The Study Of Biodegradation And Plant Cell Wall Structure Using AFM Single Molecular Recognition Technique

The main challenges of large-scale biochemical conversion involve the high costs of cellulolytic enzymes and the inefficiency in enzymatic deconstruction of polysaccharides embedded in the complex structure of the plant cell wall,leading to ongoing interests in studying the predominant mode of enzymatic hydrolysis.Having a single-molecule research on the transform mechanism of biofuel becomes a very important thing.The interaction between the modified AFM tip and the specific component will lead to the decrease of the resonance amplitude.This corresponding signal can be processed to produce recognition image.The technique using both the high-resolution AFM topography image and specific recognition image is named as PicoTREC.PicoTREC is a powerful and new-type biochemical detection technology.The ligand molecules modified on AFM tips can have interaction with on one specific component in the complex sample.We can distinguish this component,and then observe it in real-time during a series of dynamic process.In this study,complete enzymatic hydrolysis of natural cell wall substrates and pretreated biomass substrates was visualized in situ and in real time by atomic force microscopy(AFM)topography and recognition imaging.I also used the same technique to observe the generation of different polysacarides and glycoproteins on the surface of protoplast during the whole cell wall regeneration process.The main works and conclution in this thesis are as follows:1)We studied the binding kinetics of family 3 carbohydrate-biding module(CBM 3a)molecules to crystalline cellulose fibrils by AFM recognition imaging.Single molecule dynamic force spectroscopy(SMDFS)was applied to study the affinity between CBM 3a and crystalline cellulose.Quantitatively,the unbinding force between the CBM 3a and crystalline cellulose was determined as44.96±18.80 pN.The minimal effective initial CBM 3a concentration was found to be 5.1?10~-77 M.Dissection of the binding mechanism of cellulose-specific CBMs at single-molecule level may lead to better understanding of biomass-molecule interaction.2)CBM 3a molecules were modified on the AFM tips to recognize crystalline cellulose.I scanned the natural cell wall sample and their mutant,and compared their structures and components.During the hydrolysis process of plant cell wall,Endoglucanases(EG)preferentially hydrolyzes the outermost amorphous parts covered on the crystalline cellulose.Therefore,the action of EG would expose pure crystalline cellulose to be hydrolyzed more easily with exoglucanases(CBH I).The hydrolysis mode of natural cell wall sample by CBH I depends on the contact area of the sample surface.Because of the complex structure of the natural cell wall sample,it is very hard to get the specific cellulose hydrolysis information.I observed in real-time and quantitatively monitored the complete hydrolytic process on pretreated cellulose.The synergetic effect among the different enzymes can accelerate the cellulose hydrolysis rate dramatically.However,the combination of exoglucanase(CBH I)and?-glucosidases(?-G)exhibited a similar degradation capacity as did whole enzyme(contains the cellobiohydrolases and endoglucanase as its major enzyme components).3)A comprehensive dynamic analysis was developed for individual cellulase acting on single pretreated cellulose through use of functional atomic force microscopy(AFM)topography and recognition imaging.The single crystalline cellulose was divided into different regions based on the cracks on the substrate surface and was observed to either depolymerize or peel away by the jammed enzyme molecules.Then I focused on the observation and analysis of the peeling of cellulose microsfibers.After the exfoliation of one region,new cracks were produced for the enzyme molecules to immobilize.The fiber width may have a relationship with the peeling mode of the fibers.I performed a statistical height measure of the generated peaks of the peeled fibers.The height values range from 11 to 24 nm.I assume that the CBH I enzymes stop progressing along the cellulose microfibril when the peeled microfibril height exceeds 11 nm.4)I modified the AFM tips by different kinds of monoclonal antibodies of polysacarides and glycoprotein(JIM 14,JIM 13 and LM 2 for Arabinogalactan protein;.LM 5 and LM 13 for pectin),CBM 3a molecule was still used to recognize crystalline cellulose in this part of research.During the whole cell wall regeneration process,I observed the produce of different polysacarides and protein in real-time.I did not find any recognition signal of cellulose on the surface of protoplasts.However,I found the recognition signal of Arabinogalactan protein(AGP)and pectin.Depend on a series of AFM images,I calculated the average roughness value and the recognition area percentage at different time points during the whole cell wall regeneration process.By scanning the protoplacts processed by Phospholipase C(PLC),I found that pectin and AGP existed together.Comparing the recognition signal changes of cellulose,AGP and pectin,I got the order of they appeared:first is pectin,then is AGP,the last is cellulose.