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1、华中科技大学 硕士学位论文 白腐菌胞外提取液与纤维素酶复合酶解玉米秸秆的研究 姓名：杜万清 申请学位级别：硕士 专业：微生物学 指导教师：马富英 2010-05-27 华 中 科 技 大 学 硕 士 学 位 论 文 华 中 科 技 大 学 硕 士 学 位 论 文 I 摘 要 摘 要 担子菌具备一套完善的木质纤维素降解体系，在木质纤维素降解中具有一定优 势，这为构建高效的复合酶解体系提供了可能。本研究系统地评价 5 株担子菌胞外 提取液对纤维素酶水解玉米秸秆的影响，构建了 Echinodontium taxodii 2538 胞外提 取液纤维素酶复合酶解体系，并进一步研究了影响复合酶解的因素和主
2、效作用因 子，初步揭示其作用机制。 研究了 5 株担子菌固体发酵和液体深层发酵胞外提取液对不同木质纤维素含量 的玉米秸秆（酸处理、碱处理和原始玉米秸秆）酶解的影响，结果发现，固体发酵 5 天的胞外提取液与纤维素酶的复合酶液提高了秸秆的酶解，胞外提取液和纤维素酶 之间存在协同作用。45下酶解提高最显著，较单纯纤维素酶处理分别提高了 0.14 （酸处理秸秆）、0.27（碱处理秸秆）和 0.46 倍（原始秸秆）。氧气可以显著提高 复合酶液对酸处理秸秆和原始秸秆的酶解，酶解 24 h 时糖化率较单纯纤维素酶处理 分别提高了 0.63 和 0.80 倍；而对低木质素含量的碱处理秸秆的酶解没有促进作用。
3、在协同酶解中，胞外提取液对纤维素酶活性没有影响，但胞外提取液可增加秸秆可 反应多糖比率，酸处理秸秆、碱处理秸秆和原始秸秆的可反应多糖比率较处理前分 别提高了 0.87、0.24 和 0.47 倍，表明酶解抗性显著降低。因此，秸秆抗性的降低在 协同酶解中发挥了重要的作用。 为确定协同酶解的主效作用因子，初步评价了胞外提取液中胞外蛋白和小分子 物质对纤维素酶水解的影响。结果显示，E. taxodii 2538 的胞外蛋白对纤维素酶酶解 有明显抑制作用，且随蛋白浓度增加抑制作用增强；而小分子物质能促进秸秆的酶 解，在通氧条件下酶解 24 h 时糖化率较单纯纤维素酶处理提高了 0.83 倍（酸处理秸
4、秆） 、0.24 倍（碱处理秸秆）和 0.68 倍（原始秸秆） 。因此，在 E. taxodii 2538 胞外 提取液与纤维素酶的协同酶解中，小分子物质起了重要作用。 关键词：关键词：担子菌 胞外蛋白 小分子物质 协同酶解 华 中 科 技 大 学 硕 士 学 位 论 文 华 中 科 技 大 学 硕 士 学 位 论 文 II Abstract Basidiomycetes which have a complete ligninocellulose-degrading system have an advantage for the degradation of lignocellulose
5、in natural state. So, it was possible to construct a complex enzyme system with the enzymes of basidiomycetes and commercial cellulases. This work evaluated the effect of extracellular extract from basidiomycetes on enzymatic hydrolysis of corn stalks to construct the complex enzyme system with extr
6、acellular extract from Echinodontium taxodii 2538 and cellulase. Further, influence factors and main effect factor which affected complex enzymatic hydrolysis were also investigated. Simultaneously, the preliminary mechanism of enzymatic hydrolysis by combination of commercial cellulase with extrace
7、llular extract was revealed. The effect of extracellular extract from 5 strains of basidiomycetes incubated in submerged fermentation medium （SmF） and solid-state fermentation medium （SSF） was systematically evaluated. The result showed that the extracellular extract from Echinodontium taxodii 2538
8、incubated for 5 days in SSF culture increased cellulase hydrolysis of corn stalks. The synergistic action between extracellular extract and cellulase was found in complex enzymatic hydrolysis. The main influence factors which affected synergistic enzymatic hydrolysis were studied. The result showed
9、that the hydrolysis yields of corn stalks at 45 were significantly increased by 0.14-fold (acid-treated corn stalks), 0.27-fold (alkaline-treated corn stalks) and 0.27-fold (raw corn stalks) compared to that of sole cellulase hydrolysis. At the presence of oxygen, hydrolysis yields of acid-treated c
10、orn stalks and raw corn stalks which had high lignin content were further improved by complex enzyme system, and the saccharification yields were increased 0.63-fold and 0.80-fold respectively. However, alkaline-treated corn stalks with low lignin content were promoted by complex enzyme system at th
11、e presence of oxygen. In the synergistic enzymatic hydrolysis, extracellular extract from E. taxodii 2538 could not change cellulase activity. On the contrary, pretreatment of acid-treated, alkaline-treated and raw corn stalks by extracellular extract could improve the rate of reactive polysaccharid
12、es, increasing by 0.87-fold, 0.24-fold and 0.47-fold respectively compared to the control. These suggested that extracellular extract of E. taxodii 2538 could significantly decrease the enzymatic resistance of corn stalks. Therefore, the decrease of resistance play an important role in synergistic e
13、nzymatic hydrolysis. To determine the main effect factor increasing enzymatic hydrolysis, the effect of 华 中 科 技 大 学 硕 士 学 位 论 文 华 中 科 技 大 学 硕 士 学 位 论 文 III extracellular enzymes and low-molecular-weight substances from E. taxodii 2538 on cellulase hydrolysis of corn stalks were evaluated. The result
14、 showed that cellulase hydrolysis of corn stalks were inhibited by extracellular enzymes. And the inhibition was improved by the increased content of extracellular enzymes. However, low-molecular-weight substances could improve cellulase hydrolysis of corn stalks, and hydrolysis yields for 24 hours
15、in the presence of oxygen were increased respectively by 0.83-fold (acid-treated corn stalks), 0.24-fold (alkaline-treated corn stalks) and 0.68-fold (raw corn stalks) compared to control. Therefore, low-molecular-weight substances could play an important role in the synergistic hydrolysis. Keywords
16、：Basidiomycetes extracellular protein low-molecular-weight substances synergistic enzymatic hydrolysis 独创性声明独创性声明 本人声明所呈交的学位论文是我个人在导师指导下进行的研究工作及取得的研 究成果。尽我所知，除文中已经标明引用的内容外，本论文不包含任何其他个人或 集体已经发表或撰写过的研究成果。对本文的研究做出贡献的个人和集体，均已在 文中以明确方式标明。本人完全意识到，本声明的法律结果由本人承担。 学位论文作者签名： 日期： 年 月 日 学位论文版权使用授权书学位论文版权使用授权书 本学位论文作者完全了解学校有关保留、使用学位论文的规定，即：学校有权 保留并向国家有关部门或机构送交论文的复印件和电子版，允许论文被查阅和借 阅。本人授权华中科技大学可以将本学位论文的全部或部分内容编入有关数据库进 行检索，可以采用影印、缩印或扫描等复制手段保存和汇编本学位论文。 保密 ，在_年解密后适用本授权书。 不保密。 （请在以上方框内打“”） 学位论文作者
