【作者】 姚强；

【导师】 陈冠军； 刘巍峰；

【作者基本信息】 山东大学 ， 微生物学， 2007， 博士

【摘要】 存在于细胞壁中的纤维素是自然界分布最广、含量最多的一种多糖，是植物细胞壁的主要成分，也是地球上最大量的可再生碳源物质。然而，纤维素的应用研究一直进展缓慢，纤维素酶作用天然底物的低效率和实际应用中的高成本是生物质转化和有效利用的限制性瓶颈。随着分子生物学和现代实验技术的发展和应用，已基本弄清纤维素酶催化水解纤维素的“酸／碱催化”双置换作用机制，但仍然未能解决高效降解天然结晶纤维素的问题，其中主要是忽略了纤维素分子的超分子结构即聚集态结构对其降解的影响。事实上，无定形纤维素和可溶性的纤维寡糖很容易被纤维素外切酶酶解，纤维素与淀粉的结构差异才是纤维素酶解与淀粉酶解速度不同的主要原因。因此在自然界对纤维素的生物降解过程中，除了酶解机制以外可能还有其它因子和非酶组分破坏天然纤维的结晶结构来起始和协同酶解作用，比如文献报道的一些低分子量组分、短纤维生成因子、羟基自由基等等。在丝状真菌中已发现了一种含有植物膨胀因子Expansin类似序列的新蛋白Swollenin，可以破坏纤维素材料同时不产生还原糖。有研究表明Expansin促进植物细胞壁伸展和膨胀是因为其可以破坏纤维素的微纤维之间或纤维素和其它细胞壁多糖之间的氢键，虽然Expansin不具有纤维素酶活性，但它能够使滤纸、结晶纤维素、半纤维素等天然底物的纤维结构膨胀疏松，可以协同作用提高纤维素酶对微晶纤维素的水解程度，推测Swollenin也有同样的作用机制。相对于Expansin，Swollenin有可以在微生物宿主中异源表达和分子改造的优势，而Expansin的研究基本建立在蛋白纯化的基础上，异源表达困难。但是对于真菌Swollenin的研究还是刚刚开始，还未有关于其在纤维素降解中的协同作用的报道。为了阐明膨胀因子的膨胀机理和在纤维素酶酶解天然结晶纤维素过程中的协同作用机制，本文以克隆的木霉Swollenin基因为研究对象，主要的实验工作和结果如下：1．首先利用RT-PCR的方法扩增了瑞氏木霉QM9414的Swollenin基因（swol）的cDNA序列，经测序验证与已报道的序列一致，共1482bp，编码493个氨基酸；通过对Swollenin的保守域序列进行分析设计引物，从本实验室分离保存的拟康氏木霉S38中用RT-PCR的方法扩增了其Swollenin基因cDNA的部分序列；再利用SMART-RACE技术设计锚定引物，克隆了拟康氏木霉S38的Swollenin基因的全长cDNA；经过测序验证，该序列含有一个编码495个氨基酸残基的开放阅读框，将推测的拟康氏木霉S38的Swollenin的蛋白序列与瑞氏木霉SWOⅠ比对后发现同源性高达90％，因此其应该同属于SWOⅠ（swo1）；用ProtParam软件推测了瑞氏木霉、拟康氏木霉和烟曲霉的SWOⅠ蛋白的理化性质，发现它们是一类非常相似的蛋白而且可能在真菌中普遍存在并起着类似的功能作用。2．利用各种生物信息学软件，对3个已知的真菌Swollenin蛋白序列的同源性、结构域等一级结构进行了分析。结果表明其蛋白可能起始于一个典型的信号肽序列，紧连着一个与真菌纤维素酶结合域高度同源的纤维素结合域（CBD）。其后是一段Linker区连接着C末端可能的SWOⅠ催化域（CD），其中大部分序列与糖苷水解酶45族（GH45）同源并且2／3催化域的氨基酸组成明显与植物Expansin相似。两两比较Swollenin、α-Expansin和β-Expansin大约都有25％的同源性。而且发现不同真菌SWOⅠ的序列之间特别是催化域（CD）高度同源，表明了各Swollenin的催化机制基本一致。经进化树和同源模建分析发现，SWOⅠ催化域的作用机制类似于Expansin，而非GH45内切酶。不同Swollenin的CBD及Linker序列的差异可能导致了其底物结合性质的微小差别；SWOⅠ二级结构的预测发现，其以β-折叠和c-无规则卷曲为主，特别是催化域的结构最为松散，说明SWOⅠ蛋白结构非常柔性。而且催化域中有2个非常保守的约170个氨基酸的纤连蛋白Ⅲ型（fibronectin typeⅢ，FnⅢ）重复序列，这仅在原核水解酶如纤维素酶、几丁质酶和淀粉酶中发现过。FnⅢ构成的β片层被认为能够轻松地伸展和再折叠，从而使蛋白保持了较大的柔性，这对Swollenin像Expansin那样促使微纤维的滑动是很重要，类似序列还未在真核生物的酶中发现；根据SWOⅠ不同的功能域分别进行了序列分析和三维同源模建，并预测二级结构和疏水性分析来验证模型的合理性；比对分析SWOⅠ和GH45内切酶的活性中心，由于SWOⅠ缺少在翻转型酸碱催化过程中的类似GH45内切酶中D10的广义酸基团，推测Swollenin可能因此而失去了水解活性。3．由于Swollenin在木霉胞外酶系中的含量很低，异源过量表达可以为深入研究其性质机制、分子改造和体内、体外的进化研究提供技术支撑。然而来自于真菌和一些细菌的纤维素酶基因的异源表达比较困难、高效的异源表达体系稀少，因此我们将来源于瑞氏木霉QM9414和拟康氏木霉S38的Swollenin基因构建了在不同原核和真核宿主中异源表达的重组质粒，分别转化大肠杆菌、枯草芽孢杆菌、酿酒酵母和瑞氏木霉，并得到重组表达菌株。经SDS电泳和Western Blotting检测SWOⅠ在真核宿主中可以活性表达，其中来源于瑞氏木霉和拟康氏木霉的swo1在瑞氏木霉宿主中的异源表达分泌量分别为0.62g／L和0.5 g／L，是木霉自身分泌的SWOⅠ的五百倍以上。4．通过CM-scpharose离子交换层析和Mono-S高压液相离子交换层析从瑞氏木霉转化子的发酵液中分别提取纯化了来源于瑞氏木霉和拟康氏木霉swo1的重组表达蛋白，SDS电泳显示其分子量均为75kDa左右，比预测的52kDa要大。对纯化后的重组Swollenin的活性检测表明其没有β-1，4糖苷键的水解活性，有微量的β-1，3葡聚糖和木聚糖水解活性，并在作用不溶的固体纤维素底物时不产生还原糖。5．由于Swollenin可以膨胀棉纤维和改变纤维的结构，用扫描电子显微镜观察到纤维形状的改变。推测经Swollcnin作用后可以打开纤维间的交连耦合，使其纤维素结构中的结晶区域更无序。对Swollenin在纤维素酶降解不溶的结晶纤维素底物过程中的协同作用进行了初步的研究，发现Swollcnin确实能够提高纤维素酶酶解天然结晶度较高的纤维素类底物，协同度（DS）最高可达1.24。因此推测Swollcnin可以从纤维素表面疏解多糖链，使真菌纤维素酶更容易进入纤维内部和接触更多的底物，同时使微纤维表面的葡聚糖对纤维素酶的攻击更为敏感，从而提高纤维素酶对天然底物的水解活性。6．在实现与糖苷水解酶45家族具有有相同翻转型催化机制的糖苷水解酶第8家族——纤维素内切酶CclA高水平异源表达的基础上，结合蛋白质结构生物学信息，对其催化中心的相关关键氨基酸进行了系统的点突变分析。结果表明，其152位天冬氨酸（D152）突变为天冬酰胺后，CelA活性完全丧失；而第278位天冬氨酸突变后，其k_cat与野生型CelA相比没有降低，说明天冬氨酸152可能在CelA的酸碱催化中发挥重要作用，这与以前关于天冬氨酸278是广义碱残基的推测完全不同。此外，第215位酪氨酸突变后k_cat降为野生型的一半，推测其可能与定向亲核水分子有关。更多还原

