Research On The Cellulase System Of Cytophaga Hutchinsonii

With the improvement of industry and the increase of population, the problems of energy crisis and environment pollution become the main challenges for the survival and developing of our human society. Solar energy is the main energy resource for the planet. As the fixed solar energy, biomass is certainly the most abundant carbon resource and maybe the renewable energy to support our society. Nearly half of the biomass is cellulose, which we could use as the clean and renewable energy. However, for its insolubility and crystalline regions, cellulose is hard to utilize, becoming the main challenge for developing cellulose energy. Now the crystalline regions are usually destructed by physical or chemical methods, which are energy consumed and environment harmful. So the bioconversion of cellulose is urged.Cytophaga hutchinsonii is Gram-nagetive cellulolytic bacterium belonging to the phylum Bacteroidetes and wide-spread in soil. C. hutchinsonii could digest cellulose rapidly and sufficiently. The cellulases in C. hutchinsonii are all cell-associated and no cellulases are secreted outside the cell, also no reducing sugars were detected outside the cell during the digestion of cellulose. Genomic analysis revealed that in C. hutchinsonii there were no genes encoding dockerin or cohesion, which were important components of cellulosomes. C. hutchinsonii may employ a novel cellulose digest mechanism different from the two well-known strategies. Research on the novel mechanism may help us realize the bioconversion of cellulose. However we know little about this mechanism and most of the cellulases in C. hutchinsonii were unknown to us. Genomic analysis indicated C. hutchinsonii code for no exoglucanases and its cellulases contained no carbohydrate binding modules （CBM）. The research on cellulases in C. hutchinsonii was of much importance, which may help us make the novel cellulose digest mechanism clear and find some novel cellulases that could be used in industry.In this study, some cellulses in C. hutchinsonii were expressed in E. coli, characterization of the cellulases revealed that three novel cellulases were found in C. hutchinsonii.1. Research on the novel processive endoglucanase CHU₂103Generally, processive cellulases included exoglucanases and processive endoglucanases, which could digest cellulose processively with the main products of cellobiose and cellotriose and play important roles in the digestion of cellulose. Genomic analysis indicated there were no genes coding for exoglucanases in C. hutchinsonii, but there were two endoglucanases （CHU₁107 and CHU₂103） that were similar with the known processive endoglucanases. CHU₂103 consisted of 346 amino acid residues and contained a GH5 （Glycoside hydrolase） conserved domain. Different from other processive endoglucanases belonging to GH5, CHU₂103 contained no CBMs.CHU₂103 was expressed in E. coli, and active protein was obtained. We found CHU₂103 could not only digest carboxymethylcellulose （CMC） but also digest 4-nitrophenyl beta-D-cellobioside （pNPC） and cellotriose which were usually the substrates of exoglucanases and could not be digested by typical endoglucanases. It indicated CHU₂103 possessed the activity of both endoglucanase and exoglucanase. The final products of CHU₂103 hydrolyzing insoluble cellulose were cellobiose and cellotriose and 80% of the reducing ends came from the soluble fraction, indicating CHU₂103 may digest cellulose processively producing much soluble reducing sugars. When digesting CMC, CHU₂103 could decrease the viscosity of CMC solution in a short time and the final products were a series of cellodextrins with different degree of polymerization （DP）, indicating CHU₂103 hydrolyzed CMC with an endo-mode. All the above proved CHU₂103 was a processive endoglucanase. When CHU 2103 hydrolyzing cellodextrins, little glucose was produced, and when CHU₂103 hydrolyzing pNP-cellodextrins larger-molecular-weight cellodextrins were detected, indicating CHU₂103 possessed the both hydrolyzation and transglycosylation activity.Different from most reported processive endoglucanases, CHU₂103 contained only a catalytic domain without carbohydrate binding domain. However, CHU₂103 could absorb to cellulose, indicating its ability to bind to cellulose resided in the catalytic domain. Site-mutation of the conserved amino acids in the catalytic domain proved that W197 was the key amino acid for the processivity and absorption to cellulose of CHU 2103. When W197 mutated into alanine, CHU 2103 could neither absorb to cellulose nor act processively on cellulose, becoming a typical endoglucanase.The mutant strain in which chu₂103 was disrupted exhibited a decrease of CMCase activity of about 35% and 25% for the intact cells and cellular extracts, respectively, suggesting CHU₂103 is one of the main endoglucanases of C. hutchinsonii. However, Chin2103 showed no obvious growth defect on Avicel PH-101 or filter paper. So it is not essential for crystalline cellulose degradation by C. hutchinsonii.2. Research on the Ca2+-dependent processive endoglucanase CHU₁280CHU 1280 consisted of 571 amino acid