Study On The Mechanism Of Cellulose Utilization By Cytophaga Hutchinsonii

Cellulosic biomass from plants is the most abundant renewable resource on the earth. With the running out of nonrenewable fossil resources such as oil and coals, developping new renewable resources has becoming great necessary. So more and moer attention has been payed to the use of cellulosic biomass these years. However, only a small proportion of biomass is uesd resulted from the special properties of cellulose and little knowledgement about the cellulolytic microorganisms and cellulases.There are many kinds of cellulolytic microorganisms in the natural environments using different strategies to degradate cellulose. An aerobic bacterium, Cytophaga hutchinsonii, has a great ability of utilizing crystalline cellulose with an unknown mechanism. A new strategy different from the known noncomplexed cellulase system and complexed cellulosome may be used by C. hutchinsonii. The cellulose utilization mechanism of C. hutchinsonii was studied in this work. The utilization of different carbon sources and the membrane cellulases were studied. A hypothetical model of cellulose utilization by C. hutchinsonii was proposed. The major results of this thesis are as follows:1. Growth, substrates consumption and products were analyzed when C. hutchinsonii was grown on different carbon sources. The distribution of cellulases in the cell was studied at the same time.The substrates consumption and biomass yields were similar when C. hutchinsonii was grown on crystalline cellulose and regenerated amorphous cellulose (RAC); A small amount of glucose, cellobiose and cellotriose were detected from the culture of C. hutchinsonii when grown on cellulose; The resting cells of C. hutchinsonii have abilities of hydrolyzing crystalline cellulose to release cellodextrins and hydrolyzing cellodextrins directly; There is an inducing effect on the utilization of cellobiose and cellodextrins by C. hutchinsonii with cellobiose. Based on the set up cell fractionation technique, the distribution of cellulases in the cell was analyzed, which revealed the carboxymethylcellulase activity on the outer membrane was significantly induced by crystalline cellulose and the cellobiase activity on the cytoplasmic membrane was induced by cellobiose. 2. The FACE technique was set up and first used successfully in the study of cellulose utilization mechanism by C. hutchinsonii.Cellodextrins were separated well when the concentration of the separation gel was over 32% in FACE; The ANTS labled substrates were very stable in high temperature. Cellulose materials such as Avicel, RAC and Filter paper were successfully labled by ANTS. FACE can be used to analyze products from cellulose and cellodextrins released by cellulases. Moreover, it can analyze the positions of glucosidic bonds that the cellulases attack on cellodextrins.3. The membrane associated protein extraction technique was set up, based on which, the Blue Native-PAGE technique was further set up to analyze the potential prorein complexs located on the membranes of C. hutchinsonii.Different detergents were compared in aspect of their ability in dissolving proteins from membranes and maintaining their activities, and n-dodecyl-β-D-maltoside (DDM) was chosen in the protein ectraction techbnique at last. The Blue Native-PAGE technique was further set up to analyze the prorein complexs located on the membranes. This technique can separate prorein complexs keeping their activities, and the components can be identified by Mass Spectrometry.4. Three cellulases were purified from the outer membranes of C. hutchinsonii for the first time. The properties of the purified cellulases were further studied.The outer membrane proteins were separated by DEAE Sepharose Fast Flow chromatography, Q Sepharose Fast Flow chromatography and Sephacryl S-100 HR gel filtration chromatography, and three cellulases were purified. The three purified cellulases were identified by MALDI-TOF. They were CHU₁107, CHU₁075 and CHU₃84. The molecular weights are 136.2 kDa,274.8 kDa and 33.8kDa respectively. The optimum reaction pH for CMCase is all 5.0, and the optimum reaction temperatures are 45℃,40℃and 45℃respectively. The three enzymes all have activities on CMC, Lichenan, Xylan and Cellodextrins. CHU₁107 and CHU₁075 have activities on RAC and Chitin respectively. The detergents DDM and TritonX-100 can enhance the CMCase activity of CHU₁107.5. The catalytic domains of CHU₁107 and CHU₁075 were expressed successfully in E. coli BL21. The properties of catalytic domains of CHU₁107 and key amino acids in the catalytic center were further studied.CHU 1107GH5 has activities on CMC, Lichenan, Cellodextrins (DP>2), RAC, Filter paper and Avicel with cellobiose as the main product. CBM from CBH I can enhance the activity on Avicel of CHU₁107GH5. The CrI of Avicel was decreased after cultured with CHU₁107GH5 with the average DP of Avicel unchanged, which revealed CHU₁107GH5 is a processive endoglucanase. CHU₁107GH5 is homologous with the processive endoglucanase Ce15H from Saccharophagus degradans. Amino acid residues H131, H225 and E166 in the catalytic center play key roles in the catalytic activity of CHU₁107GH5 and aromatic residues Trp61 and Trp308 around the catalytic center may involved in binding to cellulose.6. The genetical manipulation techniques including random insertions to the genome by a trasposon pHimarEml and gene interuption by a suicide plasmid pLYL03 were set up. The first gene was interupted in C. hutchinsonii.A mutant library was set up through random insertions to the genome of C. hutchinsonii by pHimarEml. The function of a promoter PompA from F. johnsoniae was tested in C. hutchinsonii by a GFP expression cassete. The gene chu₁107 was interupted by a suicide plasmid pLYL03, which led to a decrease of CMCase activity on the outer membrane. The ability of utilizing cellulose by C. hutchinsonii was also decreased, which revealed CHU₁107 may play an important role in cellulose utilization by C. hutchinsonii.