentation with inoculated strain A15,the weight loss rate of rape straw was 29.17%,and the contents of cellulose and hemicellulose decreased to 29.41%and 17.18%respectively.After a 30 days simulated straw returning pot experiment,the weight loss rate of rape straw was 57.50%.The GH7 family cellulase CelI derived from strain A15 was successfully expressed and purified.The relative molecular weight of the protein was about 72×103,which simultaneously showed the dual activity of endoglucanase and exoglucanase.The optimum reaction temperature of CelI was 50℃and showed good thermal stability.The optimum pH for the endoglucanase activity of CelI was 4,and Co^(2+),Mn^(2+),Ca^(2+)could promote it,while the optimum pH for the exoglucanase activity of CelI was 5,and Mn^(2+)could significantly activate it,but Zn^(2+)and Fe^(3+)could inhibit it.[Conclusions]A high efficient rape straw degrading fungus Trichoderma viride A15 was screened from rotten rape straw soil samples.It could produce extracellular cellulase efficiently and had a good application prospect in the efficient resource utilization of rape straw.
作者
李凌波
许欢欢
夏燕维
郭传旭
杨明珠
马磊
缪有志
刘东阳
张瑞福
LI Lingbo;XU Huanhuan;XIA Yanwei;GUO Chuanxu;YANG Mingzhu;MA Lei;MIAO Youzhi;LIU Dongyang;ZHANG Ruifu(Jiangsu Provincial Key Lab of Solid Organic Waste Utilization,Nanjing Agricultural University,Nanjing 210095,China)
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2022年第4期711-720,共10页
Journal of Nanjing Agricultural University
基金
国家重点研发计划项目(2017YFD0200800)。
关键词
纤维素酶
油菜秸秆
基因克隆
异源表达
酶学特性
cellulase
rape straw
gene cloning
heterologous expression
enzymatic properties
