Study On The Reaction Mechanism Of Cellulose Hydrating Protein

As an important feedstock of bioethanol production,cellulose is difficult to be directly assimilated by microorganisms due to its complex structure.In recent years,the research on cellulose biodegradation is still in its infancy;However,there have been some hypothetic mechanisms of cellulose depolymerization.One important theory is the C₁-C_X process,which supposed that the compact structure of cellulose is loosen firstly by a certain swelling factor?C1?,and then degraded by a catalytically active hydrolase.Up to now,this theory has not been fully proved,and the research of C1 factor mainly focused on Expansins,Swollenins and EXLX1 proteins,and C₁-C_X reaction process remains unclesr.Therefore,it is of great theoretical value to the behavior of C₁-C_X.In our previous work,a novel protein named C22was isolated from the cellulose degrading strain Arthrobotrys sp.Cx1 The C22 protein belongs to the glycoside hydrolase 6 family?GH6?,and previous research showed that C22caused the hydration the filter paper,The C22 consists of a GH6 core catalytic domain?CLF?,a linker and a binding module-?CBM1?.In order to further study the C1-Cx mechanism the reaction behaviors of different C22 domains were observed in this work.Quantitative analysis mainly included:kinetics parameter measurement,detection of enzymatic activity and the determination of cellulose crystallinity and hydration values.Qualitative experiments included the observation of the morphological changes of cellulose substrates treated by C22recombinant proteins,and the binding abilities of these recombinants.The results showed that the water retention value of the filter paper treated by C22,CF and CLF increased by 25.6%,21.5%,and 14.7%,respectively,The decreased water retention value of CLF treated substrate indicating that linker plays a major role in the hydration process.The crystallinity?CrI?of the filter paper treated with the three recombinant proteins was examined by X-ray diffraction.The results showed that CrI of the treated filter paper reduced by about 13%compared to that of the untreated filter paper,and no difference was observed between substrates treated with different recombinants.This result suggested that CBM and Linker do not have the ability to destroy the crystalline structure of cellulose.The adsorption isotherms of the three recombinant proteins showed that the adsorption capacities of C22,CF and CLF on the filter paper and Avicel decreased in turn,indicating that the CBM and Linker could help the core catalytic domain to bind to the cellulose substrate.Qualitative experimental results show that,the CLF-treated cotton fiber swelled significantly,its diameter indicating that CBM and Linker could not destroy the cellulose crystal structure.Thus,we hypothesized that,during the cellulose hydration process,CBM and Linker lead to the binding of catalytic domain of C22 to the cellulose substrate,then the catalytic domain destroy the cellulose crystallinity and the water molecules entered the cellulose fiber with the help of the linker.Our work should be valuable for clarifying the cellulose C1-Cx degradation mechanism.