【Abstract】 The cellulose is the most widespread polysaccharides in nature. As a main component in plant cell wall, it is the most abundant renewable carbon source material on the earth. However, the progress on the research of cellulose application has been slow, largerly due to the low efficiency of the cellulases to hydrolyze the natural cellulosic material and the high cost in the pratical application, which constitute the bottlenecks in the conversion and the utilization of biomass. With the development and application of molecular biology and the modern research techniques, althoug the " general acid catalysis ", double-displacement functional mechanism of cellulase, has already been clear, there still exsits a question about how to efficiently degrade the natural crystallize cellulose. A key point is that the influence of the super molecular sructure of cellulose is someitmes neglected. In fact, amorphous structural cellulose and soluable oligosaccharides are easy to be degraded by cellulases, and there are not many differences between the cellulase and amylases in enzyme activity, so it is the structural differences of cellulose and starch that causes the degradation-speed difference.For biological degration of cellulose, there must be factors and non-enzyme components other than enzymes that can break the cellulose crystal strucrue to initiate and/or synergizing cellulose degradation. In this case, some low molecular weight components, short fiber born factors, and the OH have been reported. In filamentous fungi, a new kind of protein called Swollenin has been discovered which bears similar sequence as the plant swelling factor-Expansin, which can break the cellulose material without producing reducing sugars in the meantime. It has been reported that Expansin can promote the stretch and inflation of the plant cell wall because it can break the hydrogen bonding between the microfibers of the cellulose, or those between the cellulose and other cell wall polysaccharides. Although Expansin doesn’t seem to show a cellulase activity, it can make natural cellulose substrates swell and loosy, such as filter paper, crystal cellulose, and the semi-cellulose etc. So it can be helpful in enhancing hydrolyzation effects of the cellulase on the microlite cellulose, and hence it is predicted that Swollenin may also have a same mechamsm of function mechamsm. Compared with Expansin, Swollenin has another advantage in that it can be expressed heterologously in a different microorganism host, and is thus subjected to molecular engineering. Heterologous expression of Expansin has not been reported up to date. But the research about Swollenin is just in its early stage and its synergy with other components of cellulase system has not been reported. So in order to elucidate the possible synergy mechanism of this swelling factor, and its effects in the cellulase degration, the present study report on the cloning, expression and functional characterizaiton of the swollenin from Trichoderma strains. The main results are as follows:1. The cDNA sequence of the Swollenin gene(swo 1) is cloned from the T. reesei QM9414 by RT-PCR, acording to the result of DNA sequence, it has the same sequence as one reported. The sequence has 1482 bps, coding 493 amino acids. After