【作者】 赵新宇；

【导师】 冯雁；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 纤维素酶在生物质转化为生物能源的过程中具有关键性作用，其中对不溶性生物质的生物降解是生物转化过程中的瓶颈，关于纤维素酶对不溶性底物的降解机理的研究显得尤为重要。来源于嗜热菌的酶类具有极好的高温反应活性和稳定性，嗜热细菌Caldicellulosiruptor bescii中含有丰富的纤维素酶系，可以利用高木质素的草料和硬木作为唯一的碳源进行有效生长，对该菌中纤维素酶的表征及结构-功能关系分析具有重要的科研意义及潜在的应用价值。本论文对内切纤维素酶及相关纤维素结合结构域的功能进行了系统研究。C. bescii中推测的纤维素酶基因（Athe₁867）编码5个模块化结构域，依次为第9家族纤维素内切酶（CbCel9A），三个纤维素结合结构域（CBM3c-CBM3b-CBM3b）和第48家族纤维素外切酶（CbCBH48A）。本论文针对该基因展开工作并获得如下结果：（1）杂合酶的克隆、表达以及纯化。以该菌基因组为模板，通过PCR方法得到CbCel9A及其C端串联有不同数量CBM的杂合酶（CbCel9A_CBM, CbCel9A₂CBM and CbCel9A₃CBM），以pET-21b为表达载体，以E. coli BL21-CodonPlus （DE3）-RIL为宿主菌对目的基因进行表达，经过热处理和镍柱纯化得到目的蛋白。（2）含有CBM的杂合酶具有高温反应活性，最适温度为95-100oC，在85oC下孵育50min仍可保持50%以上的残余活性；而CbCel9A在高温下反应活性较低，最适温度为45-50oC，在60oC下孵育20min后残余活性仅剩20%；（3）杂合酶的最适底物为羧甲基纤维素钠（CMC），为典型的内切纤维素酶特性；随着重组CBM数量的增加，杂合酶对不溶性纤维素Avicel的活力逐渐提高；（4）含有CBM的杂合酶对Avicel以及Filter paper具有吸附性及渐进性。随着CBM数量的增加，杂合酶对底物的吸附性和渐进性增强；（5）杂合酶对再生不定型纤维素（RAC）水解的产物主要为葡萄糖、纤维二糖和纤维三糖，CbCel9A水解三糖的速率低于其它杂合酶；（6）杂合酶与外切纤维素酶CbCBH48A、内切纤维素酶FnCel5A之间存在正协同性。综合以上结果得出，与CbCel9A紧邻的CBM3c结构域起到了稳定CbCel9A结构域结构与功能的作用，使之最适温度、热稳定性以及催化活力得到提高；与CbCel9A串联的三个CBM3结构域有利于该酶对不溶性底物的吸附及水解；杂合酶与CbCBH48A等酶协同作用可以对滤纸进行有效的水解。以上研究结果对该嗜热内切纤维素酶及相关的CBM结构域的功能进行了揭示，为进一步研究与应用提供了依据。更多还原

【Abstract】 Cellulases play a key role in the biotransformation process of bio-ethanol.Thus it is of great interest and significance to study the mechanism of enzymaticdegradation of insoluble biomass. Characterization of thermostable and thermoactivecellulases from thermophilic bacteria and investigation of their structure-functionrelationship will not only shed light on the structure base of stability and catalyticmechanism at high temperature, but also provide us with new enzymes for potentialindustrial applications. This thesis reports the functional research of theendo-cellulase and its related CBM from Caldicellulosiruptor bescii.The cellulase gene （CbCelA） from C. bescii consists of five modules, whichare designated CbCel9A, three CBMs （CBM3c-CBM3b-CBM3b） and CbCBH48A.This thesis focuses on this gene and get the following results:（1） Successful cloned,expressed and purified the truncated enzymes. Four truncated enzymes whichcontained GH9module and0-3CBMs were constructed and functionallyoverexpressed in E. coli BL21-CodonPlus （DE3）–RIL, respectively. The proteinswere designated CbCel9A, CbCel9A_CBM, CbCel9A₂CBM and CbCel9A₃CBM.The recombined proteins were purified after the heat-treated and nickel affinitychromatography.（2） the enzymes with different numbers of CBMs had activity athigh temperature. The optimum temperature of enzymes are about95-100oC andthermal stability maintained a good situation even at85oC for50min, however, theoptimum temperature of CbCel9A is45-50oC, and thermal stability analysis showedthat it only remained20%activity after20min incubation at60oC.（3） The optimalsubstrate was the sodium carboxymethyl cellulose （CMC） for soluble saccharideswhich was a typical characteristics of endo-cellulase. The activity of truncatedenzymes towards microcrystalline cellulose Avicel was in an order of CbCel9A₃CBM>CbCel9A₂CBM>CbCel9A_CBM>CbCel9A;（4） The enzymeshad adsorption and processivity on filter paper and Avicel. The adsorption andprocessive ratio of the recombinant enzymes on filter paper showed the same orderabove.（5） The major oligosaccharides in the hydrolysis of the RAC were glucose,cellobiose and cellotriose. The activity of CbCel9A towards cellotriose was lowerthan the others.（6） CbCel9A_CBM and CbCel9A₂CBM synergistically workedwith cellobiohydrolase CbCbh48A from C. bescii and β-glucosidase CbBgl1A ondegradation of filter paper, and generated sole glucose. Based on the above results, theCBM3c adjacent to CbCel9A plays critical roles in maintaining themoactivity andthermostability of CbCel9A, and has positive effects on the adsorption of insolublesubstrate. The CBMs are conductive to the adsorption and processive ratio ofCbCel9A on the insoluble substrates; The enzyme has synergy with both CbCBH48Aand FnCel5A, and can hydrolyze the Filter paper efficiently. The result revealed thefunction of CbCel9A and its related CBM, and the enzymes we investigated here arevery valuable for further research and potential application.更多还原