| 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.
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