离子液纤维素酶体系高效降解秸秆
文献类型：学位论文
| 作者 | 李强 |
| 学位类别 | 博士 |
| 答辩日期 | 2010-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 咸漠 |
| 关键词 | 离子液体 生物兼容性 秸秆 酶解 高效预处理 生物转化 发酵性能 |
| 学位专业 | 环境工程 |
| 中文摘要 | 随着石油等不可再生资源的日益短缺和石化工业造成的污染问题日益严重，天然纤维素这样丰富的可再生的资源备受人们关注。然而天然纤维素中存在着较高的结晶度和分子间与分子内存在大量的氢键,导致其不溶于普通有机溶剂,这已成为纤维素材料应用的最大障碍之一。因此目前人们正在寻找新型绿色的纤维素材料的溶剂，以期破坏纤维素材料的紧密结构达到高效利用天然纤维素材料的目的。近年的研究表明离子液体可以作为纤维素的溶剂。首先，本研究建立了磷酸根离子液体/纤维素酶两步法工艺高效降解纤维素材料制备还原糖的方法。其次，纤维素酶原位降解纤维素可以减少工艺步骤，降低应用的成本，有较大的实际应用价值。但是目前还没有报道显示存在耐受高浓度离子液体的纤维素酶，离子液体中的离子会破坏纤维素酶的氢键使其失活。为了实现离子液体体系原位酶解纤维素材料，本研究采取了以下三种方式：1. 筛选生物兼容性和纤维素溶解能力好的离子液体: 本研究合成了磷酸根、盐酸根、醋酸根、硫酸根、硼酸根这五大类离子液体，从各类中选取具有代表性的离子液体进行研究纤维素溶解处理和生物兼容性的研究, 筛选到绿色的离子液体——磷酸根类的离子液体。这类离子液体处理可以使木质纤维素材料紧密的结构遭到破坏，变成更加利于酶解的松散的多孔结构，为纤维素酶提供了更多的结合位点，因此很大程度上提高了糖化率。2. 使用添加剂和纤维素酶的固定化：我们采用了吐温-80等添加剂和酶的固定化的方法来提高商品纤维素酶在离子液体中的活性和稳定性。添加剂和固定化方法可以使纤维素酶在一定离子液体体系中保持较高的稳定性，活性提高了10%。3. 分离和筛选耐受离子液体的纤维素酶：采用了多种分离方法从自然界中分离到菌株 Nocardiopsis LC-27 能够产生对离子液体有较强耐受性的酶。该酶在离子液体体系中的活性比里氏木霉纤维素酶高了 10%。通过上述这三种方式我们实现了在较高浓度下（20-30%）离子液体浓度下进行高效原位酶解纤维素材料。实现了高效降解秸秆成为还原糖，完善了纤维素转化的绿色生物化工工艺，具有较大潜在经济价值。并且在离子液体预处理和纤维素酶酶解之后利用酶解液作为碳源，采用大肠杆菌、酿酒酵母和红球菌三种微生结果表明离子液体参与的这种生物转化工艺过程对于微生物的生长和代物发酵。谢产生生物燃料没有负面的影响。总之，磷酸根离子液体和纤维素酶参与的这种绿色转化工艺有望为第二代生物燃料的生产作出贡献。 |
| 英文摘要 | With the shortage of unrenewable resource such as fossil oil and the pollution of petrochemical industry, more attention was paid on renewable resource as natural cellulose. But there are high degree of crystallinity and intra-molecule hydro-bonds in natural cellulose which result in its insolublity in normal solvents as one of the biggest obstacle of cellulose application. As a result, people are searching for new type of green solvent which can deconstruct tight structure of cellulosic materials toward natural cellulosic materials high efficient conversion.Recent research revealed that ionic liquids could be used as the solvent of cellulosic materials. Firstly, in our case, phosphate-anion based ionic liquid/cellulose two step conversion craft was investigated to obtain high efficient conversion of cellulosic materials to reducing sugars. Secondly, for in situ enzymatic hydrolysis of cellulosic materials can decrease the craft process and the cost which is favorable of practical value. But there is no report on ionic liquid tolerant cellulase for ionic liquids will deconstruct the hydro-bonds of cellulase and lead to the inactivity of the enzyme. Three kinds of stretagies were adopted to carry out in situ enzymatic hydrolysis of cellulosic materials in ionic lquid media：1. Selection of biocompatible ionic liquids with both lignocellulose solubility and cellulase activity：We synthesized five types of ionic liquids (phosphate-based, Cl-based, acetic acid-based, sulphate-based and borate-based ionic liquids). Thease ionic liquids were investigated for lignocellulose solubility and cellulase activity. At last phosphate-based ionic liquids were selected as green solvent. The tight structure of cellulosic materials was disrupted by the type of ionic liquid pretreatment and transferred to the fibres with more rupture and porous of fibres and free of trenches for cellulase adsorption which was easier to be enzymatical hydrolyzed for enhanced saccharification rate.2. Application of additives and cellulase immobilization: additives such as tween-80 and immobilized cellulase were utilized to to enhance the activity and stablilty of cellulase. The two kinds of methods can maintain the cellulase activity in certain concentration of ionic liquid media to obtain 10% enhancement of activity.3. Isolation and screening of ionic liquid tolerant cellulase: multi-methods were performed to isolate ionic liquid tolerant cellulase producing microbe. As a result, strain Nocardiopsis LC-27 was confirmed to be ionic liquid tolerant cellulose producing starin with 10% enhancement of cellulase higher than Trichoderma reesei activity was obtained. With the implement of above three kinds of stretagies, in situ enzymatic hydrolysis of cellulosic materials in high concentration of ionic liquid (20-30%) were achieved to efficiently convert straw into reducing sugars and improved the green biochemical process of cellulosic materials which is potential in viewpoint of practice. In addition, after the ionic liquid pretreatment and cellulase enzymatic hydrolysis the hydrolyzate was used as carbon source of E.coli, S. cerevisiae and R. opacus. The result elucidated that ionic liquid involved green bioconversion process didn't bring negative effects on saccharification, cell growth and metabolic production of biofuel. In conclusion, the phosphate-anion based ionic liquids and cellulase involved green process is promising for the production of second generation of biofuel. |
| 学科主题 | 生物基化学品 |
| 语种 | 中文 |
| 公开日期 | 2011-08-29 |
| 源URL | [http://ir.qibebt.ac.cn//handle/337004/328]  |
| 专题 | 青岛生物能源与过程研究所_材料生物技术研究中心 |
| 推荐引用方式 GB/T 7714 | 李强. 离子液纤维素酶体系高效降解秸秆[D]. 北京. 中国科学院研究生院. 2010. |
入库方式： OAI收割
来源：青岛生物能源与过程研究所
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