residues and contained a conserved catalytic domain belonging to GH9 without CBM. It was similar to an exoglucanase in C. thermocellum with an identity of 29%. The three dimension structure of CHU₁280 was similar to Cel D in C. thermocellum, and two of the Ca2+-binding sites in Cel D were conserved in CHU₁280. CHU₁280 was expressed in E. coli and protein with activity was obtained. We found that CHU₁280 showed no activity in the absence of Ca2+, and after CaCl2 was added into the reaction mixture CHU₁280 could hydrolyze both CMC and pNPC, indicating its activity was Ca2+-dependent.CHU₁280 could hydrolyze CMC with the products of a series of cellodextrins with different DP, and decrease the viscosity of CMC solution in a short time. When insoluble cellulose was used as the substrate, the final products were mainly cellobiose and glucose, and nearly 80% of the reducing ends produced came from the soluble fraction, indicating CHU₁280 hydrolyzed CMC with an endo-mode and could hydrolyze insoluble cellulose processively. All the above proved that CHU₁280 was a processive endoglucanase.CHU₁280 could hydrolyze cellodextrins including cellotriose, cellotetraose and cellopentrose. The main products from cellotriose were cellobiose and glucose while the products from cellotetraose were mainly cellobiose with little glucose and cellotriose and the products from cellopentrose were mainly cellobiose and cellotriose with little glucose and cellotetraose, indicating CHU₁280 was prone to hydrolyze the internal glycoside bonds in the cellulose chain and could also produce little glucose from the non-reducing ends of the cellulose chain.CHU₁280 contained no CBMs, but it could bind to cellulose. Its binding to cellulose also depended on Ca2+, indicating that the binding of Ca2+ to CHU₁280 might change the structure of CHU₁280, making it obtain the ability to bind to and hydrolyze cellulose.Generally, it was thought that Ca2+ enhanced the activity of cellulases by making them more stable. However, we found that Ca2+ made the active site mores stable while the whole conformation more unstable.3. Research on the cellodextinase CHU₂268CHU 2268 consisted of 758 amino acid residues and contained a conserved domain belonging to GH3, speculated as a β-glucosidase belonging to GH3. The three dimensional structure of CHU₂268 was similar to that of the exo-hydrolyzase Exo I from baley and that of exo-β-1,3/1,4-glucanase Exo P from Psedoalteromonas, both the similarity were more than 30%. CHU₂268 contained no signal peptide sequence predicted by SignalP, however we found the amino acid residues 1-26 were hydrophobic and might be the signal peptide.CHU₂268 was expressed in E. coli functionally. We found that CHU₂268 could hydrolyze not only pNPG but also pNPC, and the final products were both pNP and glucose. When CHU₂268 hydrolyzing pNPC, pNPG and glucose were first produced, indicating CHU₂268 hydrolyzed pNPC with an exo-mode from the non-reducing end.CHU₂268 could hydrolyze both cellobiose and cellodextrins, and all the final products were glucose. Moreover, the Km values on the different substrate were similar, indicating the optimum substrates of CHU₂268 were cellobiose and cellodextrins, proving that CHU₂268 was cellodextrinase. We found CHU₂268 hydrolyze cellodextrins also with an exo-mode producing glucose from the non-reducing ends. And larger-molecular-mass cellodextrins were detected during the hydrolyzation of cellodextrins, indicating CHU₂268 possess the transglycosylation activity.CHU₂268 could act synergistically with endoglucanases on cellulose producing glucose, and its synergy with endoglucanases was more than β-glucosidases. CHU₂268 might play important role in the degradation of cellulose by C. hutchinsonii.4. Probing the protein complexes in the membrane of C. hutchinsonii by Blue-native PAGEWe found different type of cellulases on the membrane of C. hutchinsonii, however whether these cellulases were combined and acted together on cellulose was not clear. Research on the protein on the membrane of C. hutchinsonii interacted with the cellulases might help us answer the question.Blue-native PAGE （BN-PAGE） was developed for the separation of mitochondrial membrane proteins and complexes. BN-PAGE could be used to identify physiological protein-protein interactions and in-gel activity assays for the protein complexes separated by this way retained activity. BN-PAGE was combined with SDS-PAGE, in-gel activity assays and protein MS to identify the protein complexes on the membrane of C. hutchinsonii. Three protein complexes which might participate in the utilization of glucose and amino acids were identified. And three protein complexes containing P-glucosidases were identified, one of which might contain six different proteins, these proteins need further research.Now we know more about the cellulases in C. hutchinsonii and speculate that the degradation of cellulose by C. hutchinsonii might depend on the synergic action of endoglucanases, processive endoglucanases β-glucosidases and cellodextrinases. The research on the cellulases may help to reveal the unique mechanism employed by C. hutchinsonii to digest cellulose.