【作者】 陈虹；

【导师】 张晓昱；

【作者基本信息】 华中科技大学 ， 微生物学， 2017， 硕士

【摘要】 木质素是限制秸秆、木屑等木质纤维素废弃物生物转化的关键抗性屏障。木腐真菌作为自然界中植物生物质的主要分解者,能利用胞外木质素降解酶和自由基催化途径高效降解木质素,为木质纤维素高效转化提供了一个绿色、低能耗的新途径。本研究分别体外模拟了木腐真菌胞外酶和自由基的去木质化途径,评价了不同降解过程中木质素降解规律,在此基础上,通过模拟木腐真菌的羟自由基去木质化途径实现竹木木质纤维素糖化增效。主要结果如下:利用Fenton反应模拟了木腐真菌的羟自由基去木质化途径。结果表明,模拟体系中产生的羟自由基可以高效降解木质素,木质素降解率与羟自由基浓度呈正相关,最高可达45.11±1.06%。木质素可有效抑制羟自由基的衰减,当铁浓度为2mM、H₂O₂浓度为0.5M时,含木质素体系中羟自由基含量(OD₅₃₂)较对照最高提高了102.52%。采用气相色谱-质谱联用（Gas Chromatography-Mass Spectrometer,GC-MS）分析木质素降解产物,结果显示,木质素降解产物主要由短链脂肪酸、长链及环状脂肪酸、含苯环有机酸组成,其中短链脂肪酸含量增加7倍,说明羟自由基通过将木质素高分子苯环开环进而降解为短链脂肪酸,从而实现木质素的降解。模拟木腐真菌的酶降解木质素途径,研究了漆酶（Laccase,Lac）和多功能过氧化物酶（Versatile Peroxidase,VP）两种主要木质素降解酶对木质素的体外降解。结果表明,高酶负荷可促进木质素降解产生酚类物质,当Lac酶负荷为1000U/g、VP酶负荷为80U/g时,Lac和VP降解木质素体系中可溶性总酚含量分别为非酶处理体系的2.4倍和1.3倍;胞外酶降解也可导致木质素的再聚合,Lac和VP处理后木质素的重均分子量（Weight-average Molecular Weight,Mw）最高分别增加了69.32%和61.16%。此外,亚油酸能促进VP体外降解体系中脂质过氧化物的产生,减轻木质素再聚合。模拟羟自由基去木质化反应增效毛竹木质纤维素的酶解糖化研究结果表明,模拟体系中,羟自由基含量随H₂O₂和铁浓度升高而升高,并与毛竹酶解产糖呈正相关。处理后毛竹酶解产糖显著提高,游离铁较螯合铁的促进效果更加显著,预处理后毛竹酶解48h产糖量比对照最高提高了3.74倍。模拟体系可有效降解毛竹木质素,促进毛竹酶解糖化,当铁离子浓度为4mM、H₂O₂浓度为4M时,毛竹木质素最大降解率为22.33%。综上所述,模拟真菌羟自由基去木质化途径可以有效降解毛竹木质素,并实现木质纤维素的糖化增效。由此提出了一个清洁温和的增效木质纤维素生物转化的策略,在木质纤维素转化为生物能源及化工产品等领域具有一定应用潜力。更多还原

【Abstract】 Lignin is the key barrier that limits the biotransformation of lignocellulosic wastes,such as straw and sawdust.As a major decomposer of plant biomass in wood,wood-rot fungi can efficiently degrade lignin by extracellular lignin degrading enzymes and free radicals,providing a new way of green and low energy consumption for efficient conversion of lignocellulose.In this study,we studied the delignification pathways of extracellular enzymes and free radicals secreted by wood rot fungi,and evaluated the degradation of lignin during different degradation processes.On this basis,by simulating the hydroxyl radical producing pathway of wood-rot fungi to achieve delignification and glycosylation rate increasing of bamboo.The main conclusions are as follows:The Fenton reaction was used to simulate the hydroxyl radical producing pathway of wood-rot fungi for lignin degradation.As the results showing,the hydroxyl radicals produced by the simulated system can degrade lignin efficiently,and the lignin degradation rate is positively correlated with the concentration of hydroxyl radicals,up to 45.11±1.06%.Lignin could effectively inhibit the decay of hydroxyl radicals.When the iron concentration was 2mM and the concentration of H₂O₂ was 0.5M,the content of hydroxyl radicals(OD₅₃₂)in the lignin-containing system was 102.52%higher than that of the control.The results of Gas Chromatography-Mass Spectrometry（GC-MS）analysis showed that lignin degradation products were mainly composed of short-chain fatty acids,long-chain and cyclic fatty acids and benzene ring organic acids,among which the content of short-chain fatty acid increased by 7 times,indicating that hydroxyl radicals made high molecular benzene rings of lignin open and then degraded them into short chain fatty acids to achieve lignin degradation.The degradation of lignin by laccase（Lac）and Versatile Peroxidase（VP）were studied to simulate the enzyme delignification pathway of wood-rot fungi.The results showed that the high enzyme load could promote the degradation of lignin to produce phenolics.When the Lac enzyme load was 1000U/g and the VP enzyme load was 80U/g,the content of soluble phenolics in degrading system were 2.4 times and 1.3 times of that in non-enzymatic treatment system.Enzymes’treatments could also lead to the re-polymerization of lignin,and the weight-average Molecular Weight（Mw）of lignin increased by 69.32%and 61.16%,respectively,after Lac and VP treatment.In addition,linoleic acid can promote the production of lipid peroxides in the in vitro degradation system of VP to reduce the lignin repolymerization.The hydroxyl radical delignification pathway of wood-rot fungi was simulated to enhance enzymatic hydrolysis of lignocellulose.The results showed that the content of hydroxyl radicals in the simulated system increased with the increase of H₂O₂ and iron concentration,and positively correlated with the sugar yield of bamboo.After treatment,the sugar yield of bamboo increased significantly,and the effect of free iron was more significant than that of chelated iron.After pretreatment,the sugar yield of bamboo was increased by 3.74 times compared with the control.The simulated system can effectively degrade lignin of bamboo to promote the enzymatic hydrolysis.when the concentration of iron is 4mM and the concentration of H₂O₂ is 4M,the maximum degradation rate of bamboo lignin is 22.33%.In summary,the in vitro simulation of fungal hydroxyl free radical delignification pathway can effectively degrade lignin of bamboo and promoting the hydrolysis of lignocellulose.This study presents a strategy for clean and mildly enhanced lignocellulosic biotransformation,which has certain potential in the field of lignocellulose conversion to bioenergy and chemical products.更多还原