analyzing the swollenin conservative area sequence, we designed the primer and clonded a part of cDNA sequence of swollenin gene from T. pseudokoningii S38 which had been separated by our labrary,and then cloned the complete cDNA of this gene by the anchor primer which designded through the method of SMART-RACE. Its open reading frame codes 495 amino acids and its sequence has 90% similarity with the swol gene from T. reesei, so it is same as swol gene. Using the software ProtParam, we analyzed the physical and chemical characters of the swo genes from T. reesei 、 T. pseudokoningii and A. fumigatus, and found that they were very similar, indeed one type of protein, and may have a widespread existence in the fungi and also very similar function.2. By various bio-informatics software, we analyzed the primary structure of the three swollenin protein sequences from fungi that have already been known , including protein domains, conserved patterns and functional sites etc. These protein are predicted to have an N-terminal signal sequence followed by a cellulose binding domain (CBD), which has high similarity sequence with that of cellulase, connected by a linker region. The major part of the remaining sequence found to have sequence similarity with the family 45 hydrolases in a BLAST database search was a possible catalyst domain (CD), among them 2/3 amino acidses have obviously similarity as plant Expansin. The identity between swollenin and individual a-orβ-expansin in pairwise comparisons is about 25 % over an area of about 200 amino acids. The sequences of different fungi SWOI especially their catalyst domain (CD) have high identity, which indicates swollenin have identical catalyst function in the different fungi, and their function mechanism are similar to Expansion, but the difference of their cellulose binding domain(CBD) may cause the slightly difference of their combination. The secondary structure of SWOI is mainly β-pleated sheet and c-random coil, especially the structure of CBD is the most loosy, which indicates the protein SWOI is very gentle. In the catalyst area, two high conservative repeated sequence called fibronectin type III (FnIII) coding 170 amino acids have been found in prokaryotic hydrolases such as cellulases, chitinases and amylases, but thus far not in fungal enzymes. β-pleated sheet composed of this repeated sequence which can easily launch and fold again to make the protein able to stretch, that is probably important to urge the gliding of micro-fiber like Expansin and Swollenin. Finally we respectively analyzed the swollenin conservative area and homology moldling its structure according to the SWOI different function area, and then we verified the rationality of the model through analyzing its second structure and hydrophobicity parameters etc. Compared the enzyme activity area between SWOI and GH45, we found that swo is lack of a acid catalyst of the D10 in response to "general acid catalysis", so we calculated that Swollenin has an inflation function without hydrolysis activity.3. Because the content of Swollenin in the mixtures of Trichoderma extracellular enzymes is very low, heterologous over expression can be helpful to the