| Plant biomass is one of the most abundant renewable resources on earth.The effective translation of solid-state biomass into C02 and other micromolecule is key to maintain the carbon cycle on earth.Lignin,hemicellulose and cellulose are three major ingredients that constitute plant biomass.Among them,crystalline cellulose is act like the framework which decelerates the degration of biomass and hinders its industrialized application.To accelerate the effective use of plant biomass,we must fully understand the ultrastructure of natural cellulose which finally forms biomass recalcitrance.Besides,the studies on how biomass recalcitrance influences physical,chemical and biological treatments,the sole function of the abundant cellulase act during its hydrolysis that secrete by microorganism are also significant.In this paper,with the help of visualization,we make a thorough inquiry into the relationship between pretreatments and structural of cellulosic substrates from ultrastructural level.We also utilized bioinformatics to analyze the degrading-process of natural cellulose hydrolyzed by T.reeseithree major endoglucanases.The main points and results are as follows.(1)Ultrastructure of natural cellulose indicates that the damage of quarternary structure should be the key step to destroy biomass recalcitrance.Three common treatments like dilute acid,T.reesei cellulase and C.thermocellum LQR1 are used to hydrolyze CF11 cotton fibre.The structure of surface microfibril was destroyed after hydrolysis while inner microfibril gathered tightly as control group.However,dilute acid treatments can generate some small gaps between bunches of microfibril while cellulase and microorganism didn't generate.Hydrolyzing the hydrolyzed CF11 substrates using T.reesei cellulase and C.thermocellum LQR1,dilute acid increased conversion rate which indicates dilute acid may open ways for cellulase touching inner microfibril by destroying quarternary structure of CF11 substrate;the percent conversion of hydrolyzed substrates degraded by C.thermocellum LQR1 achieved 75?80%which shows great ability in hydrolyzing crystalline cellulose without destroying quarternary structure of substrates.(2)The characteristics of T.reesei three major endoglucanases and the analysis of synergy among endoglucanases.In this paper,we expressed T.reesei three major endoglucanases by utilizing E.coli heterologous expression-system.Physicochemical properties including enzyme activity,optimum reaction conditions show that endoglucanases which expressed by heterologous expression-system have no difference with endoglucanases expressed by wild types.Taking advantage of bioinformatic method,we also contrastive analyzed the kinds,numbers,conservatism of key AAs in each of glycoside hydrolase(GH)family which contains EG I,EG II,EGIII,trying to explore the differences between them.Besides,researches on soluble substrates hydrolysis showed a greater percent conversion which indicates the synergistic reaction among three endoglucanases.Different substrates would also change the percent-conversion and degree of synergy,indicating specific cellulose-hydrolyzing mechanism.(3)The analysis of EG?,EG?,EG? in hydrolyzing natural crystalline cellulose.Using EG?,EG?,EG? hydrolyzing insolubility,combining with visualizable method,observed substrates' surface structure.The surface of hydrolyzed substrate generated numbers of particles which is the residual microfibril resisting enzymolysis.By determining the length of particles,each endoglucanases has a specific range of particle length indicating EG?,EG ?,EG? have specific binding site.According to microscopic images,we think EG? may have ability in swelling crystalline cellulose while EG ? mainly hydrolyzes amorphous cellulose which suggests ability in hydrolyzing lateral microfibril and hardly degrades crystalline cellulose.EG ? has a same function with EG? in swelling crystalline cellulose,but the binding site should be know from EG I. |
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