futher study of the activity, molecular engineering and evolution of enzyme. While heterologous expressing some cellulase from fungi or bacteria is difficult, we constructed a recombinant plasmid containing swollenin gene from T. reesei QM9414 and T. pseudokoningii S38 in order to heterologous express them in the prokaryotic or eukaryotic host. These plasmids were respectively transformed into E. coli, B. subtilis, S. cerebisiae and T. reesei. SDS-PAGE and Western Blotting proved that SWOI expressed in eukaryotic host is active, and their production in the T. reesei host can be up to 62 g/L or 0.55 g/L which is several hundredfolds of swol secreted by itself.4. We purified the recombinant expressed protein swollenin from Trichoderma through the ion exchange chromatography of the CM-sepharose and the Mono-S HPLC and the result of SDS-PAGE shows that its molecular weight is about 75 kDa, and the purified swollenin had a slight activity towardsβ-1,3-glucan, mannan and xylan, but no activity towards hydroxyethyl cellulose. The detected enzyme activities were very low and there is no reducing sugar produced in their action on solid cellulose substrates.5. The experiment proved the Swollenin can swell cotton fiber and change the structure of fiber and this change can be observed by the scan electronic microscope. It is calculated that the swollenin can open a hand between fiber and make the crystallize district in its cellulose structure more amorphously. It is discovered that Swollenin really can help the cellulase hydrate the natural high-crystallinity cellulose substrate and its degree of synergism (DS) can reach to 1.24. So it is concluded that Swollenin can remove many sugar chains from the cellulose surface and therefore make fungi cellulase more easily to get into the inner part of fiber and touch more bottom things, in the meantime glucan chain in the microfiber surface is more sensitive to the attack of cellulase, and thus help the cellulase hydrate natural cellulose substrate.6. The thermophilic endoglucanase CelA from Clostridium thermocellum, one of the family 8 inverting glycosidases sharing the same catalystic mechanism as the family 45 glycosidases, was successfully and highly expressed in Bacillus subtilis. In combination with its crystal structural information, potentially important residues at the catalytic site were systematically changed by site-directed mutagenesis in order to dissect the catalytic mechanism of CelA. The enzyme was isolated by Ni2+-affinity purification, and results showed that site-directed substitution of D278 with an asparagine or an alanine residue surprisingly showed no dramatic decrease in apparent kcat value. Further substitutions of two other potentially critical residues, Y215 and D152, resulted in a 2-fold decrease in apparent kcat value for Y215P and complete loss of activity for D152N. Therefore, Asp278 may not play the important role in CelA catalysis as has been suggested previously by structural analyses. On the other hand, D152 plays a crucial role in the catalysis of hydrolysis and Y215 probably functions only in coordinating the position of the nucleophilic water molecule in an orientation proper for catalysis.更多还原